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Sample records for poliovirus rna-dependent rna

  1. Multiple isoelectric forms of poliovirus RNA-dependent RNA polymerase: Evidence for phosphorylation

    International Nuclear Information System (INIS)

    Ransone, L.J.; Dasgupta, A.

    1989-01-01

    Poliovirus-specific RNA-dependent RNA polymerase (3Dpol) was purified to apparent homogeneity. A single polypeptide of an apparent molecular weight of 63,000 catalyzes the synthesis of dimeric and monomeric RNA products in response to the poliovirion RNA template. Analysis of purified 3Dpol by two-dimensional electrophoresis showed multiple forms of 3Dpol, suggesting posttranslational modification of the protein in virus-infected cells. The two major forms of 3Dpol appear to have approximate pI values of 7.1 and 7.4. Incubation of purified 3Dpol with calf intestinal phosphatase resulted in almost complete disappearance of the pI 7.1 form and a concomitant increase in the intensity of the pI 7.4 form of 3Dpol. Addition of 32P-labeled Pi during infection of HeLa cells with poliovirus resulted in specific labeling of 3Dpol and 3CD, a viral protein which contains the entire 3Dpol sequence. Both 3Dpol and 3CD appear to be phosphorylated at serine residues. Ribosomal salt washes prepared from both mock- and poliovirus-infected cells contain phosphatases capable of dephosphorylating quantitatively the phosphorylated form (pI 7.1) of 3Dpol

  2. Improved crystallization of the coxsackievirus B3 RNA-dependent RNA polymerase

    Energy Technology Data Exchange (ETDEWEB)

    Jabafi, Ilham; Selisko, Barbara; Coutard, Bruno; De Palma, Armando M.; Neyts, Johan; Egloff, Marie-Pierre; Grisel, Sacha; Dalle, Karen; Campanacci, Valerie; Spinelli, Silvia; Cambillau, Christian; Canard, Bruno; Gruez, Arnaud, E-mail: arnaud.gruez@maem.uhp-nancy.fr [Centre National de la Recherche Scientifique and Universités d’Aix-Marseille I et II, UMR 6098, Architecture et Fonction des Macromolécules Biologiques, Ecole Supérieure d’Ingénieurs de Luminy-Case 925, 163 Avenue de Luminy, 13288 Marseille CEDEX 9 (France)

    2007-06-01

    The first crystal of a coxsackievirus RNA-dependent RNA polymerase is reported. The Picornaviridae virus family contains a large number of human pathogens such as poliovirus, hepatitis A virus and rhinoviruses. Amongst the viruses belonging to the genus Enterovirus, several serotypes of coxsackievirus coexist for which neither vaccine nor therapy is available. Coxsackievirus B3 is involved in the development of acute myocarditis and dilated cardiomyopathy and is thought to be an important cause of sudden death in young adults. Here, the first crystal of a coxsackievirus RNA-dependent RNA polymerase is reported. Standard crystallization methods yielded crystals that were poorly suited to X-ray diffraction studies, with one axis being completely disordered. Crystallization was improved by testing crystallization solutions from commercial screens as additives. This approach yielded crystals that diffracted to 2.1 Å resolution and that were suitable for structure determination.

  3. Improved crystallization of the coxsackievirus B3 RNA-dependent RNA polymerase

    International Nuclear Information System (INIS)

    Jabafi, Ilham; Selisko, Barbara; Coutard, Bruno; De Palma, Armando M.; Neyts, Johan; Egloff, Marie-Pierre; Grisel, Sacha; Dalle, Karen; Campanacci, Valerie; Spinelli, Silvia; Cambillau, Christian; Canard, Bruno; Gruez, Arnaud

    2007-01-01

    The first crystal of a coxsackievirus RNA-dependent RNA polymerase is reported. The Picornaviridae virus family contains a large number of human pathogens such as poliovirus, hepatitis A virus and rhinoviruses. Amongst the viruses belonging to the genus Enterovirus, several serotypes of coxsackievirus coexist for which neither vaccine nor therapy is available. Coxsackievirus B3 is involved in the development of acute myocarditis and dilated cardiomyopathy and is thought to be an important cause of sudden death in young adults. Here, the first crystal of a coxsackievirus RNA-dependent RNA polymerase is reported. Standard crystallization methods yielded crystals that were poorly suited to X-ray diffraction studies, with one axis being completely disordered. Crystallization was improved by testing crystallization solutions from commercial screens as additives. This approach yielded crystals that diffracted to 2.1 Å resolution and that were suitable for structure determination

  4. The RNA Template Channel of the RNA-Dependent RNA Polymerase as a Target for Development of Antiviral Therapy of Multiple Genera within a Virus Family

    NARCIS (Netherlands)

    van der Linden, Lonneke; Vives-Adrián, Laia; Selisko, Barbara; Ferrer-Orta, Cristina; Liu, Xinran; Lanke, Kjerstin; Ulferts, Rachel; De Palma, Armando M; Tanchis, Federica; Goris, Nesya; Lefebvre, David; De Clercq, Kris; Leyssen, Pieter; Lacroix, Céline; Pürstinger, Gerhard; Coutard, Bruno; Canard, Bruno; Boehr, David D; Arnold, Jamie J; Cameron, Craig E; Verdaguer, Nuria; Neyts, Johan; van Kuppeveld, Frank J M

    2015-01-01

    The genus Enterovirus of the family Picornaviridae contains many important human pathogens (e.g., poliovirus, coxsackievirus, rhinovirus, and enterovirus 71) for which no antiviral drugs are available. The viral RNA-dependent RNA polymerase is an attractive target for antiviral therapy.

  5. RNA binding and replication by the poliovirus RNA polymerase

    International Nuclear Information System (INIS)

    Oberste, M.S.

    1988-01-01

    RNA binding and RNA synthesis by the poliovirus RNA-dependent RNA polymerase were studied in vitro using purified polymerase. Templates for binding and RNA synthesis studies were natural RNAs, homopolymeric RNAs, or subgenomic poliovirus-specific RNAs synthesized in vitro from cDNA clones using SP6 or T7 RNA polymerases. The binding of the purified polymerase to poliovirion and other RNAs was studied using a protein-RNA nitrocellulose filter binding assay. A cellular poly(A)-binding protein was found in the viral polymerase preparations, but was easily separated from the polymerase by chromatography on poly(A) Sepharose. The binding of purified polymerase to 32 P-labeled ribohomopolymeric RNAs was examined, and the order of binding observed was poly(G) >>> poly(U) > poly(C) > poly(A). The K a for polymerase binding to poliovirion RNA and to a full-length negative strand transcript was about 1 x 10 9 M -1 . The polymerase binds to a subgenomic RNAs which contain the 3' end of the genome with a K a similar to that for virion RNA, but binds less well to 18S rRNA, globin mRNA, and subgenomic RNAs which lack portions of the 3' noncoding region

  6. Nucleobase but not Sugar Fidelity is Maintained in the Sabin I RNA-Dependent RNA Polymerase.

    Science.gov (United States)

    Liu, Xinran; Musser, Derek M; Lee, Cheri A; Yang, Xiaorong; Arnold, Jamie J; Cameron, Craig E; Boehr, David D

    2015-10-26

    The Sabin I poliovirus live, attenuated vaccine strain encodes for four amino acid changes (i.e., D53N, Y73H, K250E, and T362I) in the RNA-dependent RNA polymerase (RdRp). We have previously shown that the T362I substitution leads to a lower fidelity RdRp, and viruses encoding this variant are attenuated in a mouse model of poliovirus. Given these results, it was surprising that the nucleotide incorporation rate and nucleobase fidelity of the Sabin I RdRp is similar to that of wild-type enzyme, although the Sabin I RdRp is less selective against nucleotides with modified sugar groups. We suggest that the other Sabin amino acid changes (i.e., D53N, Y73H, K250E) help to re-establish nucleotide incorporation rates and nucleotide discrimination near wild-type levels, which may be a requirement for the propagation of the virus and its efficacy as a vaccine strain. These results also suggest that the nucleobase fidelity of the Sabin I RdRp likely does not contribute to viral attenuation.

  7. Nucleobase but not Sugar Fidelity is Maintained in the Sabin I RNA-Dependent RNA Polymerase

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    Xinran Liu

    2015-10-01

    Full Text Available The Sabin I poliovirus live, attenuated vaccine strain encodes for four amino acid changes (i.e., D53N, Y73H, K250E, and T362I in the RNA-dependent RNA polymerase (RdRp. We have previously shown that the T362I substitution leads to a lower fidelity RdRp, and viruses encoding this variant are attenuated in a mouse model of poliovirus. Given these results, it was surprising that the nucleotide incorporation rate and nucleobase fidelity of the Sabin I RdRp is similar to that of wild-type enzyme, although the Sabin I RdRp is less selective against nucleotides with modified sugar groups. We suggest that the other Sabin amino acid changes (i.e., D53N, Y73H, K250E help to re-establish nucleotide incorporation rates and nucleotide discrimination near wild-type levels, which may be a requirement for the propagation of the virus and its efficacy as a vaccine strain. These results also suggest that the nucleobase fidelity of the Sabin I RdRp likely does not contribute to viral attenuation.

  8. RNA-dependent RNA polymerases from cowpea mosaic virus-infected cowpea leaves

    NARCIS (Netherlands)

    Dorssers, L.

    1983-01-01

    The aim of the research described in this thesis was the purification and identification of the RNA-dependent RNA polymerase engaged in replicating viral RNA in cowpea mosaic virus (CPMV)- infected cowpea leaves.

    Previously, an RNA-dependent RNA polymerase produced upon infection of

  9. miRNA-dependent translational repression in the Drosophila ovary.

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    John Reich

    Full Text Available The Drosophila ovary is a tissue rich in post-transcriptional regulation of gene expression. Many of the regulatory factors are proteins identified via genetic screens. The more recent discovery of microRNAs, which in other animals and tissues appear to regulate translation of a large fraction of all mRNAs, raised the possibility that they too might act during oogenesis. However, there has been no direct demonstration of microRNA-dependent translational repression in the ovary.Here, quantitative analyses of transcript and protein levels of transgenes with or without synthetic miR-312 binding sites show that the binding sites do confer translational repression. This effect is dependent on the ability of the cells to produce microRNAs. By comparison with microRNA-dependent translational repression in other cell types, the regulated mRNAs and the protein factors that mediate repression were expected to be enriched in sponge bodies, subcellular structures with extensive similarities to the P bodies found in other cells. However, no such enrichment was observed.Our results reveal the variety of post-transcriptional regulatory mechanisms that operate in the Drosophila ovary, and have implications for the mechanisms of miRNA-dependent translational control used in the ovary.

  10. Inhibition of poliovirus RNA synthesis by brefeldin A.

    OpenAIRE

    Maynell, L A; Kirkegaard, K; Klymkowsky, M W

    1992-01-01

    Brefeldin A (BFA), a fungal metabolite that blocks transport of newly synthesized proteins from the endoplasmic reticulum, was found to inhibit poliovirus replication 10(5)- to 10(6)-fold. BFA does not inhibit entry of poliovirus into the cell or translation of viral RNA. Poliovirus RNA synthesis, however, is completely inhibited by BFA. A specific class of membranous vesicles, with which the poliovirus replication complex is physically associated, is known to proliferate in poliovirus-infect...

  11. RNA-dependent RNA polymerase 1 in potato (Solanum tuberosum) and its relationship to other plant RNA-dependent RNA polymerases.

    Science.gov (United States)

    Hunter, Lydia J R; Brockington, Samuel F; Murphy, Alex M; Pate, Adrienne E; Gruden, Kristina; MacFarlane, Stuart A; Palukaitis, Peter; Carr, John P

    2016-03-16

    Cellular RNA-dependent RNA polymerases (RDRs) catalyze synthesis of double-stranded RNAs that can serve to initiate or amplify RNA silencing. Arabidopsis thaliana has six RDR genes; RDRs 1, 2 and 6 have roles in anti-viral RNA silencing. RDR6 is constitutively expressed but RDR1 expression is elevated following plant treatment with defensive phytohormones. RDR1 also contributes to basal virus resistance. RDR1 has been studied in several species including A. thaliana, tobacco (Nicotiana tabacum), N. benthamiana, N. attenuata and tomato (Solanum lycopersicum) but not to our knowledge in potato (S. tuberosum). StRDR1 was identified and shown to be salicylic acid-responsive. StRDR1 transcript accumulation decreased in transgenic potato plants constitutively expressing a hairpin construct and these plants were challenged with three viruses: potato virus Y, potato virus X, and tobacco mosaic virus. Suppression of StRDR1 gene expression did not increase the susceptibility of potato to these viruses. Phylogenetic analysis of RDR genes present in potato and in a range of other plant species identified a new RDR gene family, not present in potato and found only in Rosids (but apparently lost in the Rosid A. thaliana) for which we propose the name RDR7.

  12. In vitro transcription of Sonchus yellow net virus RNA by a virus-associated RNA-dependent RNA polymerase

    NARCIS (Netherlands)

    Flore, P.H.

    1986-01-01

    The aim of the investigation presented in this thesis was to elucidate the nature of the RNA- dependent RNA polymerase, thought to be associated with Sonchus yellow net virus (SYNV), a rhabdovirus infecting plants. This research was initiated to shed light on the

  13. Evolution of Tertiary Structure of Viral RNA Dependent Polymerases

    Czech Academy of Sciences Publication Activity Database

    Černý, Jiří; Černá, B.; Valdés, James J.; Grubhoffer, Libor; Růžek, Daniel

    2014-01-01

    Roč. 9, č. 5 (2014), e96070 E-ISSN 1932-6203 R&D Projects: GA ČR GAP502/11/2116; GA ČR GAP302/12/2490; GA MŠk(CZ) EE2.3.30.0032 Institutional support: RVO:60077344 Keywords : Q-BETA replicase * C virus RNA * crystal structure Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.234, year: 2014

  14. Structural Analysis of Monomeric RNA-Dependent Polymerases: Evolutionary and Therapeutic Implications.

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    Rodrigo Jácome

    Full Text Available The crystal structures of monomeric RNA-dependent RNA polymerases and reverse transcriptases of more than 20 different viruses are available in the Protein Data Bank. They all share the characteristic right-hand shape of DNA- and RNA polymerases formed by the fingers, palm and thumb subdomains, and, in many cases, "fingertips" that extend from the fingers towards the thumb subdomain, giving the viral enzyme a closed right-hand appearance. Six conserved structural motifs that contain key residues for the proper functioning of the enzyme have been identified in all these RNA-dependent polymerases. These enzymes share a two divalent metal-ion mechanism of polymerization in which two conserved aspartate residues coordinate the interactions with the metal ions to catalyze the nucleotidyl transfer reaction. The recent availability of crystal structures of polymerases of the Orthomyxoviridae and Bunyaviridae families allowed us to make pairwise comparisons of the tertiary structures of polymerases belonging to the four main RNA viral groups, which has led to a phylogenetic tree in which single-stranded negative RNA viral polymerases have been included for the first time. This has also allowed us to use a homology-based structural prediction approach to develop a general three-dimensional model of the Ebola virus RNA-dependent RNA polymerase. Our model includes several of the conserved structural motifs and residues described in other viral RNA-dependent RNA polymerases that define the catalytic and highly conserved palm subdomain, as well as portions of the fingers and thumb subdomains. The results presented here help to understand the current use and apparent success of antivirals, i.e. Brincidofovir, Lamivudine and Favipiravir, originally aimed at other types of polymerases, to counteract the Ebola virus infection.

  15. Biochemical characterization of a recombinant Japanese encephalitis virus RNA-dependent RNA polymerase

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    Kim Chan-Mi

    2007-07-01

    Full Text Available Abstract Background Japanese encephalitis virus (JEV NS5 is a viral nonstructural protein that carries both methyltransferase and RNA-dependent RNA polymerase (RdRp domains. It is a key component of the viral RNA replicase complex that presumably includes other viral nonstructural and cellular proteins. The biochemical properties of JEV NS5 have not been characterized due to the lack of a robust in vitro RdRp assay system, and the molecular mechanisms for the initiation of RNA synthesis by JEV NS5 remain to be elucidated. Results To characterize the biochemical properties of JEV RdRp, we expressed in Escherichia coli and purified an enzymatically active full-length recombinant JEV NS5 protein with a hexahistidine tag at the N-terminus. The purified NS5 protein, but not the mutant NS5 protein with an Ala substitution at the first Asp of the RdRp-conserved GDD motif, exhibited template- and primer-dependent RNA synthesis activity using a poly(A RNA template. The NS5 protein was able to use both plus- and minus-strand 3'-untranslated regions of the JEV genome as templates in the absence of a primer, with the latter RNA being a better template. Analysis of the RNA synthesis initiation site using the 3'-end 83 nucleotides of the JEV genome as a minimal RNA template revealed that the NS5 protein specifically initiates RNA synthesis from an internal site, U81, at the two nucleotides upstream of the 3'-end of the template. Conclusion As a first step toward the understanding of the molecular mechanisms for JEV RNA replication and ultimately for the in vitro reconstitution of viral RNA replicase complex, we for the first time established an in vitro JEV RdRp assay system with a functional full-length recombinant JEV NS5 protein and characterized the mechanisms of RNA synthesis from nonviral and viral RNA templates. The full-length recombinant JEV NS5 will be useful for the elucidation of the structure-function relationship of this enzyme and for the

  16. A Structural Overview of RNA-Dependent RNA Polymerases from the Flaviviridae Family

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    Jiqin Wu

    2015-06-01

    Full Text Available RNA-dependent RNA polymerases (RdRPs from the Flaviviridae family are representatives of viral polymerases that carry out RNA synthesis through a de novo initiation mechanism. They share a ≈ 600-residue polymerase core that displays a canonical viral RdRP architecture resembling an encircled right hand with palm, fingers, and thumb domains surrounding the active site. Polymerase catalytic motifs A–E in the palm and motifs F/G in the fingers are shared by all viral RdRPs with sequence and/or structural conservations regardless of the mechanism of initiation. Different from RdRPs carrying out primer-dependent initiation, Flaviviridae and other de novo RdRPs utilize a priming element often integrated in the thumb domain to facilitate primer-independent initiation. Upon the transition to the elongation phase, this priming element needs to undergo currently unresolved conformational rearrangements to accommodate the growth of the template-product RNA duplex. In the genera of Flavivirus and Pestivirus, the polymerase module in the C-terminal part of the RdRP protein may be regulated in cis by the N-terminal region of the same polypeptide. Either being a methyltransferase in Flavivirus or a functionally unclarified module in Pestivirus, this region could play auxiliary roles for the canonical folding and/or the catalysis of the polymerase, through defined intra-molecular interactions.

  17. Enzymatic activities of the GB virus-B RNA-dependent RNA polymerase

    International Nuclear Information System (INIS)

    Ranjith-Kumar, C.T.; Santos, Jan Lee; Gutshall, Lester L.; Johnston, Victor K.; Juili, L.-G.; Kim, M.-J.; Porter, David J.; Maley, Derrick; Greenwood, Cathy; Earnshaw, David L.; Baker, Audrey; Gu Baohua; Silverman, Carol; Sarisky, Robert T.; Kao Cheng

    2003-01-01

    The GB virus-B (GBV-B) nonstructural protein 5B (NS5B) encodes an RNA-dependent RNA polymerase (RdRp) with greater than 50% sequence similarity to the hepatitis C virus (HCV) NS5B. Recombinant GBV-B NS5B was reported to possess RdRp activity (W. Zhong et al., 2000, J. Viral Hepat. 7, 335-342). In this study, the GBV-B RdRp was examined more thoroughly for different RNA synthesis activities, including primer-extension, de novo initiation, template switch, terminal nucleotide addition, and template specificity. The results can be compared with previous characterizations of the HCV RdRp. The two RdRps share similarities in terms of metal ion and template preference, the abilities to add nontemplated nucleotides, perform both de novo initiation and extension from a primer, and switch templates. However, several differences in RNA synthesis between the GBV-B and HCV RdRps were observed, including (i) optimal temperatures for activity, (ii) ranges of Mn 2+ concentration tolerated for activity, and (iii) cation requirements for de novo RNA synthesis and terminal transferase activity. To assess whether the recombinant GBV-B RdRp may represent a relevant surrogate system for testing HCV antiviral agents, two compounds demonstrated to be active at nanomolar concentrations against HCV NS5B were tested on the GBV RdRp. A chain terminating nucleotide analog could prevent RNA synthesis, while a nonnucleoside HCV inhibitor was unable to affect RNA synthesis by the GBV RdRp

  18. RNA-dependent RNA polymerase: Addressing Zika outbreak by a phylogeny-based drug target study.

    Science.gov (United States)

    Stephen, Preyesh; Lin, Sheng-Xiang

    2018-01-01

    Since the first major outbreak of Zika virus (ZIKV) in 2007, ZIKV is spreading explosively through South and Central America, and recent reports in highly populated developing countries alarm the possibility of a more catastrophic outbreak. ZIKV infection in pregnant women leads to embryonic microcephaly and Guillain-Barré syndrome in adults. At present, there is limited understanding of the infectious mechanism, and no approved therapy has been reported. Despite the withdrawal of public health emergency, the WHO still considers the ZIKV as a highly significant and long-term public health challenge that the situation has to be addressed rapidly. Non-structural protein 5 is essential for capping and replication of viral RNA and comprises a methyltransferase and RNA-dependent RNA polymerase (RdRp) domain. We used molecular modeling to obtain the structure of ZIKV RdRp, and by molecular docking and phylogeny analysis, we here demonstrate the potential sites for drug screening. Two metal binding sites and an NS3-interacting region in ZIKV RdRp are demonstrated as potential drug screening sites. The docked structures reveal a remarkable degree of conservation at the substrate binding site and the potential drug screening sites. A phylogeny-based approach is provided for an emergency preparedness, where similar class of ligands could target phylogenetically related proteins. © 2017 John Wiley & Sons A/S.

  19. The RNA template channel of the RNA-dependent RNA polymerase as a target for development of antiviral therapy of multiple genera within a virus family.

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    Lonneke van der Linden

    2015-03-01

    Full Text Available The genus Enterovirus of the family Picornaviridae contains many important human pathogens (e.g., poliovirus, coxsackievirus, rhinovirus, and enterovirus 71 for which no antiviral drugs are available. The viral RNA-dependent RNA polymerase is an attractive target for antiviral therapy. Nucleoside-based inhibitors have broad-spectrum activity but often exhibit off-target effects. Most non-nucleoside inhibitors (NNIs target surface cavities, which are structurally more flexible than the nucleotide-binding pocket, and hence have a more narrow spectrum of activity and are more prone to resistance development. Here, we report a novel NNI, GPC-N114 (2,2'-[(4-chloro-1,2-phenylenebis(oxy]bis(5-nitro-benzonitrile with broad-spectrum activity against enteroviruses and cardioviruses (another genus in the picornavirus family. Surprisingly, coxsackievirus B3 (CVB3 and poliovirus displayed a high genetic barrier to resistance against GPC-N114. By contrast, EMCV, a cardiovirus, rapidly acquired resistance due to mutations in 3Dpol. In vitro polymerase activity assays showed that GPC-N114 i inhibited the elongation activity of recombinant CVB3 and EMCV 3Dpol, (ii had reduced activity against EMCV 3Dpol with the resistance mutations, and (iii was most efficient in inhibiting 3Dpol when added before the RNA template-primer duplex. Elucidation of a crystal structure of the inhibitor bound to CVB3 3Dpol confirmed the RNA-binding channel as the target for GPC-N114. Docking studies of the compound into the crystal structures of the compound-resistant EMCV 3Dpol mutants suggested that the resistant phenotype is due to subtle changes that interfere with the binding of GPC-N114 but not of the RNA template-primer. In conclusion, this study presents the first NNI that targets the RNA template channel of the picornavirus polymerase and identifies a new pocket that can be used for the design of broad-spectrum inhibitors. Moreover, this study provides important new insight

  20. Poliovirus RNA polymerase: in vitro enzymatic activities, fidelity of replication, and characterization of a temperature-sensitive RNA-negative mutant

    International Nuclear Information System (INIS)

    Stokes, M.A.M.

    1985-01-01

    The in vitro activities of the purified poliovirus RNA polymerase were investigated in this study. The polymerase was shown to be a strict RNA dependent RNA polymerase. It only copied RNA templates but used either a DNA or RNA primer to initiate RNA synthesis. Partially purified polymerase has some DNA polymerase activities. Additional purification of the enzyme and studies with a mutant poliovirus RNA polymerase indicated that the DNA polymerase activities were due to a cellular polymerase. The fidelity of RNA replication in vitro by the purified poliovirus RNA polymerase was studied by measuring the rate of misincorporation of noncomplementary ribonucleotide monophosphates on synthetic homopolymeric RNA templates. The results showed that the ratio of noncomplementary to complementary ribonucleotides incorporated was 1-5 x 10 -3 . The viral polymerase of a poliovirus temperature sensitive RNA-negative mutant, Ts 10, was isolated. This study confirmed that the mutant was viable 33 0 , but was RNA negative at 39 0 . Characterization of the Ts 10 polymerase showed it was significantly more sensitive to heat inactivation than was the old-type polymerase. Highly purified poliovirions were found to contain several noncapsid proteins. At least two of these proteins were labeled by [ 35 S]methionine infected cells and appeared to be virally encoded proteins. One of these proteins was immunoprecipitated by anti-3B/sup vpg/ antiserum. This protein had the approximate Mr = 50,000 and appeared to be one of the previously identified 3B/sup vpg/ precursor proteins

  1. Potent host-directed small-molecule inhibitors of myxovirus RNA-dependent RNA-polymerases.

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    Stefanie A Krumm

    Full Text Available Therapeutic targeting of host cell factors required for virus replication rather than of pathogen components opens new perspectives to counteract virus infections. Anticipated advantages of this approach include a heightened barrier against the development of viral resistance and a broadened pathogen target spectrum. Myxoviruses are predominantly associated with acute disease and thus are particularly attractive for this approach since treatment time can be kept limited. To identify inhibitor candidates, we have analyzed hit compounds that emerged from a large-scale high-throughput screen for their ability to block replication of members of both the orthomyxovirus and paramyxovirus families. This has returned a compound class with broad anti-viral activity including potent inhibition of different influenza virus and paramyxovirus strains. After hit-to-lead chemistry, inhibitory concentrations are in the nanomolar range in the context of immortalized cell lines and human PBMCs. The compound shows high metabolic stability when exposed to human S-9 hepatocyte subcellular fractions. Antiviral activity is host-cell species specific and most pronounced in cells of higher mammalian origin, supporting a host-cell target. While the compound induces a temporary cell cycle arrest, host mRNA and protein biosynthesis are largely unaffected and treated cells maintain full metabolic activity. Viral replication is blocked at a post-entry step and resembles the inhibition profile of a known inhibitor of viral RNA-dependent RNA-polymerase (RdRp activity. Direct assessment of RdRp activity in the presence of the reagent reveals strong inhibition both in the context of viral infection and in reporter-based minireplicon assays. In toto, we have identified a compound class with broad viral target range that blocks host factors required for viral RdRp activity. Viral adaptation attempts did not induce resistance after prolonged exposure, in contrast to rapid

  2. The Crystal Structure of the RNA-Dependent RNA Polymerase from Human Rhinovirus: A Dual Function Target for Common Cold Antiviral Therapy

    Energy Technology Data Exchange (ETDEWEB)

    Love, Robert A.; Maegley, Karen A.; Yu, Xiu; Ferre, RoseAnn; Lingardo, Laura K.; Diehl, Wade; Parge, Hans E.; Dragovich, Peter S.; Fuhrman, Shella A. (Pfizer)

    2010-11-16

    Human rhinoviruses (HRV), the predominant members of the Picornaviridae family of positive-strand RNA viruses, are the major causative agents of the common cold. Given the lack of effective treatments for rhinoviral infections, virally encoded proteins have become attractive therapeutic targets. The HRV genome encodes an RNA-dependent RNA polymerase (RdRp) denoted 3D{sup pol}, which is responsible for replicating the viral genome and for synthesizing a protein primer used in the replication. Here the crystal structures for three viral serotypes (1B, 14, and 16) of HRV 3D{sup pol} have been determined. The three structures are very similar to one another, and to the closely related poliovirus (PV) 3D{sup pol} enzyme. Because the reported PV crystal structure shows significant disorder, HRV 3D{sup pol} provides the first complete view of a picornaviral RdRp. The folding topology of HRV 3D{sup pol} also resembles that of RdRps from hepatitis C virus (HCV) and rabbit hemorrhagic disease virus (RHDV) despite very low sequence homology.

  3. Cyclophilin B stimulates RNA synthesis by the HCV RNA dependent RNA polymerase.

    Science.gov (United States)

    Heck, Julie A; Meng, Xiao; Frick, David N

    2009-04-01

    Cyclophilins are cellular peptidyl isomerases that have been implicated in regulating hepatitis C virus (HCV) replication. Cyclophilin B (CypB) is a target of cyclosporin A (CsA), an immunosuppressive drug recently shown to suppress HCV replication in cell culture. Watashi et al. recently demonstrated that CypB is important for efficient HCV replication, and proposed that it mediates the anti-HCV effects of CsA through an interaction with NS5B [Watashi K, Ishii N, Hijikata M, Inoue D, Murata T, Miyanari Y, et al. Cyclophilin B is a functional regulator of hepatitis C virus RNA polymerase. Mol Cell 2005;19:111-22]. We examined the effects of purified CypB proteins on the enzymatic activity of NS5B. Recombinant CypB purified from insect cells directly stimulated NS5B-catalyzed RNA synthesis. CypB increased RNA synthesis by NS5B derived from genotype 1a, 1b, and 2a HCV strains. Stimulation appears to arise from an increase in productive RNA binding. NS5B residue Pro540, a previously proposed target of CypB peptidyl-prolyl isomerase activity, is not required for stimulation of RNA synthesis.

  4. Regulation of RNA-dependent RNA polymerase 1 and isochorismate synthase gene expression in Arabidopsis.

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    Lydia J R Hunter

    Full Text Available RNA-dependent RNA polymerases (RDRs function in anti-viral silencing in Arabidopsis thaliana and other plants. Salicylic acid (SA, an important defensive signal, increases RDR1 gene expression, suggesting that RDR1 contributes to SA-induced virus resistance. In Nicotiana attenuata RDR1 also regulates plant-insect interactions and is induced by another important signal, jasmonic acid (JA. Despite its importance in defense RDR1 regulation has not been investigated in detail.In Arabidopsis, SA-induced RDR1 expression was dependent on 'NON-EXPRESSER OF PATHOGENESIS-RELATED GENES 1', indicating regulation involves the same mechanism controlling many other SA- defense-related genes, including pathogenesis-related 1 (PR1. Isochorismate synthase 1 (ICS1 is required for SA biosynthesis. In defensive signal transduction RDR1 lies downstream of ICS1. However, supplying exogenous SA to ics1-mutant plants did not induce RDR1 or PR1 expression to the same extent as seen in wild type plants. Analysing ICS1 gene expression using transgenic plants expressing ICS1 promoter:reporter gene (β-glucuronidase constructs and by measuring steady-state ICS1 transcript levels showed that SA positively regulates ICS1. In contrast, ICS2, which is expressed at lower levels than ICS1, is unaffected by SA. The wound-response hormone JA affects expression of Arabidopsis RDR1 but jasmonate-induced expression is independent of CORONATINE-INSENSITIVE 1, which conditions expression of many other JA-responsive genes. Transiently increased RDR1 expression following tobacco mosaic virus inoculation was due to wounding and was not a direct effect of infection. RDR1 gene expression was induced by ethylene and by abscisic acid (an important regulator of drought resistance. However, rdr1-mutant plants showed normal responses to drought.RDR1 is regulated by a much broader range of phytohormones than previously thought, indicating that it plays roles beyond those already suggested in virus

  5. Primer-dependent and primer-independent initiation of double stranded RNA synthesis by purified arabidopsis RNA-dependent RNA polymerases RDR2 and RDR6

    DEFF Research Database (Denmark)

    Devert, Anthony; Fabre, Nicolas; Floris, Maina Huguette Joséphine

    2015-01-01

    ) targeted by RNA silencing. The dsRNA is subsequently cleaved by the ribonuclease DICER-like into secondary small interfering RNAs (siRNAs) that reinforce and/or maintain the silenced state of the target RNA. Models of RNA silencing propose that RDRs could use primer-independent and primer......Cellular RNA-dependent RNA polymerases (RDRs) are fundamental components of RNA silencing in plants and many other eukaryotes. In Arabidopsis thaliana genetic studies have demonstrated that RDR2 and RDR6 are involved in the synthesis of double stranded RNA (dsRNA) from single stranded RNA (ssRNA......-dependent initiation to generate dsRNA from a transcript targeted by primary siRNA or microRNA (miRNA). However, the biochemical activities of RDR proteins are still partly understood. Here, we obtained active recombinant RDR2 and RDR6 in a purified form. We demonstrate that RDR2 and RDR6 have primer...

  6. MicroRNA-dependent regulation of transcription in non-small cell lung cancer.

    Directory of Open Access Journals (Sweden)

    Sonia Molina-Pinelo

    Full Text Available Squamous cell lung cancer (SCC and adenocarcinoma are the most common histological subtypes of non-small cell lung cancer (NSCLC, and have been traditionally managed in the clinic as a single entity. Increasing evidence, however, illustrates the biological diversity of these two histological subgroups of lung cancer, and supports the need to improve our understanding of the molecular basis beyond the different phenotypes if we aim to develop more specific and individualized targeted therapy. The purpose of this study was to identify microRNA (miRNA-dependent transcriptional regulation differences between SCC and adenocarcinoma histological lung cancer subtypes. In this work, paired miRNA (667 miRNAs by TaqMan Low Density Arrays (TLDA and mRNA profiling (Whole Genome 44 K array G112A, Agilent was performed in tumor samples of 44 NSCLC patients. Nine miRNAs and 56 mRNAs were found to be differentially expressed in SCC versus adenocarcinoma samples. Eleven of these 56 mRNA were predicted as targets of the miRNAs identified to be differently expressed in these two histological conditions. Of them, 6 miRNAs (miR-149, miR-205, miR-375, miR-378, miR-422a and miR-708 and 9 target genes (CEACAM6, CGN, CLDN3, ABCC3, MLPH, ACSL5, TMEM45B, MUC1 were validated by quantitative PCR in an independent cohort of 41 lung cancer patients. Furthermore, the inverse correlation between mRNAs and microRNAs expression was also validated. These results suggest miRNA-dependent transcriptional regulation differences play an important role in determining key hallmarks of NSCLC, and may provide new biomarkers for personalized treatment strategies.

  7. Direct measurement of the poliovirus RNA polymerase error frequency in vitro

    International Nuclear Information System (INIS)

    Ward, C.D.; Stokes, M.A.M.; Flanegan, J.B.

    1988-01-01

    The fidelity of RNA replication by the poliovirus-RNA-dependent RNA polymerase was examined by copying homopolymeric RNA templates in vitro. The poliovirus RNA polymerase was extensively purified and used to copy poly(A), poly(C), or poly(I) templates with equimolar concentrations of noncomplementary and complementary ribonucleotides. The error frequency was expressed as the amount of a noncomplementary nucleotide incorporated divided by the total amount of complementary and noncomplementary nucleotide incorporated. The polymerase error frequencies were very high, depending on the specific reaction conditions. The activity of the polymerase on poly(U) and poly(G) was too low to measure error frequencies on these templates. A fivefold increase in the error frequency was observed when the reaction conditions were changed from 3.0 mM Mg 2+ (pH 7.0) to 7.0 mM Mg 2+ (pH 8.0). This increase in the error frequency correlates with an eightfold increase in the elongation rate that was observed under the same conditions in a previous study

  8. Poliovirus RNA synthesis in vitro: structural elements and antibody inhibition

    International Nuclear Information System (INIS)

    Semler, B.L.; Hanecak, R.; Dorner, L.F.; Anderson, C.W.; Wimmer, E.

    1983-01-01

    The poliovirus RNA polymerase complex has been analyzed by immunoautoradiography using antibody probes derived from purified replicase (P3) region viral polypeptides. Antibody preparations made against the polio RNA polymerase, P3-4b, detected a previously unreported cellular protein that copurifies with the RNA polymerase. An IgG fraction purified from rabbit antiserum to polypeptide P3-2, a precursor fo the RNA polymerase, specifically inhibits poliovirus RNA synthesis in vitro. The authors have also immunoprecipitated a 60,000-dalton protein (P3-4a) with antiserum to protein P3-4b and have determined the precise genomic map position of this protein by automated Edman degradation. Protein P3-4a originates by cleavage of the RNA polymerase precursor at a glutamine-glucine amino acid pair not previously reported to be a viral cleavage site

  9. Purification, crystallization and preliminary X-ray diffraction analysis of the RNA-dependent RNA polymerase from Thosea asigna virus

    International Nuclear Information System (INIS)

    Ferrero, Diego; Buxaderas, Mònica; Rodriguez, José F.; Verdaguer, Núria

    2012-01-01

    The RNA-dependent RNA polymerase of Thosea asigna virus has been purified and crystallized in two different crystal forms. Preliminary characterization of P2 1 2 1 2 and C222 1 crystals is reported. Co-crystallization experiments in the presence of lutetium produced a heavy-atom derivative suitable for structure determination. Thosea asigna virus (TaV) is a positive-sense, single-stranded RNA (ssRNA) virus that belongs to the Permutotetravirus genera within the recently created Permutotetraviridae family. The genome of TaV consists of an RNA segment of about 5.700 nucleotides with two open reading frames, encoding for the replicase and capsid protein. The particular TaV replicase does not contain N7-methyl transferase and helicase domains but includes a structurally unique RNA-dependent RNA polymerase (RdRp) with a sequence permutation in the domain where the active site is anchored. This architecture is also found in double-stranded RNA viruses of the Birnaviridae family. Here we report the purification and preliminary crystallographic studies TaV RdRp. The enzyme was crystallized by the sitting-drop vapour diffusion method using PEG 8K and lithium sulfate as precipitants. Two different crystal forms were obtained: native RdRp crystallized in space group P2 1 2 1 2 and diffracts up to 2.1 Å and the RdRp-Lu 3+ derivative co-crystals belong to the C222 1 space group, diffracting to 3.0 Å resolution. The structure of TaV RdRp represents the first structure of a non-canonical RdRp from ssRNA viruses

  10. Looking for inhibitors of the dengue virus NS5 RNA-dependent RNA-polymerase using a molecular docking approach

    Directory of Open Access Journals (Sweden)

    Galiano V

    2016-10-01

    Full Text Available Vicente Galiano,1 Pablo Garcia-Valtanen,2 Vicente Micol,3,4 José Antonio Encinar3 1Physics and Computer Architecture Department, Miguel Hernández University (UMH, Elche, Spain; 2Experimental Therapeutics Laboratory, Hanson and Sansom Institute for Health Research, School of Pharmacy and Medical Science, University of South Australia, Adelaide, Australia; 3Molecular and Cell Biology Institute, Miguel Hernández University (UMH, Elche, Spain; 4CIBER: CB12/03/30038, Physiopathology of the Obesity and Nutrition, CIBERobn, Instituto de Salud Carlos III, Palma de Mallorca, Spain Abstract: The dengue virus (DENV nonstructural protein 5 (NS5 contains both an N-terminal methyltransferase domain and a C-terminal RNA-dependent RNA polymerase domain. Polymerase activity is responsible for viral RNA synthesis by a de novo initiation mechanism and represents an attractive target for antiviral therapy. The incidence of DENV has grown rapidly and it is now estimated that half of the human population is at risk of becoming infected with this virus. Despite this, there are no effective drugs to treat DENV infections. The present in silico study aimed at finding new inhibitors of the NS5 RNA-dependent RNA polymerase of the four serotypes of DENV. We used a chemical library comprising 372,792 nonnucleotide compounds (around 325,319 natural compounds to perform molecular docking experiments against a binding site of the RNA template tunnel of the virus polymerase. Compounds with high negative free energy variation (ΔG <-10.5 kcal/mol were selected as putative inhibitors. Additional filters for favorable druggability and good absorption, distribution, metabolism, excretion, and toxicity were applied. Finally, after the screening process was completed, we identified 39 compounds as lead DENV polymerase inhibitor candidates. Potentially, these compounds could act as efficient DENV polymerase inhibitors in vitro and in vivo. Keywords: virtual screening, molecular

  11. A Broad RNA Virus Survey Reveals Both miRNA Dependence and Functional Sequestration

    DEFF Research Database (Denmark)

    Scheel, Troels K H; Luna, Joseph M; Liniger, Matthias

    2016-01-01

    , critically depended on the interaction of cellular miR-17 and let-7 with the viral 3' UTR. Unlike canonical miRNA interactions, miR-17 and let-7 binding enhanced pestivirus translation and RNA stability. miR-17 sequestration by pestiviruses conferred reduced AGO binding and functional de...... immunoprecipitation (CLIP) of the Argonaute (AGO) proteins to characterize strengths and specificities of miRNA interactions in the context of 15 different RNA virus infections, including several clinically relevant pathogens. Notably, replication of pestiviruses, a major threat to milk and meat industries...

  12. Neuronal phosphorylated RNA-dependent protein kinase in Creutzfeldt-Jakob disease.

    LENUS (Irish Health Repository)

    Paquet, Claire

    2009-02-01

    The mechanisms of neuronal apoptosis in Creutzfeldt-Jakob disease (CJD) and their relationship to accumulated prion protein (PrP) are unclear. A recent cell culture study showed that intracytoplasmic PrP may induce phosphorylated RNA-dependent protein kinase (PKR(p))-mediated cell stress. The double-stranded RNA protein kinase PKR is a proapoptotic and stress kinase that accumulates in degenerating neurons in Alzheimer disease. To determine whether neuronal apoptosis in human CJD is associated with activation of the PKR(p) signaling pathway, we assessed in situ end labeling and immunocytochemistry for PrP, glial fibrillary acidic protein, CD68, activated caspase 3, and phosphorylated PKR (Thr451) in samples of frontal, occipital, and temporal cortex, striatum, and cerebellum from 6 patients with sporadic CJD and 5 controls. Neuronal immunostaining for activated PKR was found in all CJD cases. The most staining was in nuclei and, in contrast to findings in Alzheimer disease, cytoplasmic labeling was not detected. Both the number and distribution of PKR(p)-positive neurons correlated closely with the extent of neuronal apoptosis, spongiosis, astrocytosis, and microglial activation and with the phenotype and disease severity. There was no correlation with the type, topography, or amount of extracellular PrP deposits. These findings suggest that neuronal apoptosis in human CJD may result from PKR(p)-mediated cell stress and are consistent with recent studies supporting a pathogenic role for intracellular or transmembrane PrP.

  13. Toscana virus NSs protein promotes degradation of double-stranded RNA-dependent protein kinase.

    Science.gov (United States)

    Kalveram, Birte; Ikegami, Tetsuro

    2013-04-01

    Toscana virus (TOSV), which is transmitted by Phlebotomus spp. sandflies, is a major etiologic agent of aseptic meningitis and encephalitis in the Mediterranean. Like other members of the genus Phlebovirus of the family Bunyaviridae, TOSV encodes a nonstructural protein (NSs) in its small RNA segment. Although the NSs of Rift Valley fever virus (RVFV) has been identified as an important virulence factor, which suppresses host general transcription, inhibits transcription from the beta interferon promoter, and promotes the proteasomal degradation of double-stranded RNA-dependent protein kinase (PKR), little is known about the functions of NSs proteins encoded by less-pathogenic members of this genus. In this study we report that TOSV is able to downregulate PKR with similar efficiency as RVFV, while infection with the other phleboviruses-i.e., Punta Toro virus, sandfly fever Sicilian virus, or Frijoles virus-has no effect on cellular PKR levels. In contrast to RVFV, however, cellular transcription remains unaffected during TOSV infection. TOSV NSs protein promotes the proteasome-dependent downregulation of PKR and is able to interact with kinase-inactive PKR in infected cells.

  14. StralSV: assessment of sequence variability within similar 3D structures and application to polio RNA-dependent RNA polymerase

    Energy Technology Data Exchange (ETDEWEB)

    Zemla, A; Lang, D; Kostova, T; Andino, R; Zhou, C

    2010-11-29

    Most of the currently used methods for protein function prediction rely on sequence-based comparisons between a query protein and those for which a functional annotation is provided. A serious limitation of sequence similarity-based approaches for identifying residue conservation among proteins is the low confidence in assigning residue-residue correspondences among proteins when the level of sequence identity between the compared proteins is poor. Multiple sequence alignment methods are more satisfactory - still, they cannot provide reliable results at low levels of sequence identity. Our goal in the current work was to develop an algorithm that could overcome these difficulties and facilitate the identification of structurally (and possibly functionally) relevant residue-residue correspondences between compared protein structures. Here we present StralSV, a new algorithm for detecting closely related structure fragments and quantifying residue frequency from tight local structure alignments. We apply StralSV in a study of the RNA-dependent RNA polymerase of poliovirus and demonstrate that the algorithm can be used to determine regions of the protein that are relatively unique or that shared structural similarity with structures that are distantly related. By quantifying residue frequencies among many residue-residue pairs extracted from local alignments, one can infer potential structural or functional importance of specific residues that are determined to be highly conserved or that deviate from a consensus. We further demonstrate that considerable detailed structural and phylogenetic information can be derived from StralSV analyses. StralSV is a new structure-based algorithm for identifying and aligning structure fragments that have similarity to a reference protein. StralSV analysis can be used to quantify residue-residue correspondences and identify residues that may be of particular structural or functional importance, as well as unusual or unexpected

  15. Structural explanation for the role of Mn2+ in the activity of phi6 RNA-dependent RNA polymerase.

    Science.gov (United States)

    Poranen, Minna M; Salgado, Paula S; Koivunen, Minni R L; Wright, Sam; Bamford, Dennis H; Stuart, David I; Grimes, Jonathan M

    2008-11-01

    The biological role of manganese (Mn(2+)) has been a long-standing puzzle, since at low concentrations it activates several polymerases whilst at higher concentrations it inhibits. Viral RNA polymerases possess a common architecture, reminiscent of a closed right hand. The RNA-dependent RNA polymerase (RdRp) of bacteriophage 6 is one of the best understood examples of this important class of polymerases. We have probed the role of Mn(2+) by biochemical, biophysical and structural analyses of the wild-type enzyme and of a mutant form with an altered Mn(2+)-binding site (E491 to Q). The E491Q mutant has much reduced affinity for Mn(2+), reduced RNA binding and a compromised elongation rate. Loss of Mn(2+) binding structurally stabilizes the enzyme. These data and a re-examination of the structures of other viral RNA polymerases clarify the role of manganese in the activation of polymerization: Mn(2+) coordination of a catalytic aspartate is necessary to allow the active site to properly engage with the triphosphates of the incoming NTPs. The structural flexibility caused by Mn(2+) is also important for the enzyme dynamics, explaining the requirement for manganese throughout RNA polymerization.

  16. Structural and functional characterisation of Aichi virus RNA dependent RNA polymerase

    Czech Academy of Sciences Publication Activity Database

    Dubánková, Anna; Humpolíčková, Jana; Šilhán, Jan; Bäumlová, Adriana; Chalupská, Dominika; Klíma, Martin; Bouřa, Evžen

    2017-01-01

    Roč. 15, č. 1 (2017), s. 7-8 ISSN 2336-7202. [Mezioborové setkání mladých biologů, biochemiků a chemiků /17./. 30.05.2017-01.06.2017, Milovy] Institutional support: RVO:61388963 Keywords : Aichi virus * RNA replication Subject RIV: CE - Biochemistry

  17. Characterization of purified Sindbis virus nsP4 RNA-dependent RNA polymerase activity in vitro

    International Nuclear Information System (INIS)

    Rubach, Jon K.; Wasik, Brian R.; Rupp, Jonathan C.; Kuhn, Richard J.; Hardy, Richard W.; Smith, Janet L.

    2009-01-01

    The Sindbis virus RNA-dependent RNA polymerase (nsP4) is responsible for the replication of the viral RNA genome. In infected cells, nsP4 is localized in a replication complex along with the other viral non-structural proteins. nsP4 has been difficult to homogenously purify from infected cells due to its interactions with the other replication proteins and the fact that its N-terminal residue, a tyrosine, causes the protein to be rapidly turned over in cells. We report the successful expression and purification of Sindbis nsP4 in a bacterial system, in which nsP4 is expressed as an N-terminal SUMO fusion protein. After purification the SUMO tag is removed, resulting in the isolation of full-length nsP4 possessing the authentic N-terminal tyrosine. This purified enzyme is able to produce minus-strand RNA de novo from plus-strand templates, as well as terminally add adenosine residues to the 3' end of an RNA substrate. In the presence of the partially processed viral replicase polyprotein, P123, purified nsP4 is able to synthesize discrete template length minus-strand RNA products. Mutations in the 3' CSE or poly(A) tail of viral template RNA prevent RNA synthesis by the replicase complex containing purified nsP4, consistent with previously reported template requirements for minus-strand RNA synthesis. Optimal reaction conditions were determined by investigating the effects of time, pH, and the concentrations of nsP4, P123 and magnesium on the synthesis of RNA

  18. dsRNA-Dependent Protein Kinase PKR and its Role in Stress, Signaling and HCV Infection

    Directory of Open Access Journals (Sweden)

    Eliane F. Meurs

    2012-10-01

    Full Text Available The double-stranded RNA-dependent protein kinase PKR plays multiple roles in cells, in response to different stress situations. As a member of the interferon (IFN‑Stimulated Genes, PKR was initially recognized as an actor in the antiviral action of IFN, due to its ability to control translation, through phosphorylation, of the alpha subunit of eukaryotic initiation factor 2 (eIF2a. As such, PKR participates in the generation of stress granules, or autophagy and a number of viruses have designed strategies to inhibit its action. However, PKR deficient mice resist most viral infections, indicating that PKR may play other roles in the cell other than just acting as an antiviral agent. Indeed, PKR regulates several signaling pathways, either as an adapter protein and/or using its kinase activity. Here we review the role of PKR as an eIF2a kinase, its participation in the regulation of the NF-kB, p38MAPK and insulin pathways, and we focus on its role during infection with the hepatitis C virus (HCV. PKR binds the HCV IRES RNA, cooperates with some functions of the HCV core protein and may represent a target for NS5A or E2. Novel data points out for a role of PKR as a pro-HCV agent, both as an adapter protein and as an eIF2a-kinase, and in cooperation with the di-ubiquitin-like protein ISG15. Developing pharmaceutical inhibitors of PKR may help in resolving some viral infections as well as stress-related damages.

  19. Poliovirus Polymerase Leu420 Facilitates RNA Recombination and Ribavirin Resistance

    Science.gov (United States)

    Kempf, Brian J.; Peersen, Olve B.

    2016-01-01

    ABSTRACT RNA recombination is important in the formation of picornavirus species groups and the ongoing evolution of viruses within species groups. In this study, we examined the structure and function of poliovirus polymerase, 3Dpol, as it relates to RNA recombination. Recombination occurs when nascent RNA products exchange one viral RNA template for another during RNA replication. Because recombination is a natural aspect of picornavirus replication, we hypothesized that some features of 3Dpol may exist, in part, to facilitate RNA recombination. Furthermore, we reasoned that alanine substitution mutations that disrupt 3Dpol-RNA interactions within the polymerase elongation complex might increase and/or decrease the magnitudes of recombination. We found that an L420A mutation in 3Dpol decreased the frequency of RNA recombination, whereas alanine substitutions at other sites in 3Dpol increased the frequency of recombination. The 3Dpol Leu420 side chain interacts with a ribose in the nascent RNA product 3 nucleotides from the active site of the polymerase. Notably, the L420A mutation that reduced recombination also rendered the virus more susceptible to inhibition by ribavirin, coincident with the accumulation of ribavirin-induced G→A and C→U mutations in viral RNA. We conclude that 3Dpol Leu420 is critically important for RNA recombination and that RNA recombination contributes to ribavirin resistance. IMPORTANCE Recombination contributes to the formation of picornavirus species groups and the emergence of circulating vaccine-derived polioviruses (cVDPVs). The recombinant viruses that arise in nature are occasionally more fit than either parental strain, especially when the two partners in recombination are closely related, i.e., members of characteristic species groups, such as enterovirus species groups A to H or rhinovirus species groups A to C. Our study shows that RNA recombination requires conserved features of the viral polymerase. Furthermore, a

  20. Evf2 lncRNA/BRG1/DLX1 interactions reveal RNA-dependent inhibition of chromatin remodeling.

    Science.gov (United States)

    Cajigas, Ivelisse; Leib, David E; Cochrane, Jesse; Luo, Hao; Swyter, Kelsey R; Chen, Sean; Clark, Brian S; Thompson, James; Yates, John R; Kingston, Robert E; Kohtz, Jhumku D

    2015-08-01

    Transcription-regulating long non-coding RNAs (lncRNAs) have the potential to control the site-specific expression of thousands of target genes. Previously, we showed that Evf2, the first described ultraconserved lncRNA, increases the association of transcriptional activators (DLX homeodomain proteins) with key DNA enhancers but represses gene expression. In this report, mass spectrometry shows that the Evf2-DLX1 ribonucleoprotein (RNP) contains the SWI/SNF-related chromatin remodelers Brahma-related gene 1 (BRG1, SMARCA4) and Brahma-associated factor (BAF170, SMARCC2) in the developing mouse forebrain. Evf2 RNA colocalizes with BRG1 in nuclear clouds and increases BRG1 association with key DNA regulatory enhancers in the developing forebrain. While BRG1 directly interacts with DLX1 and Evf2 through distinct binding sites, Evf2 directly inhibits BRG1 ATPase and chromatin remodeling activities. In vitro studies show that both RNA-BRG1 binding and RNA inhibition of BRG1 ATPase/remodeling activity are promiscuous, suggesting that context is a crucial factor in RNA-dependent chromatin remodeling inhibition. Together, these experiments support a model in which RNAs convert an active enhancer to a repressed enhancer by directly inhibiting chromatin remodeling activity, and address the apparent paradox of RNA-mediated stabilization of transcriptional activators at enhancers with a repressive outcome. The importance of BRG1/RNA and BRG1/homeodomain interactions in neurodevelopmental disorders is underscored by the finding that mutations in Coffin-Siris syndrome, a human intellectual disability disorder, localize to the BRG1 RNA-binding and DLX1-binding domains. © 2015. Published by The Company of Biologists Ltd.

  1. MicroRNA-134 regulates poliovirus replication by IRES targeting

    OpenAIRE

    Bakre, Abhijeet A.; Shim, Byoung-Shik; Tripp, Ralph A.

    2017-01-01

    Global poliovirus eradication efforts include high vaccination coverage with live oral polio vaccine (OPV), surveillance for acute flaccid paralysis, and OPV “mop-up” campaigns. An important objective involves host-directed strategies to reduce PV replication to diminish viral shedding in OPV recipients. In this study, we show that microRNA-134-5p (miR-134) can regulate Sabin-1 replication but not Sabin-2 or Sabin-3 via direct interaction with the PV 5′UTR. Hypochromicity data showed miR-134 ...

  2. Primary structure, gene organization and polypeptide expression of poliovirus RNA

    Energy Technology Data Exchange (ETDEWEB)

    Kitamura, N. (State Univ. of New York, Stony Brook); Semler, B.L.; Rothberg, P.G.

    1981-06-18

    The primary structure of the poliovirus genome has been determined. The RNA molecule is 7433 nucleotides long, polyadenylated at the 3' terminus, and covalently linked to a small protein (VPg) at the 5' terminus. An open reading frame of 2207 consecutive triplets spans over 89% of the nucleotide sequence and codes for the viral polyprotein NCVPOO. Twelve viral polypeptides have been mapped by amino acid sequence analysis and were found to be proteolytic cleavage products of the polyprotein, cleavages occurring predominantly at Gln-Gly pairs.

  3. NS5B RNA dependent RNA polymerase inhibitors: the promising approach to treat hepatitis C virus infections.

    Science.gov (United States)

    Deore, R R; Chern, J-W

    2010-01-01

    Hepatitis C virus (HCV), a causative agent for non-A and non-B hepatitis, has infected approximately 3% of world's population. The current treatment option of ribavirin in combination with pegylated interferon possesses lower sustained virological response rates, and has serious disadvantages. Unfortunately, no prophylactic vaccine has been approved yet. Therefore, there is an unmet clinical need for more effective and safe anti-HCV drugs. HCV NS5B RNA dependent RNA polymerase is currently pursued as the most popular target to develop safe anti-HCV agents, as it is not expressed in uninfected cells. More than 25 pharmaceutical companies and some research groups have developed ≈50 structurally diverse scaffolds to inhibit NS5B. Here we provide comprehensive account of the drug development process of these scaffolds. NS5B polymerase inhibitors have been broadly classified in nucleoside and non nucleoside inhibitors and are sub classified according to their mechanism of action and structural diversities. With some additional considerations about the inhibitor bound NS5B enzyme X-ray crystal structure information and pharmacological aspects of the inhibitors, this review summarizes the lead identification, structure activity relationship (SAR) studies leading to the most potent NS5B inhibitors with subgenomic replicon activity.

  4. Heat shock 70 protein interaction with Turnip mosaic virus RNA-dependent RNA polymerase within virus-induced membrane vesicles

    International Nuclear Information System (INIS)

    Dufresne, Philippe J.; Thivierge, Karine; Cotton, Sophie; Beauchemin, Chantal; Ide, Christine; Ubalijoro, Eliane; Laliberte, Jean-Francois; Fortin, Marc G.

    2008-01-01

    Tandem affinity purification was used in Arabidopsis thaliana to identify cellular interactors of Turnip mosaic virus (TuMV) RNA-dependent RNA polymerase (RdRp). The heat shock cognate 70-3 (Hsc70-3) and poly(A)-binding (PABP) host proteins were recovered and shown to interact with the RdRp in vitro. As previously shown for PABP, Hsc70-3 was redistributed to nuclear and membranous fractions in infected plants and both RdRp interactors were co-immunoprecipitated from a membrane-enriched extract using RdRp-specific antibodies. Fluorescently tagged RdRp and Hsc70-3 localized to the cytoplasm and the nucleus when expressed alone or in combination in Nicotiana benthamiana. However, they were redistributed to large perinuclear ER-derived vesicles when co-expressed with the membrane binding 6K-VPg-Pro protein of TuMV. The association of Hsc70-3 with the RdRp could possibly take place in membrane-derived replication complexes. Thus, Hsc70-3 and PABP2 are potentially integral components of the replicase complex and could have important roles to play in the regulation of potyviral RdRp functions

  5. Inhibition of dengue virus replication by novel inhibitors of RNA-dependent RNA polymerase and protease activities.

    Science.gov (United States)

    Pelliccia, Sveva; Wu, Yu-Hsuan; Coluccia, Antonio; La Regina, Giuseppe; Tseng, Chin-Kai; Famiglini, Valeria; Masci, Domiziana; Hiscott, John; Lee, Jin-Ching; Silvestri, Romano

    2017-12-01

    Dengue virus (DENV) is the leading mosquito-transmitted viral infection in the world. With more than 390 million new infections annually, and up to 1 million clinical cases with severe disease manifestations, there continues to be a need to develop new antiviral agents against dengue infection. In addition, there is no approved anti-DENV agents for treating DENV-infected patients. In the present study, we identified new compounds with anti-DENV replication activity by targeting viral replication enzymes - NS5, RNA-dependent RNA polymerase (RdRp) and NS3 protease, using cell-based reporter assay. Subsequently, we performed an enzyme-based assay to clarify the action of these compounds against DENV RdRp or NS3 protease activity. Moreover, these compounds exhibited anti-DENV activity in vivo in the ICR-suckling DENV-infected mouse model. Combination drug treatment exhibited a synergistic inhibition of DENV replication. These results describe novel prototypical small anti-DENV molecules for further development through compound modification and provide potential antivirals for treating DENV infection and DENV-related diseases.

  6. Agrobacterium-mediated transformation of grapefruit with the wild-type and mutant RNA-dependent RNA polymerase genes of Citrus tristeza virus

    Science.gov (United States)

    Citrus paradisi Macf. cv. Duncan was transformed with constructs coding for the wild-type and mutant RNA-dependent RNA polymerase (RdRp) of Citrus tristeza virus (CTV) for exploring replicase-mediated pathogen-derived resistance (RM-PDR). The RdRp gene was amplified from CTV genome and used to gener...

  7. Rift Valley fever virus NSs inhibits host transcription independently of the degradation of dsRNA-dependent Protein Kinase PKR

    OpenAIRE

    Kalveram, Birte; Lihoradova, Olga; Indran, Sabarish V.; Lokugamage, Nandadeva; Head, Jennifer A.; Ikegami, Tetsuro

    2012-01-01

    Rift Valley fever virus (RVFV) encodes one major virulence factor, the NSs protein. NSs suppresses host general transcription, including interferon (IFN)-β mRNA synthesis, and promotes degradation of the dsRNA-dependent protein kinase (PKR). We generated a novel RVFV mutant (rMP12-NSsR173A) specifically lacking the function to promote PKR degradation. rMP12-NSsR173A infection induces early phosphorylation of eIF2α through PKR activation, while retaining the function to inhibit host general tr...

  8. Zinc Salts Block Hepatitis E Virus Replication by Inhibiting the Activity of Viral RNA-Dependent RNA Polymerase.

    Science.gov (United States)

    Kaushik, Nidhi; Subramani, Chandru; Anang, Saumya; Muthumohan, Rajagopalan; Shalimar; Nayak, Baibaswata; Ranjith-Kumar, C T; Surjit, Milan

    2017-11-01

    Hepatitis E virus (HEV) causes an acute, self-limiting hepatitis in healthy individuals and leads to chronic disease in immunocompromised individuals. HEV infection in pregnant women results in a more severe outcome, with the mortality rate going up to 30%. Though the virus usually causes sporadic infection, epidemics have been reported in developing and resource-starved countries. No specific antiviral exists against HEV. A combination of interferon and ribavirin therapy has been used to control the disease with some success. Zinc is an essential micronutrient that plays crucial roles in multiple cellular processes. Zinc salts are known to be effective in reducing infections caused by few viruses. Here, we investigated the effect of zinc salts on HEV replication. In a human hepatoma cell (Huh7) culture model, zinc salts inhibited the replication of genotype 1 (g-1) and g-3 HEV replicons and g-1 HEV infectious genomic RNA in a dose-dependent manner. Analysis of a replication-defective mutant of g-1 HEV genomic RNA under similar conditions ruled out the possibility of zinc salts acting on replication-independent processes. An ORF4-Huh7 cell line-based infection model of g-1 HEV further confirmed the above observations. Zinc salts did not show any effect on the entry of g-1 HEV into the host cell. Furthermore, our data reveal that zinc salts directly inhibit the activity of viral RNA-dependent RNA polymerase (RdRp), leading to inhibition of viral replication. Taken together, these studies unravel the ability of zinc salts in inhibiting HEV replication, suggesting their possible therapeutic value in controlling HEV infection. IMPORTANCE Hepatitis E virus (HEV) is a public health concern in resource-starved countries due to frequent outbreaks. It is also emerging as a health concern in developed countries owing to its ability to cause acute and chronic infection in organ transplant and immunocompromised individuals. Although antivirals such as ribavirin have been used

  9. A Novel, Highly Selective Inhibitor of Pestivirus Replication That Targets the Viral RNA-Dependent RNA Polymerase

    Science.gov (United States)

    Paeshuyse, Jan; Leyssen, Pieter; Mabery, Eric; Boddeker, Nina; Vrancken, Robert; Froeyen, Matheus; Ansari, Israrul H.; Dutartre, Hélène; Rozenski, Jef; Gil, Laura H. V. G.; Letellier, Carine; Lanford, Robert; Canard, Bruno; Koenen, Frank; Kerkhofs, Pierre; Donis, Ruben O.; Herdewijn, Piet; Watson, Julia; De Clercq, Erik; Puerstinger, Gerhard; Neyts, Johan

    2006-01-01

    We report on the highly potent and selective antipestivirus activity of 5-[(4-bromophenyl)methyl]-2-phenyl-5H-imidazo[4,5-c]pyridine (BPIP). The 50% effective concentration (EC50) for inhibition of bovine viral diarrhea virus (BVDV)-induced cytopathic effect formation was 0.04 ± 0.01 μM. Comparable reduction of viral RNA synthesis (EC50 = 0.12 ± 0.02 μM) and production of infectious virus (EC50 = 0.074 ± 0.003 μM) were observed. The selectivity index (ratio of 50% cytostatic concentration/EC50) of BPIP was ∼2,000. BPIP was inactive against the hepatitis C virus subgenomic replicon and yellow fever virus but demonstrated weak activity against GB virus. Drug-resistant mutants were at least 300-fold less susceptible to BPIP than wild-type virus; showed cross-resistance to N-propyl-N-[2-(2H-1,2,4-triazino[5,6-b]indol-3-ylthio)ethyl]-1-propanamine (VP32947), and carried the F224S mutation in the viral RNA-dependent RNA polymerase (RdRp). When the F224S mutation was introduced into an infectious clone, the drug-resistant phenotype was obtained. BPIP did not inhibit the in vitro activity of recombinant BVDV RdRp, but did inhibit the activity of replication complexes (RCs). Computational docking revealed that F224 is located at the top of the finger domain of the polymerase. Docking of BPIP in the crystal structure of the BVDV RdRp revealed aromatic ring stacking, some hydrophobic contacts, and a hydrogen bond. Since two structurally unrelated compounds, i.e., BPIP and VP32947, target the same region of the BVDV RdRp, this position may be expected to be critical in the functioning of the polymerase or assembly of the RC. The potential of BPIP for the treatment of pestivirus and hepacivirus infections is discussed. PMID:16352539

  10. Homology Modeling and Analysis of Structure Predictions of the Bovine Rhinitis B Virus RNA Dependent RNA Polymerase (RdRp

    Directory of Open Access Journals (Sweden)

    Devendra K. Rai

    2012-07-01

    Full Text Available Bovine Rhinitis B Virus (BRBV is a picornavirus responsible for mild respiratory infection of cattle. It is probably the least characterized among the aphthoviruses. BRBV is the closest relative known to Foot and Mouth Disease virus (FMDV with a ~43% identical polyprotein sequence and as much as 67% identical sequence for the RNA dependent RNA polymerase (RdRp, which is also known as 3D polymerase (3Dpol. In the present study we carried out phylogenetic analysis, structure based sequence alignment and prediction of three-dimensional structure of BRBV 3Dpol using a combination of different computational tools. Model structures of BRBV 3Dpol were verified for their stereochemical quality and accuracy. The BRBV 3Dpol structure predicted by SWISS-MODEL exhibited highest scores in terms of stereochemical quality and accuracy, which were in the range of 2Å resolution crystal structures. The active site, nucleic acid binding site and overall structure were observed to be in agreement with the crystal structure of unliganded as well as template/primer (T/P, nucleotide tri-phosphate (NTP and pyrophosphate (PPi bound FMDV 3Dpol (PDB, 1U09 and 2E9Z. The closest proximity of BRBV and FMDV 3Dpol as compared to human rhinovirus type 16 (HRV-16 and rabbit hemorrhagic disease virus (RHDV 3Dpols is also substantiated by phylogeny analysis and root-mean square deviation (RMSD between C-α traces of the polymerase structures. The absence of positively charged α-helix at C terminal, significant differences in non-covalent interactions especially salt bridges and CH-pi interactions around T/P channel of BRBV 3Dpol compared to FMDV 3Dpol, indicate that despite a very high homology to FMDV 3Dpol, BRBV 3Dpol may adopt a different mechanism for handling its substrates and adapting to physiological requirements. Our findings will be valuable in the

  11. microRNA dependent and independent deregulation of long non-coding RNAs by an oncogenic herpesvirus.

    Directory of Open Access Journals (Sweden)

    Sunantha Sethuraman

    2017-07-01

    Full Text Available Kaposi's sarcoma (KS is a highly prevalent cancer in AIDS patients, especially in sub-Saharan Africa. Kaposi's sarcoma-associated herpesvirus (KSHV is the etiological agent of KS and other cancers like Primary Effusion Lymphoma (PEL. In KS and PEL, all tumors harbor latent KSHV episomes and express latency-associated viral proteins and microRNAs (miRNAs. The exact molecular mechanisms by which latent KSHV drives tumorigenesis are not completely understood. Recent developments have highlighted the importance of aberrant long non-coding RNA (lncRNA expression in cancer. Deregulation of lncRNAs by miRNAs is a newly described phenomenon. We hypothesized that KSHV-encoded miRNAs deregulate human lncRNAs to drive tumorigenesis. We performed lncRNA expression profiling of endothelial cells infected with wt and miRNA-deleted KSHV and identified 126 lncRNAs as putative viral miRNA targets. Here we show that KSHV deregulates host lncRNAs in both a miRNA-dependent fashion by direct interaction and in a miRNA-independent fashion through latency-associated proteins. Several lncRNAs that were previously implicated in cancer, including MEG3, ANRIL and UCA1, are deregulated by KSHV. Our results also demonstrate that KSHV-mediated UCA1 deregulation contributes to increased proliferation and migration of endothelial cells.

  12. RNA-dependent RNA polymerase of hepatitis C virus binds to its coding region RNA stem-loop structure, 5BSL3.2, and its negative strand.

    Science.gov (United States)

    Kanamori, Hiroshi; Yuhashi, Kazuhito; Ohnishi, Shin; Koike, Kazuhiko; Kodama, Tatsuhiko

    2010-05-01

    The hepatitis C virus NS5B RNA-dependent RNA polymerase (RdRp) is a key enzyme involved in viral replication. Interaction between NS5B RdRp and the viral RNA sequence is likely to be an important step in viral RNA replication. The C-terminal half of the NS5B-coding sequence, which contains the important cis-acting replication element, has been identified as an NS5B-binding sequence. In the present study, we confirm the specific binding of NS5B to one of the RNA stem-loop structures in the region, 5BSL3.2. In addition, we show that NS5B binds to the complementary strand of 5BSL3.2 (5BSL3.2N). The bulge structure of 5BSL3.2N was shown to be indispensable for tight binding to NS5B. In vitro RdRp activity was inhibited by 5BSL3.2N, indicating the importance of the RNA element in the polymerization by RdRp. These results suggest the involvement of the RNA stem-loop structure of the negative strand in the replication process.

  13. Mutational analysis of Sep-tRNA:Cys-tRNA synthase reveals critical residues for tRNA-dependent cysteine formation.

    Science.gov (United States)

    Helgadóttir, Sunna; Sinapah, Sylvie; Söll, Dieter; Ling, Jiqiang

    2012-01-02

    In methanogenic archaea, Sep-tRNA:Cys-tRNA synthase (SepCysS) converts Sep-tRNA(Cys) to Cys-tRNA(Cys). The mechanism of tRNA-dependent cysteine formation remains unclear due to the lack of functional studies. In this work, we mutated 19 conserved residues in Methanocaldococcus jannaschii SepCysS, and employed an in vivo system to determine the activity of the resulting variants. Our results show that three active-site cysteines (Cys39, Cys42 and Cys247) are essential for SepCysS activity. In addition, combined with structural modeling, our mutational and functional analyses also reveal multiple residues that are important for the binding of PLP, Sep and tRNA. Our work thus represents the first systematic functional analysis of conserved residues in archaeal SepCysSs, providing insights into the catalytic and substrate binding mechanisms of this poorly characterized enzyme. Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  14. Structure of Hepatitis E Virion-Sized Particle Reveals an RNA-Dependent Viral Assembly Pathway

    Energy Technology Data Exchange (ETDEWEB)

    Xing, L.; Wall, J.; Li, T.-C.; Mayazaki, N.; Simon, M. N.; Moore, M.; Wang, C.-Y.; Takeda, N.; Wakita, T.; Miyamura, T.; Cheng, R. H.

    2010-10-22

    Hepatitis E virus (HEV) induces acute hepatitis in humans with a high fatality rate in pregnant women. There is a need for anti-HEV research to understand the assembly process of HEV native capsid. Here, we produced a large virion-sized and a small T=1 capsid by expressing the HEV capsid protein in insect cells with and without the N-terminal 111 residues, respectively, for comparative structural analysis. The virion-sized capsid demonstrates a T=3 icosahedral lattice and contains RNA fragment in contrast to the RNA-free T=1 capsid. However, both capsids shared common decameric organization. The in vitro assembly further demonstrated that HEV capsid protein had the intrinsic ability to form decameric intermediate. Our data suggest that RNA binding is the extrinsic factor essential for the assembly of HEV native capsids.

  15. Expression analysis of argonaute, Dicer-like, and RNA-dependent ...

    Indian Academy of Sciences (India)

    DEFANG GAN

    the first report of expression analysis of all CsDCL, CsAGO and CsRDR family genes in cucumber under .... tissues and organs were detected using online data and semi- ...... Wheeler B. S. 2013 Small RNAs, big impact: small RNA pathways.

  16. RNA-Dependent Intergenerational Inheritance of Enhanced Synaptic Plasticity after Environmental Enrichment

    Directory of Open Access Journals (Sweden)

    Eva Benito

    2018-04-01

    Full Text Available Summary: Physical exercise in combination with cognitive training is known to enhance synaptic plasticity, learning, and memory and lower the risk for various complex diseases including Alzheimer’s disease. Here, we show that exposure of adult male mice to an environmental enrichment paradigm leads to enhancement of synaptic plasticity and cognition also in the next generation. We show that this effect is mediated through sperm RNA and especially miRs 212/132. In conclusion, our study reports intergenerational inheritance of an acquired cognitive benefit and points to specific miRs as candidates mechanistically involved in this type of transmission. : Environmental enrichment (EE, a combination of physical and mental exercise, has been shown to increase cognitive abilities in mice and in humans. Benito et al. find that offspring of male mice subjected to EE also show this increase. This effect is dependent on sperm RNA and involves microRNA212/132. Keywords: epigenetics, brain, microRNA, memory, intergenerational, transgenerational, exercise, environmental enrichment, cognition

  17. Environmental cues induce a long noncoding RNA-dependent remodeling of the nucleolus.

    Science.gov (United States)

    Jacob, Mathieu D; Audas, Timothy E; Uniacke, James; Trinkle-Mulcahy, Laura; Lee, Stephen

    2013-09-01

    The nucleolus is a plurifunctional organelle in which structure and function are intimately linked. Its structural plasticity has long been appreciated, particularly in response to transcriptional inhibition and other cellular stresses, although the mechanism and physiological relevance of these phenomena are unclear. Using MCF-7 and other mammalian cell lines, we describe a structural and functional adaptation of the nucleolus, triggered by heat shock or physiological acidosis, that depends on the expression of ribosomal intergenic spacer long noncoding RNA (IGS lncRNA). At the heart of this process is the de novo formation of a large subnucleolar structure, termed the detention center (DC). The DC is a spatially and dynamically distinct region, characterized by an 8-anilino-1-naphthalenesulfonate-positive hydrophobic signature. Its formation is accompanied by redistribution of nucleolar factors and arrest in ribosomal biogenesis. Silencing of regulatory IGS lncRNA prevents the creation of this structure and allows the nucleolus to retain its tripartite organization and transcriptional activity. Signal termination causes a decrease in IGS transcript levels and a return to the active nucleolar conformation. We propose that the induction of IGS lncRNA by environmental signals operates as a molecular switch that regulates the structure and function of the nucleolus.

  18. The 3D protein of duck hepatitis A virus type 1 binds to a viral genomic 3' UTR and shows RNA-dependent RNA polymerase activity.

    Science.gov (United States)

    Zhang, Yu; Cao, Qianda; Wang, Mingshu; Jia, Renyong; Chen, Shun; Zhu, Dekang; Liu, Mafeng; Sun, Kunfeng; Yang, Qiao; Wu, Ying; Zhao, Xinxin; Chen, Xiaoyue; Cheng, Anchun

    2017-12-01

    To explore the RNA-dependent RNA polymerase (RdRP) function of the 3D protein of duck hepatitis A virus type 1 (DHAV-1), the gene was cloned into the pET-32a(+) vector for prokaryotic expression. The 3' untranslated region (3' UTR) of DHAV-1 together with a T7 promoter was cloned into the pMD19-T vector for in vitro transcription of 3' UTR RNA, which was further used as a template in RNA-dependent RNA polymerization. In this study, three methods were applied to analyze the RdRP function of the 3D protein: (1) ammonium molybdate spectrophotometry to detect pyrophosphate produced during polymerization; (2) quantitative reverse transcription PCR (RT-qPCR) to investigate the changes in RNA quantity during polymerization; and (3) electrophoresis mobility shift assay to examine the interaction between the 3D protein and 3' UTR. The results showed the 3D protein was successfully expressed in bacteria culture supernatant in a soluble form, which could be purified by affinity chromatography. In 3D enzymatic activity assays, pyrophosphate and RNA were produced, the amounts of which increased based on approximative kinetics, and binding of the 3D protein to the 3' UTR was observed. These results indicate that prokaryotically expressed soluble DHAV-13D protein can bind to a viral genomic 3' UTR and exhibit RdRP activity.

  19. Evidence for Importance of tRNA-dependent Cytokinin Biosynthetic Pathway in the Moss Physcomitrella patens

    Czech Academy of Sciences Publication Activity Database

    Yevdakova, N.A.; Motyka, Václav; Malbeck, Jiří; Trávníčková, Alena; Novák, Ondřej; Strnad, Miroslav; von Schwartzenberg, K.

    2008-01-01

    Roč. 27, č. 3 (2008), s. 271-281 ISSN 0721-7595 R&D Projects: GA MŠk(CZ) LC06034; GA ČR GA206/05/0894; GA AV ČR IAA600380701 Institutional research plan: CEZ:AV0Z50380511 Keywords : Physcomitrella * ove mutant * Cytokinin biosynthesis * tRNA-isopentenyl transferase Subject RIV: ED - Physiology Impact factor: 2.109, year: 2008

  20. RNA-Dependent Intergenerational Inheritance of Enhanced Synaptic Plasticity after Environmental Enrichment.

    Science.gov (United States)

    Benito, Eva; Kerimoglu, Cemil; Ramachandran, Binu; Pena-Centeno, Tonatiuh; Jain, Gaurav; Stilling, Roman Manuel; Islam, Md Rezaul; Capece, Vincenzo; Zhou, Qihui; Edbauer, Dieter; Dean, Camin; Fischer, André

    2018-04-10

    Physical exercise in combination with cognitive training is known to enhance synaptic plasticity, learning, and memory and lower the risk for various complex diseases including Alzheimer's disease. Here, we show that exposure of adult male mice to an environmental enrichment paradigm leads to enhancement of synaptic plasticity and cognition also in the next generation. We show that this effect is mediated through sperm RNA and especially miRs 212/132. In conclusion, our study reports intergenerational inheritance of an acquired cognitive benefit and points to specific miRs as candidates mechanistically involved in this type of transmission. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

  1. Immunopurification of the suppressor tRNA dependent rabbit β-globin readthrough protein

    International Nuclear Information System (INIS)

    Hatfield, D.; Thorgeirsson, S.S.; Copeland, T.D.; Oroszlan, S.; Bustin, M.

    1988-01-01

    In mammalian cells, the rabbit β-globin readthrough protein is the only known example of a naturally occurring readthrough protein which does not involve a viral system. To provide an efficient means for its isolation, detection, and study, the authors elicited specific antibodies against this unique protein. The 22 amino acid peptide corresponding to the readthrough portion of this protein was synthesized, coupled to keyhole limpet hemocyanin, and injected into sheep. Specific antibodies to the peptide were produced as demonstrated by the enzyme-linked immunosorbent assay technique and by immunoblotting. The antibodies did not react with globin. The rabbit β-globin readthrough protein was separated from globin and other reticulocyte proteins by polyacrylamide gel electrophoresis and visualized by silver staining or by labeling with [ 35 S] methionine. Incorporation of [ 35 S] methionine into the readthrough protein was significantly enhanced upon addition of an opal suppressor tRNA to reticulocyte lysates. Immunoblotting revealed that the readthrough protein also occurs in lysates without added suppressor tRNA. The antibodies were purified on an affi-gel column which had been coupled with the peptide antigen. The readthrough protein was then purified from reticulocytes by immunoaffinity chromatography and by high-performance liquid chromatography. The results provide conclusive evidence that the β-globin readthrough protein is naturally occurring in rabbit reticulocytes

  2. Rift Valley fever virus NSs inhibits host transcription independently of the degradation of dsRNA-dependent protein kinase PKR.

    Science.gov (United States)

    Kalveram, Birte; Lihoradova, Olga; Indran, Sabarish V; Lokugamage, Nandadeva; Head, Jennifer A; Ikegami, Tetsuro

    2013-01-20

    Rift Valley fever virus (RVFV) encodes one major virulence factor, the NSs protein. NSs suppresses host general transcription, including interferon (IFN)-β mRNA synthesis, and promotes degradation of the dsRNA-dependent protein kinase (PKR). We generated a novel RVFV mutant (rMP12-NSsR173A) specifically lacking the function to promote PKR degradation. rMP12-NSsR173A infection induces early phosphorylation of eIF2α through PKR activation, while retaining the function to inhibit host general transcription including IFN-β gene inhibition. MP-12 NSs but not R173A NSs binds to wt PKR. R173A NSs formed filamentous structure in nucleus in a mosaic pattern, which was distinct from MP-12 NSs filament pattern. Due to early phosphorylation of eIF2α, rMP12-NSsR173A could not efficiently accumulate viral proteins. Our results suggest that NSs-mediated host general transcription suppression occurs independently of PKR degradation, while the PKR degradation is important to inhibit the phosphorylation of eIF2α in infected cells undergoing host general transcription suppression. Copyright © 2012 Elsevier Inc. All rights reserved.

  3. A multi-step strategy to obtain crystals of the dengue virus RNA-dependent RNA polymerase that diffract to high resolution

    International Nuclear Information System (INIS)

    Yap, Thai Leong; Chen, Yen Liang; Xu, Ting; Wen, Daying; Vasudevan, Subhash G.; Lescar, Julien

    2007-01-01

    Crystals of the RNA-dependent RNA polymerase catalytic domain from the dengue virus NS5 protein have been obtained using a strategy that included expression screening of naturally occurring serotype variants of the protein, the addition of divalent metal ions and crystal dehydration. These crystals diffract to 1.85 Å resolution and are thus suitable for a structure-based drug-design program. Dengue virus, a member of the Flaviviridae genus, causes dengue fever, an important emerging disease with several million infections occurring annually for which no effective therapy exists. The viral RNA-dependent RNA polymerase NS5 plays an important role in virus replication and represents an interesting target for the development of specific antiviral compounds. Crystals that diffract to 1.85 Å resolution that are suitable for three-dimensional structure determination and thus for a structure-based drug-design program have been obtained using a strategy that included expression screening of naturally occurring serotype variants of the protein, the addition of divalent metal ions and crystal dehydration

  4. A multi-step strategy to obtain crystals of the dengue virus RNA-dependent RNA polymerase that diffract to high resolution

    Energy Technology Data Exchange (ETDEWEB)

    Yap, Thai Leong [Novartis Institute for Tropical Diseases, 10 Biopolis Road, Chromos Building, Singapore 138670 (Singapore); School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 (Singapore); Chen, Yen Liang; Xu, Ting; Wen, Daying; Vasudevan, Subhash G. [Novartis Institute for Tropical Diseases, 10 Biopolis Road, Chromos Building, Singapore 138670 (Singapore); Lescar, Julien, E-mail: julien@ntu.edu.sg [Novartis Institute for Tropical Diseases, 10 Biopolis Road, Chromos Building, Singapore 138670 (Singapore); School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 (Singapore)

    2007-02-01

    Crystals of the RNA-dependent RNA polymerase catalytic domain from the dengue virus NS5 protein have been obtained using a strategy that included expression screening of naturally occurring serotype variants of the protein, the addition of divalent metal ions and crystal dehydration. These crystals diffract to 1.85 Å resolution and are thus suitable for a structure-based drug-design program. Dengue virus, a member of the Flaviviridae genus, causes dengue fever, an important emerging disease with several million infections occurring annually for which no effective therapy exists. The viral RNA-dependent RNA polymerase NS5 plays an important role in virus replication and represents an interesting target for the development of specific antiviral compounds. Crystals that diffract to 1.85 Å resolution that are suitable for three-dimensional structure determination and thus for a structure-based drug-design program have been obtained using a strategy that included expression screening of naturally occurring serotype variants of the protein, the addition of divalent metal ions and crystal dehydration.

  5. Purification and properties of poliovirus RNA polymerase expressed in Escherichia coli

    International Nuclear Information System (INIS)

    Plotch, S.J.; Palant, O.; Gluzman, Y.

    1989-01-01

    A cDNA clone encoding the RNA polymerase of poliovirus has been expressed in Escherichia coli under the transcriptional control of a T7 bacteriophage promoter. This poliovirus enzyme was designed to contain only a single additional amino acid, the N-terminal methionine. The recombinant enzyme has been purified to near homogeneity, and polyclonal antibodies have been prepared against it. The enzyme exhibits poly(A)-dependent oligo(U)-primed ply(U) polymerase activity as well as RNA polymerase activity. In the presence of an oligo(U) primer, the enzyme catalyzes the synthesis of a full-length copy of either poliovirus or globin RNA templates. In the absence of added primer, RNA products up to twice the length of the template are synthesized. When incubated in the presence of a single nucleoside triphosphate, [α- 32 P]UTP, the enzyme catalyzes the incorporation of radioactive label into template RNA. These results are discussed in light of previously proposed models of poliovirus RNA synthesis in vitro

  6. The modeled structure of the RNA dependent RNA polymerase of GBV-C Virus suggests a role for motif E in Flaviviridae RNA polymerases

    Directory of Open Access Journals (Sweden)

    Dutartre Hélène

    2005-10-01

    Full Text Available Abstract Background The Flaviviridae virus family includes major human and animal pathogens. The RNA dependent RNA polymerase (RdRp plays a central role in the replication process, and thus is a validated target for antiviral drugs. Despite the increasing structural and enzymatic characterization of viral RdRps, detailed molecular replication mechanisms remain unclear. The hepatitis C virus (HCV is a major human pathogen difficult to study in cultured cells. The bovine viral diarrhea virus (BVDV is often used as a surrogate model to screen antiviral drugs against HCV. The structure of BVDV RdRp has been recently published. It presents several differences relative to HCV RdRp. These differences raise questions about the relevance of BVDV as a surrogate model, and cast novel interest on the "GB" virus C (GBV-C. Indeed, GBV-C is genetically closer to HCV than BVDV, and can lead to productive infection of cultured cells. There is no structural data for the GBV-C RdRp yet. Results We show in this study that the GBV-C RdRp is closest to the HCV RdRp. We report a 3D model of the GBV-C RdRp, developed using sequence-to-structure threading and comparative modeling based on the atomic coordinates of the HCV RdRp structure. Analysis of the predicted structural features in the phylogenetic context of the RNA polymerase family allows rationalizing most of the experimental data available. Both available structures and our model are explored to examine the catalytic cleft, allosteric and substrate binding sites. Conclusion Computational methods were used to infer evolutionary relationships and to predict the structure of a viral RNA polymerase. Docking a GTP molecule into the structure allows defining a GTP binding pocket in the GBV-C RdRp, such as that of BVDV. The resulting model suggests a new proposition for the mechanism of RNA synthesis, and may prove useful to design new experiments to implement our knowledge on the initiation mechanism of RNA

  7. In vitro replication of poliovirus

    International Nuclear Information System (INIS)

    Lubinski, J.M.

    1986-01-01

    Poliovirus is a member of the Picornaviridae whose genome is a single stranded RNA molecule of positive polarity surrounded by a proteinaceous capsid. Replication of poliovirus occurs via negative strand intermediates in infected cells using a virally encoded RNA-dependent RNA polymerase and host cell proteins. The authors have exploited the fact that complete cDNA copies of the viral genome when transfected onto susceptible cells generate virus. Utilizing the bacteriophage SP6 DNA dependent RNA polymerase system to synthesize negative strands in vitro and using these in an in vitro reaction the authors have generated full length infectious plus strands. Mutagenesis of the 5' and 3' ends of the negative and positive strands demonstrated that replication could occur either de novo or be extensions of the templates from their 3' ends or from nicks occurring during replication. The appearance of dimeric RNA molecules generated in these reactions was not dependent upon the same protein required for de novo initiation. Full length dimeric RNA molecules using a 5' 32 P end-labelled oligo uridylic acid primer and positive strand template were demonstrated in vitro containing only the 35,000 Mr host protein and the viral RNA-dependent RNA polymerase. A model for generating positive strands without protein priming by cleavage of dimeric RNA molecules was proposed

  8. Cytoplasmic viral RNA-dependent RNA polymerase disrupts the intracellular splicing machinery by entering the nucleus and interfering with Prp8.

    Directory of Open Access Journals (Sweden)

    Yen-Chin Liu

    2014-06-01

    Full Text Available The primary role of cytoplasmic viral RNA-dependent RNA polymerase (RdRp is viral genome replication in the cellular cytoplasm. However, picornaviral RdRp denoted 3D polymerase (3D(pol also enters the host nucleus, where its function remains unclear. In this study, we describe a novel mechanism of viral attack in which 3D(pol enters the nucleus through the nuclear localization signal (NLS and targets the pre-mRNA processing factor 8 (Prp8 to block pre-mRNA splicing and mRNA synthesis. The fingers domain of 3D(pol associates with the C-terminal region of Prp8, which contains the Jab1/MPN domain, and interferes in the second catalytic step, resulting in the accumulation of the lariat form of the splicing intermediate. Endogenous pre-mRNAs trapped by the Prp8-3D(pol complex in enterovirus-infected cells were identified and classed into groups associated with cell growth, proliferation, and differentiation. Our results suggest that picornaviral RdRp disrupts pre-mRNA splicing processes, that differs from viral protease shutting off cellular transcription and translation which contributes to the pathogenesis of viral infection.

  9. Endogenous short RNAs generated by Dicer 2 and RNA-dependent RNA polymerase 1 regulate mRNAs in the basal fungus Mucor circinelloides

    Science.gov (United States)

    Nicolas, Francisco Esteban; Moxon, Simon; de Haro, Juan P.; Calo, Silvia; Grigoriev, Igor V.; Torres-Martínez, Santiago; Moulton, Vincent; Ruiz-Vázquez, Rosa M.; Dalmay, Tamas

    2010-01-01

    Endogenous short RNAs (esRNAs) play diverse roles in eukaryotes and usually are produced from double-stranded RNA (dsRNA) by Dicer. esRNAs are grouped into different classes based on biogenesis and function but not all classes are present in all three eukaryotic kingdoms. The esRNA register of fungi is poorly described compared to other eukaryotes and it is not clear what esRNA classes are present in this kingdom and whether they regulate the expression of protein coding genes. However, evidence that some dicer mutant fungi display altered phenotypes suggests that esRNAs play an important role in fungi. Here, we show that the basal fungus Mucor circinelloides produces new classes of esRNAs that map to exons and regulate the expression of many protein coding genes. The largest class of these exonic-siRNAs (ex-siRNAs) are generated by RNA-dependent RNA Polymerase 1 (RdRP1) and dicer-like 2 (DCL2) and target the mRNAs of protein coding genes from which they were produced. Our results expand the range of esRNAs in eukaryotes and reveal a new role for esRNAs in fungi. PMID:20427422

  10. Endogenous short RNAs generated by Dicer 2 and RNA-dependent RNA polymerase 1 regulate mRNAs in the basal fungus Mucor circinelloides

    Energy Technology Data Exchange (ETDEWEB)

    Grigoriev, Igor; Nicolas, Francisco; Moxon, Simon; Haro, Juan de; Calo, Silvia; Torres-Martinez, Santiago; Moulton, Vincent; Ruiz-Vazquez, Rosa; Dalmay, Tamas

    2011-09-01

    Endogenous short RNAs (esRNAs) play diverse roles in eukaryotes and usually are produced from double-stranded RNA (dsRNA) by Dicer. esRNAs are grouped into different classes based on biogenesis and function but not all classes are present in all three eukaryotic kingdoms. The esRNA register of fungi is poorly described compared to other eukaryotes and it is not clear what esRNA classes are present in this kingdom and whether they regulate the expression of protein coding genes. However, evidence that some dicer mutant fungi display altered phenotypes suggests that esRNAs play an important role in fungi. Here, we show that the basal fungus Mucor circinelloides produces new classes of esRNAs that map to exons and regulate the expression of many protein coding genes. The largest class of these exonic-siRNAs (ex-siRNAs) are generated by RNA-dependent RNA Polymerase 1 (RdRP1) and dicer-like 2 (DCL2) and target the mRNAs of protein coding genes from which they were produced. Our results expand the range of esRNAs in eukaryotes and reveal a new role for esRNAs in fungi

  11. Activation of double-stranded RNA-dependent protein kinase inhibits proliferation of pancreatic β-cells

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Shan-Shan [Key Laboratory of Human Functional Genomics of Jiangsu Province, Nanjing Medical University, Nanjing (China); Department of Biochemistry and Molecular Biology, Nanjing Medical University, Nanjing (China); Jiang, Teng [Department of Neurology, Qingdao Municipal Hospital, Nanjing Medical University, Nanjing (China); Wang, Yi; Gu, Li-Ze [Key Laboratory of Human Functional Genomics of Jiangsu Province, Nanjing Medical University, Nanjing (China); Department of Biochemistry and Molecular Biology, Nanjing Medical University, Nanjing (China); Wu, Hui-Wen [Laboratory Center for Basic Medical Sciences, Nanjing Medical University, Nanjing (China); Tan, Lan [Department of Neurology, Qingdao Municipal Hospital, Nanjing Medical University, Nanjing (China); Guo, Jun, E-mail: Guoj@njmu.edu.cn [Key Laboratory of Human Functional Genomics of Jiangsu Province, Nanjing Medical University, Nanjing (China); Department of Biochemistry and Molecular Biology, Nanjing Medical University, Nanjing (China)

    2014-01-17

    Highlights: •PKR can be activated by glucolipitoxicity and pro-inflammatory cytokines in β-cells. •Activated PKR inhibited β-cell proliferation by arresting cell cycle at G1 phase. •Activated PKR fully abrogated the pro-proliferative effects of IGF-I on β-cells. -- Abstract: Double-stranded RNA-dependent protein kinase (PKR) is revealed to participate in the development of insulin resistance in peripheral tissues in type 2 diabetes (T2DM). Meanwhile, PKR is also characterized as a critical regulator of cell proliferation. To date, no study has focused on the impact of PKR on the proliferation of pancreatic β-cells. Here, we adopted insulinoma cell lines and mice islet β-cells to investigate: (1) the effects of glucolipotoxicity and pro-inflammatory cytokines on PKR activation; (2) the effects of PKR on proliferation of pancreatic β-cells and its underlying mechanisms; (3) the actions of PKR on pro-proliferative effects of IGF-I and its underlying pathway. Our results provided the first evidence that PKR can be activated by glucolipitoxicity and pro-inflammatory cytokines in pancreatic β-cells, and activated PKR significantly inhibited cell proliferation by arresting cell cycle at G1 phase. Reductions in cyclin D1 and D2 as well as increases in p27 and p53 were associated with the anti-proliferative effects of PKR, and proteasome-dependent degradation took part in the reduction of cyclin D1 and D2. Besides, PKR activation abrogated the pro-proliferative effects of IGF-I by activating JNK and disrupting IRS1/PI3K/Akt signaling pathway. These findings indicate that the anti-proliferative actions of PKR on pancreatic β-cells may contribute to the pathogenesis of T2DM.

  12. Activation of double-stranded RNA-dependent protein kinase inhibits proliferation of pancreatic β-cells

    International Nuclear Information System (INIS)

    Chen, Shan-Shan; Jiang, Teng; Wang, Yi; Gu, Li-Ze; Wu, Hui-Wen; Tan, Lan; Guo, Jun

    2014-01-01

    Highlights: •PKR can be activated by glucolipitoxicity and pro-inflammatory cytokines in β-cells. •Activated PKR inhibited β-cell proliferation by arresting cell cycle at G1 phase. •Activated PKR fully abrogated the pro-proliferative effects of IGF-I on β-cells. -- Abstract: Double-stranded RNA-dependent protein kinase (PKR) is revealed to participate in the development of insulin resistance in peripheral tissues in type 2 diabetes (T2DM). Meanwhile, PKR is also characterized as a critical regulator of cell proliferation. To date, no study has focused on the impact of PKR on the proliferation of pancreatic β-cells. Here, we adopted insulinoma cell lines and mice islet β-cells to investigate: (1) the effects of glucolipotoxicity and pro-inflammatory cytokines on PKR activation; (2) the effects of PKR on proliferation of pancreatic β-cells and its underlying mechanisms; (3) the actions of PKR on pro-proliferative effects of IGF-I and its underlying pathway. Our results provided the first evidence that PKR can be activated by glucolipitoxicity and pro-inflammatory cytokines in pancreatic β-cells, and activated PKR significantly inhibited cell proliferation by arresting cell cycle at G1 phase. Reductions in cyclin D1 and D2 as well as increases in p27 and p53 were associated with the anti-proliferative effects of PKR, and proteasome-dependent degradation took part in the reduction of cyclin D1 and D2. Besides, PKR activation abrogated the pro-proliferative effects of IGF-I by activating JNK and disrupting IRS1/PI3K/Akt signaling pathway. These findings indicate that the anti-proliferative actions of PKR on pancreatic β-cells may contribute to the pathogenesis of T2DM

  13. The internal initiation of translation in bovine viral diarrhea virus RNA depends on the presence of an RNA pseudoknot upstream of the initiation codon

    Directory of Open Access Journals (Sweden)

    Moes Lorin

    2007-11-01

    Full Text Available Abstract Background Bovine viral diarrhea virus (BVDV is the prototype representative of the pestivirus genus in the Flaviviridae family. It has been shown that the initiation of translation of BVDV RNA occurs by an internal ribosome entry mechanism mediated by the 5' untranslated region of the viral RNA 1. The 5' and 3' boundaries of the IRES of the cytopathic BVDV NADL have been mapped and it has been suggested that the IRES extends into the coding of the BVDV polyprotein 2. A putative pseudoknot structure has been recognized in the BVDV 5'UTR in close proximity to the AUG start codon. A pseudoknot structure is characteristic for flavivirus IRESes and in the case of the closely related classical swine fever virus (CSFV and the more distantly related Hepatitis C virus (HCV pseudoknot function in translation has been demonstrated. Results To characterize the BVDV IRESes in detail, we studied the BVDV translational initiation by transfection of dicistronic expression plasmids into mammalian cells. A region coding for the amino terminus of the BVDV SD-1 polyprotein contributes considerably to efficient initiation of translation. The translation efficiency mediated by the IRES of BVDV strains NADL and SD-1 approximates the poliovirus type I IRES directed translation in BHK cells. Compared to the poliovirus IRES increased expression levels are mediated by the BVDV IRES of strain SD-1 in murine cell lines, while lower levels are observed in human cell lines. Site directed mutagenesis revealed that a RNA pseudoknot upstream of the initiator AUG is an important structural element for IRES function. Mutants with impaired ability to base pair in stem I or II lost their translational activity. In mutants with repaired base pairing either in stem 1 or in stem 2 full translational activity was restored. Thus, the BVDV IRES translation is dependent on the pseudoknot integrity. These features of the pestivirus IRES are reminiscent of those of the classical

  14. The internal initiation of translation in bovine viral diarrhea virus RNA depends on the presence of an RNA pseudoknot upstream of the initiation codon.

    Science.gov (United States)

    Moes, Lorin; Wirth, Manfred

    2007-11-22

    Bovine viral diarrhea virus (BVDV) is the prototype representative of the pestivirus genus in the Flaviviridae family. It has been shown that the initiation of translation of BVDV RNA occurs by an internal ribosome entry mechanism mediated by the 5' untranslated region of the viral RNA 1. The 5' and 3' boundaries of the IRES of the cytopathic BVDV NADL have been mapped and it has been suggested that the IRES extends into the coding of the BVDV polyprotein 2. A putative pseudoknot structure has been recognized in the BVDV 5'UTR in close proximity to the AUG start codon. A pseudoknot structure is characteristic for flavivirus IRESes and in the case of the closely related classical swine fever virus (CSFV) and the more distantly related Hepatitis C virus (HCV) pseudoknot function in translation has been demonstrated. To characterize the BVDV IRESes in detail, we studied the BVDV translational initiation by transfection of dicistronic expression plasmids into mammalian cells. A region coding for the amino terminus of the BVDV SD-1 polyprotein contributes considerably to efficient initiation of translation. The translation efficiency mediated by the IRES of BVDV strains NADL and SD-1 approximates the poliovirus type I IRES directed translation in BHK cells. Compared to the poliovirus IRES increased expression levels are mediated by the BVDV IRES of strain SD-1 in murine cell lines, while lower levels are observed in human cell lines. Site directed mutagenesis revealed that a RNA pseudoknot upstream of the initiator AUG is an important structural element for IRES function. Mutants with impaired ability to base pair in stem I or II lost their translational activity. In mutants with repaired base pairing either in stem 1 or in stem 2 full translational activity was restored. Thus, the BVDV IRES translation is dependent on the pseudoknot integrity. These features of the pestivirus IRES are reminiscent of those of the classical swine fever virus, a pestivirus, and the

  15. tRNA-dependent cysteine biosynthetic pathway represents a strategy to increase cysteine contents by preventing it from thermal degradation: thermal adaptation of methanogenic archaea ancestor.

    Science.gov (United States)

    Qu, Ge; Wang, Wei; Chen, Ling-Ling; Qian, Shao-Song; Zhang, Hong-Yu

    2009-10-01

    Although cysteine (Cys) is beneficial to stabilize protein structures, it is not prevalent in thermophiles. For instance, the Cys contents in most thermophilic archaea are only around 0.7%. However, methanogenic archaea, no matter thermophilic or not, contain relatively abundant Cys, which remains elusive for a long time. Recently, Klipcan et al. correlated this intriguing property of methanogenic archaea with their unique tRNA-dependent Cys biosynthetic pathway. But, the deep reasons underlying the correlation are ambiguous. Considering the facts that free Cys is thermally labile and the tRNA-dependent Cys biosynthesis avoids the use of free Cys, we speculate that the unique Cys biosynthetic pathway represents a strategy to increase Cys contents by preventing it from thermal degradation, which may be relevant to the thermal adaptation of methanogenic archaea ancestor.

  16. Using the Hepatitis C Virus RNA-Dependent RNA Polymerase as a Model to Understand Viral Polymerase Structure, Function and Dynamics

    Directory of Open Access Journals (Sweden)

    Ester Sesmero

    2015-07-01

    Full Text Available Viral polymerases replicate and transcribe the genomes of several viruses of global health concern such as Hepatitis C virus (HCV, human immunodeficiency virus (HIV and Ebola virus. For this reason they are key targets for therapies to treat viral infections. Although there is little sequence similarity across the different types of viral polymerases, all of them present a right-hand shape and certain structural motifs that are highly conserved. These features allow their functional properties to be compared, with the goal of broadly applying the knowledge acquired from studying specific viral polymerases to other viral polymerases about which less is known. Here we review the structural and functional properties of the HCV RNA-dependent RNA polymerase (NS5B in order to understand the fundamental processes underlying the replication of viral genomes. We discuss recent insights into the process by which RNA replication occurs in NS5B as well as the role that conformational changes play in this process.

  17. Development of a challenge-protective vaccine concept by modification of the viral RNA-dependent RNA polymerase of canine distemper virus.

    Science.gov (United States)

    Silin, D; Lyubomska, O; Ludlow, M; Duprex, W P; Rima, B K

    2007-12-01

    We demonstrate that insertion of the open reading frame of enhanced green fluorescent protein (EGFP) into the coding sequence for the second hinge region of the viral L (large) protein (RNA-dependent RNA polymerase) attenuates a wild-type canine distemper virus. Moreover, we show that single intranasal immunization with this recombinant virus provides significant protection against challenge with the virulent parental virus. Protection against wild-type challenge was gained either after recovery of cellular immunity postimmunization or after development of neutralizing antibodies. Insertion of EGFP seems to result in overattenuation of the virus, while our previous experiments demonstrated that the insertion of an epitope tag into a similar position did not affect L protein function. Thus, a desirable level of attenuation could be reached by manipulating the length of the insert (in the second hinge region of the L protein), providing additional tools for optimization of controlled attenuation. This strategy for controlled attenuation may be useful for a "quick response" in vaccine development against well-known and "new" viral infections and could be combined efficiently with other strategies of vaccine development and delivery systems.

  18. Identification of constrained peptides that bind to and preferentially inhibit the activity of the hepatitis C viral RNA-dependent RNA polymerase

    International Nuclear Information System (INIS)

    Amin, Anthony; Zaccardi, Joe; Mullen, Stanley; Olland, Stephane; Orlowski, Mark; Feld, Boris; Labonte, Patrick; Mak, Paul

    2003-01-01

    A class of disulfide constrained peptides containing a core motif FPWG was identified from a screen of phage displayed library using the HCV RNA-dependent RNA polymerase (NS5B) as a bait. Surface plasmon resonance studies showed that three highly purified synthetic constrained peptides bound to immobilized NS5B with estimated K d values ranging from 30 to 60 μM. In addition, these peptides inhibited the NS5B activity in vitro with IC 50 ranging from 6 to 48 μM, whereas in contrast they had no inhibitory effect on the enzymatic activities of calf thymus polymerase α, human polymerase β, RSV polymerase, and HIV reverse transcriptase in vitro. Two peptides demonstrated conformation-dependent inhibition since their synthetic linear versions were not inhibitory in the NS5B assay. A constrained peptide with the minimum core motif FPWG retained selective inhibition of NS5B activity with an IC 50 of 50 μM. Alanine scan analyses of a representative constrained peptide, FPWGNTW, indicated that residues F1 and W7 were critical for the inhibitory effect of this peptide, although residues P2 and N5 had some measurable inhibitory effect as well. Further analyses of the mechanism of inhibition indicated that these peptides inhibited the formation of preelongation complexes required for the elongation reaction. However, once the preelongation complex was formed, its activity was refractory to peptide inhibition. Furthermore, the constrained peptide FPWGNTW inhibited de novo initiated RNA synthesis by NS5B from a poly(rC) template. These data indicate that the peptides confer selective inhibition of NS5B activity by binding to the enzyme and perturbing an early step preceding the processive elongation step of RNA synthesis

  19. The use of RNA-dependent RNA polymerase for the taxonomic assignment of Picorna-like viruses (order Picornavirales infecting Apis mellifera L. populations

    Directory of Open Access Journals (Sweden)

    Schroeder Declan C

    2008-01-01

    Full Text Available Abstract Background Single-stranded RNA viruses, infectious to the European honeybee, Apis mellifera L. are known to reside at low levels in colonies, with typically no apparent signs of infection observed in the honeybees. Reverse transcription-PCR (RT-PCR of regions of the RNA-dependent RNA polymerase (RdRp is often used to diagnose their presence in apiaries and also to classify the type of virus detected. Results Analysis of RdRp conserved domains was undertaken on members of the newly defined order, the Picornavirales; focusing in particular on the amino acid residues and motifs known to be conserved. Consensus sequences were compiled using partial and complete honeybee virus sequences published to date. Certain members within the iflaviruses, deformed wing virus (DWV, Kakugo virus (KV and Varroa destructor virus (VDV; and the dicistroviruses, acute bee paralysis virus (ABPV, Israeli paralysis virus (IAPV and Kashmir bee virus (KBV, shared greater than 98% and 92% homology across the RdRp conserved domains, respectively. Conclusion RdRp was validated as a suitable taxonomic marker for the assignment of members of the order Picornavirales, with the potential for use independent of other genetic or phenotypic markers. Despite the current use of the RdRp as a genetic marker for the detection of specific honeybee viruses, we provide overwhelming evidence that care should be taken with the primer set design. We demonstrated that DWV, VDV and KV, or ABPV, IAPV and KBV, respectively are all recent descendents or variants of each other, meaning caution should be applied when assigning presence or absence to any of these viruses when using current RdRp primer sets. Moreover, it is more likely that some primer sets (regardless of what gene is used are too specific and thus are underestimating the diversity of honeybee viruses.

  20. MicroRNA and Transcriptomic Profiling Showed miRNA-Dependent Impairment of Systemic Regulation and Synthesis of Biomolecules in Rag2 KO Mice

    Directory of Open Access Journals (Sweden)

    Abu Musa Md Talimur Reza

    2018-02-01

    Full Text Available The Rag2 knockout (KO mouse is a well-established immune-compromised animal model for biomedical research. A comparative study identified the deregulated expression of microRNAs (miRNAs and messenger RNAs (mRNAs in Rag2 KO mice. However, the interaction between deregulated genes and miRNAs in the alteration of systemic (cardiac, renal, hepatic, nervous, and hematopoietic regulations and the synthesis of biomolecules (such as l-tryptophan, serotonin, melatonin, dopamine, alcohol, noradrenaline, putrescine, and acetate are unclear. In this study, we analyzed both miRNA and mRNA expression microarray data from Rag2 KO and wild type mice to investigate the possible role of miRNAs in systemic regulation and biomolecule synthesis. A notable finding obtained from this analysis is that the upregulation of several genes which are target molecules of the downregulated miRNAs in Rag2 KO mice, can potentially trigger the degradation of l-tryptophan, thereby leading to the systemic impairment and alteration of biomolecules synthesis as well as changes in behavioral patterns (such as stress and fear responses, and social recognition memory in Rag2 gene-depleted mice. These findings were either not observed or not explicitly described in other published Rag2 KO transcriptome analyses. In conclusion, we have provided an indication of miRNA-dependent regulations of clinical and pathological conditions in cardiac, renal, hepatic, nervous, and hematopoietic systems in Rag2 KO mice. These results may significantly contribute to the prediction of clinical disease caused by Rag2 deficiency.

  1. MicroRNA and Transcriptomic Profiling Showed miRNA-Dependent Impairment of Systemic Regulation and Synthesis of Biomolecules in Rag2 KO Mice.

    Science.gov (United States)

    Reza, Abu Musa Md Talimur; Choi, Yun-Jung; Kim, Jin-Hoi

    2018-02-27

    The Rag2 knockout (KO) mouse is a well-established immune-compromised animal model for biomedical research. A comparative study identified the deregulated expression of microRNAs (miRNAs) and messenger RNAs (mRNAs) in Rag2 KO mice. However, the interaction between deregulated genes and miRNAs in the alteration of systemic (cardiac, renal, hepatic, nervous, and hematopoietic) regulations and the synthesis of biomolecules (such as l-tryptophan, serotonin, melatonin, dopamine, alcohol, noradrenaline, putrescine, and acetate) are unclear. In this study, we analyzed both miRNA and mRNA expression microarray data from Rag2 KO and wild type mice to investigate the possible role of miRNAs in systemic regulation and biomolecule synthesis. A notable finding obtained from this analysis is that the upregulation of several genes which are target molecules of the downregulated miRNAs in Rag2 KO mice, can potentially trigger the degradation of l-tryptophan, thereby leading to the systemic impairment and alteration of biomolecules synthesis as well as changes in behavioral patterns (such as stress and fear responses, and social recognition memory) in Rag2 gene-depleted mice. These findings were either not observed or not explicitly described in other published Rag2 KO transcriptome analyses. In conclusion, we have provided an indication of miRNA-dependent regulations of clinical and pathological conditions in cardiac, renal, hepatic, nervous, and hematopoietic systems in Rag2 KO mice. These results may significantly contribute to the prediction of clinical disease caused by Rag2 deficiency.

  2. Avian reovirus L2 genome segment sequences and predicted structure/function of the encoded RNA-dependent RNA polymerase protein

    Directory of Open Access Journals (Sweden)

    Xu Wanhong

    2008-12-01

    Full Text Available Abstract Background The orthoreoviruses are infectious agents that possess a genome comprised of 10 double-stranded RNA segments encased in two concentric protein capsids. Like virtually all RNA viruses, an RNA-dependent RNA polymerase (RdRp enzyme is required for viral propagation. RdRp sequences have been determined for the prototype mammalian orthoreoviruses and for several other closely-related reoviruses, including aquareoviruses, but have not yet been reported for any avian orthoreoviruses. Results We determined the L2 genome segment nucleotide sequences, which encode the RdRp proteins, of two different avian reoviruses, strains ARV138 and ARV176 in order to define conserved and variable regions within reovirus RdRp proteins and to better delineate structure/function of this important enzyme. The ARV138 L2 genome segment was 3829 base pairs long, whereas the ARV176 L2 segment was 3830 nucleotides long. Both segments were predicted to encode λB RdRp proteins 1259 amino acids in length. Alignments of these newly-determined ARV genome segments, and their corresponding proteins, were performed with all currently available homologous mammalian reovirus (MRV and aquareovirus (AqRV genome segment and protein sequences. There was ~55% amino acid identity between ARV λB and MRV λ3 proteins, making the RdRp protein the most highly conserved of currently known orthoreovirus proteins, and there was ~28% identity between ARV λB and homologous MRV and AqRV RdRp proteins. Predictive structure/function mapping of identical and conserved residues within the known MRV λ3 atomic structure indicated most identical amino acids and conservative substitutions were located near and within predicted catalytic domains and lining RdRp channels, whereas non-identical amino acids were generally located on the molecule's surfaces. Conclusion The ARV λB and MRV λ3 proteins showed the highest ARV:MRV identity values (~55% amongst all currently known ARV and MRV

  3. In vitro synthesis of minus-strand RNA by an isolated cereal yellow dwarf virus RNA-dependent RNA polymerase requires VPg and a stem-loop structure at the 3' end of the virus RNA.

    Science.gov (United States)

    Osman, Toba A M; Coutts, Robert H A; Buck, Kenneth W

    2006-11-01

    Cereal yellow dwarf virus (CYDV) RNA has a 5'-terminal genome-linked protein (VPg). We have expressed the VPg region of the CYDV genome in bacteria and used the purified protein (bVPg) to raise an antiserum which was able to detect free VPg in extracts of CYDV-infected oat plants. A template-dependent RNA-dependent RNA polymerase (RdRp) has been produced from a CYDV membrane-bound RNA polymerase by treatment with BAL 31 nuclease. The RdRp was template specific, being able to utilize templates from CYDV plus- and minus-strand RNAs but not those of three unrelated viruses, Red clover necrotic mosaic virus, Cucumber mosaic virus, and Tobacco mosaic virus. RNA synthesis catalyzed by the RdRp required a 3'-terminal GU sequence and the presence of bVPg. Additionally, synthesis of minus-strand RNA on a plus-strand RNA template required the presence of a putative stem-loop structure near the 3' terminus of CYDV RNA. The base-paired stem, a single-nucleotide (A) bulge in the stem, and the sequence of a tetraloop were all required for the template activity. Evidence was produced showing that minus-strand synthesis in vitro was initiated by priming by bVPg at the 3' end of the template. The data are consistent with a model in which the RdRp binds to the stem-loop structure which positions the active site to recognize the 3'-terminal GU sequence for initiation of RNA synthesis by the addition of an A residue to VPg.

  4. The functional half-life of an mRNA depends on the ribosome spacing in an early coding region

    DEFF Research Database (Denmark)

    Pedersen, Margit; Nissen, Søren; Mitarai, Namiko

    2011-01-01

    Bacterial mRNAs are translated by closely spaced ribosomes and degraded from the 5'-end, with half-lives of around 2 min at 37 °C in most cases. Ribosome-free or "naked" mRNA is known to be readily degraded, but the initial event that inactivates the mRNA functionally has not been fully described...

  5. Membrane fractions active in poliovirus RNA replication contain VPg precursor polypeptides

    International Nuclear Information System (INIS)

    Takegami, T.; Semler, B.L.; Anderson, C.W.; Wimmer, E.

    1983-01-01

    The poliovirus specific polypeptide P3-9 is of special interest for studies of viral RNA replication because it contains a hydrophobic region and, separated by only seven amino acids from that region, the amino acid sequence of the genome-linked protein VPg. Membraneous complexes of poliovirus-infected HeLa cells that contain poliovirus RNA replicating proteins have been analyzed for the presence of P3-9 by immunoprecipitation. Incubation of a membrane fraction rich in P3-9 with proteinase leaves the C-terminal 69 amino acids of P3-9 intact, an observation suggesting that this portion is protected by its association with the cellular membrane. These studies have also revealed two hitherto undescribed viral polypeptides consisting of amino acid sequences of the P2 andf P3 regions of the polyprotein. Sequence analysis by stepwise Edman degradation show that these proteins are 3b/9 (M/sub r/77,000) and X/9 (M/sub r/50,000). 3b/9 and X/9 are membrane bound and are turned over rapidly and may be direct precursors to proteins P2-X and P3-9 of the RNA replication complex. P2-X, a polypeptide void of hydrophobic amino acid sequences but also found associated with membranes, is rapidly degraded when the membraneous complex is treated with trypsin. It is speculated that P2-X is associated with membranes by its affinity to the N-terminus of P3-9

  6. Identification of novel miRNAs and miRNA dependent developmental shifts of gene expression in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Shuhua Zhan

    Full Text Available microRNAs (miRNAs are small, endogenous RNAs of 20 approximately 25 nucleotides, processed from stem-loop regions of longer RNA precursors. Plant miRNAs act as negative regulators of target mRNAs predominately by slicing target transcripts, and a number of miRNAs play important roles in development. We analyzed a number of published datasets from Arabidopsis thaliana to characterize novel miRNAs, novel miRNA targets, and miRNA-regulated developmental changes in gene expression. These data include microarray profiling data and small RNA (sRNA deep sequencing data derived from miRNA biogenesis/transport mutants, microarray profiling data of mRNAs in a developmental series, and computational predictions of conserved genomic stem-loop structures. Our conservative analyses identified five novel mature miRNAs and seven miRNA targets, including one novel target gene. Two complementary miRNAs that target distinct mRNAs were encoded by one gene. We found that genes targeted by known miRNAs, and genes up-regulated or down-regulated in miRNA mutant inflorescences, are highly expressed in the wild type inflorescence. In addition, transcripts upregulated within the mutant inflorescences were abundant in wild type leaves and shoot meristems and low in pollen and seed. Downregulated transcripts were abundant in wild type pollen and seed and low in shoot meristems, roots and leaves. Thus, disrupting miRNA function causes the inflorescence transcriptome to resemble the leaf and meristem and to differ from pollen and seed. Applications of our computational approach to other species and the use of more liberal criteria than reported here will further expand the number of identified miRNAs and miRNA targets. Our findings suggest that miRNAs have a global role in promoting vegetative to reproductive transitions in A. thaliana.

  7. Initiation of poliovirus plus-strand RNA synthesis in a membrane complex of infected HeLa cells

    International Nuclear Information System (INIS)

    Takeda, N.; Kuhn, R.J.; Yang, C.F.; Takegami, T.; Wimmer, E.

    1986-01-01

    An in vitro poliovirus RNA-synthesizing system derived from a crude membrance fraction of infected HeLa cells was used to analyze the mechanism of initiation of poliovirus plus-strand RNA synthesis. This system contains an activity that synthesizes the nucleotidyl proteins VPg-pU and VPg-pUpU. These molecules represent the 5'-terminal structure of nascent RNA molecules and of virion RNA. The membranous replication complex is also capable of synthesizing mucleotidyl proteins containing nine or more of the poliovirus 5'-proximal nucleotides as assayed by the formation of the RNase T 1 -resistant oligonucleotide VPg-pUUAAAACAGp or by fingerprint analysis of the in vitro-synthesized 32 P-RNA. Incubation of preformed VPg-pUpU with unlabeled nucleoside triphosphates resulted in the formation of VPg-pUUAAAACAGp. This reaction, which appeared to be an elongation of VPg-pUpU, was stimulated by the addition of a soluble fraction (S-10) obtained from uninfected HeLa cells. Preformed VPg-pU could be chased into VPg-pUpU in the presence of UTP. The data are consistent with a model that VPg-pU can function as a primer for poliovirus plus-strand RNA synthesis in the membranous replication complex and that the elongation reaction may be stimulated by a host cellular factor

  8. Inhibition of Poliovirus-Induced Cleavage of Cellular Protein PCBP2 Reduces the Levels of Viral RNA Replication

    Science.gov (United States)

    Chase, Amanda J.; Daijogo, Sarah

    2014-01-01

    ABSTRACT Due to their small genome size, picornaviruses must utilize host proteins to mediate cap-independent translation and viral RNA replication. The host RNA-binding protein poly(rC) binding protein 2 (PCBP2) is involved in both processes in poliovirus infected cells. It has been shown that the viral proteinase 3CD cleaves PCBP2 and contributes to viral translation inhibition. However, cleaved PCBP2 remains active in viral RNA replication. This would suggest that both cleaved and intact forms of PCBP2 have a role in the viral RNA replication cycle. The picornavirus genome must act as a template for both translation and RNA replication. However, a template that is actively being translated cannot function as a template for RNA replication, suggesting that there is a switch in template usage from translation to RNA replication. We demonstrate that the cleavage of PCBP2 by the poliovirus 3CD proteinase is a necessary step for efficient viral RNA replication and, as such, may be important for mediating a switch in template usage from translation to RNA replication. IMPORTANCE Poliovirus, like all positive-strand RNA viruses that replicate in the cytoplasm of eukaryotic cells, uses its genomic RNA as a template for both viral protein synthesis and RNA replication. Given that these processes cannot occur simultaneously on the same template, poliovirus has evolved a mechanism(s) to facilitate the switch from using templates for translation to using them for RNA synthesis. This study explores one possible scenario for how the virus alters the functions of a host cell RNA binding protein to mediate, in part, this important transition. PMID:24371074

  9. Negative charge and membrane-tethered viral 3B cooperate to recruit viral RNA dependent RNA polymerase 3D(pol)

    Czech Academy of Sciences Publication Activity Database

    Dubánková, Anna; Humpolíčková, Jana; Klíma, Martin; Bouřa, Evžen

    2017-01-01

    Roč. 7, Dec 11 (2017), č. článku 17309. ISSN 2045-2322 R&D Projects: GA ČR GJ15-21030Y Institutional support: RVO:61388963 Keywords : 4-kinase III beta * in-vitro reconstitution * poliovirus protein 3AB Subject RIV: CE - Biochemistry OBOR OECD: Biochemistry and molecular biology Impact factor: 4.259, year: 2016 https://www.nature.com/articles/s41598-017-17621-6

  10. MicroRNA-dependent regulation of metamorphosis and identification of microRNAs in the red flour beetle, Tribolium castaneum.

    Science.gov (United States)

    Wu, Wei; Xiong, Wenfeng; Li, Chengjun; Zhai, Mengfan; Li, Yao; Ma, Fei; Li, Bin

    2017-10-01

    To date, although some microRNAs (miRNAs) have been discovered in the holometabolism insect Tribolium castaneum, large numbers of miRNAs still require investigation. Knocking down Dicer-1 (Dcr-1) and Argonaute-1 (Ago-1) in late larvae impaired miRNA synthesis, affected the juvenile hormone pathway by up-regulating Methoprene-tolerant (Met) and Krüppel-homolog1 (Kr-h1) transcript levels, and resulted in a series of defects in T. castaneum development and metamorphosis. Thus, high-throughput Illumina/Solexa sequencing was performed with a mixed sample of eight key developmental stages of T. castaneum. In total, 1154 unique miRNAs were discovered containing 274 conserved miRNAs belong to 68 miRNA families, 108 known candidate miRNAs and 772 novel miRNAs. Genome locus analysis showed that miRNA clusters are more abundant in T. castaneum than other species. The results indicated that RNAi of Dcr-1 and Ago-1 in T. castaneum resulted in miRNA-induced metamorphosis defects. Furthermore, large numbers of novel miRNAs were discovered in T. castaneum and localized to T. castaneum genome loci. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. A critical role of a cellular membrane traffic protein in poliovirus RNA replication.

    Directory of Open Access Journals (Sweden)

    George A Belov

    2008-11-01

    Full Text Available Replication of many RNA viruses is accompanied by extensive remodeling of intracellular membranes. In poliovirus-infected cells, ER and Golgi stacks disappear, while new clusters of vesicle-like structures form sites for viral RNA synthesis. Virus replication is inhibited by brefeldin A (BFA, implicating some components(s of the cellular secretory pathway in virus growth. Formation of characteristic vesicles induced by expression of viral proteins was not inhibited by BFA, but they were functionally deficient. GBF1, a guanine nucleotide exchange factor for the small cellular GTPases, Arf, is responsible for the sensitivity of virus infection to BFA, and is required for virus replication. Knockdown of GBF1 expression inhibited virus replication, which was rescued by catalytically active protein with an intact N-terminal sequence. We identified a mutation in GBF1 that allows growth of poliovirus in the presence of BFA. Interaction between GBF1 and viral protein 3A determined the outcome of infection in the presence of BFA.

  12. Viral precursor protein P3 and its processed products perform discrete and essential functions in the poliovirus RNA replication complex

    Science.gov (United States)

    The differential use of protein precursors and their products is a key strategy used during poliovirus replication. To characterize the role of protein precursors during replication, we examined the complementation profiles of mutants that inhibited 3D polymerase or 3C-RNA binding activity. We showe...

  13. Optimal packaging of FIV genomic RNA depends upon a conserved long-range interaction and a palindromic sequence within gag.

    Science.gov (United States)

    Rizvi, Tahir A; Kenyon, Julia C; Ali, Jahabar; Aktar, Suriya J; Phillip, Pretty S; Ghazawi, Akela; Mustafa, Farah; Lever, Andrew M L

    2010-10-15

    The feline immunodeficiency virus (FIV) is a lentivirus that is related to human immunodeficiency virus (HIV), causing a similar pathology in cats. It is a potential small animal model for AIDS and the FIV-based vectors are also being pursued for human gene therapy. Previous studies have mapped the FIV packaging signal (ψ) to two or more discontinuous regions within the 5' 511 nt of the genomic RNA and structural analyses have determined its secondary structure. The 5' and 3' sequences within ψ region interact through extensive long-range interactions (LRIs), including a conserved heptanucleotide interaction between R/U5 and gag. Other secondary structural elements identified include a conserved 150 nt stem-loop (SL2) and a small palindromic stem-loop within gag open reading frame that might act as a viral dimerization initiation site. We have performed extensive mutational analysis of these sequences and structures and ascertained their importance in FIV packaging using a trans-complementation assay. Disrupting the conserved heptanucleotide LRI to prevent base pairing between R/U5 and gag reduced packaging by 2.8-5.5 fold. Restoration of pairing using an alternative, non-wild type (wt) LRI sequence restored RNA packaging and propagation to wt levels, suggesting that it is the structure of the LRI, rather than its sequence, that is important for FIV packaging. Disrupting the palindrome within gag reduced packaging by 1.5-3-fold, but substitution with a different palindromic sequence did not restore packaging completely, suggesting that the sequence of this region as well as its palindromic nature is important. Mutation of individual regions of SL2 did not have a pronounced effect on FIV packaging, suggesting that either it is the structure of SL2 as a whole that is necessary for optimal packaging, or that there is redundancy within this structure. The mutational analysis presented here has further validated the previously predicted RNA secondary structure of FIV

  14. Citrus psorosis virus RNA 1 is of negative polarity and potentially encodes in its complementary strand a 24K protein of unknown function and 280K putative RNA dependent RNA polymerase.

    Science.gov (United States)

    Naum-Onganía, Gabriela; Gago-Zachert, Selma; Peña, Eduardo; Grau, Oscar; Garcia, Maria Laura

    2003-10-01

    Citrus psorosis virus (CPsV), the type member of genus Ophiovirus, has three genomic RNAs. Complete sequencing of CPsV RNA 1 revealed a size of 8184 nucleotides and Northern blot hybridization with chain specific probes showed that its non-coding strand is preferentially encapsidated. The complementary strand of RNA 1 contains two open reading frames (ORFs) separated by a 109-nt intergenic region, one located near the 5'-end potentially encoding a 24K protein of unknown function, and another of 280K containing the core polymerase motifs characteristic of viral RNA-dependent RNA polymerases (RdRp). Comparison of the core RdRp motifs of negative-stranded RNA viruses, supports grouping CPsV, Ranunculus white mottle virus (RWMV) and Mirafiori lettuce virus (MiLV) within the same genus (Ophiovirus), constituting a monophyletic group separated from all other negative-stranded RNA viruses. Furthermore, RNAs 1 of MiLV, CPsV and RWMV are similar in size and those of MiLV and CPsV also in genomic organization and sequence.

  15. Histone Methylation and microRNA-dependent Regulation of Epigenetic Activities in Neural Progenitor Self-Renewal and Differentiation.

    Science.gov (United States)

    Cacci, Emanuele; Negri, Rodolfo; Biagioni, Stefano; Lupo, Giuseppe

    2017-01-01

    Neural stem/progenitor cell (NSPC) self-renewal and differentiation in the developing and the adult brain are controlled by extra-cellular signals and by the inherent competence of NSPCs to produce appropriate responses. Stage-dependent responsiveness of NSPCs to extrinsic cues is orchestrated at the epigenetic level. Epigenetic mechanisms such as DNA methylation, histone modifications and non-coding RNA-mediated regulation control crucial aspects of NSPC development and function, and are also implicated in pathological conditions. While their roles in the regulation of stem cell fate have been largely explored in pluripotent stem cell models, the epigenetic signature of NSPCs is also key to determine their multipotency as well as their progressive bias towards specific differentiation outcomes. Here we review recent developments in this field, focusing on the roles of histone methylation marks and the protein complexes controlling their deposition in NSPCs of the developing cerebral cortex and the adult subventricular zone. In this context, we describe how bivalent promoters, carrying antagonistic epigenetic modifications, feature during multiple steps of neural development, from neural lineage specification to neuronal differentiation. Furthermore, we discuss the emerging cross-talk between epigenetic regulators and microRNAs, and how the interplay between these different layers of regulation can finely tune the expression of genes controlling NSPC maintenance and differentiation. In particular, we highlight recent advances in the identification of astrocyte-enriched microRNAs and their function in cell fate choices of NSPCs differentiating towards glial lineages.

  16. Schizophrenia-Related Microdeletion Impairs Emotional Memory through MicroRNA-Dependent Disruption of Thalamic Inputs to the Amygdala

    Directory of Open Access Journals (Sweden)

    Tae-Yeon Eom

    2017-05-01

    Full Text Available Individuals with 22q11.2 deletion syndrome (22q11DS are at high risk of developing psychiatric diseases such as schizophrenia. Individuals with 22q11DS and schizophrenia are impaired in emotional memory, anticipating, recalling, and assigning a correct context to emotions. The neuronal circuits responsible for these emotional memory deficits are unknown. Here, we show that 22q11DS mouse models have disrupted synaptic transmission at thalamic inputs to the lateral amygdala (thalamo-LA projections. This synaptic deficit is caused by haploinsufficiency of the 22q11DS gene Dgcr8, which is involved in microRNA processing, and is mediated by the increased dopamine receptor Drd2 levels in the thalamus and by reduced probability of glutamate release from thalamic inputs. This deficit in thalamo-LA synaptic transmission is sufficient to cause fear memory deficits. Our results suggest that dysregulation of the Dgcr8–Drd2 mechanism at thalamic inputs to the amygdala underlies emotional memory deficits in 22q11DS.

  17. Schizophrenia-Related Microdeletion Impairs Emotional Memory through MicroRNA-Dependent Disruption of Thalamic Inputs to the Amygdala.

    Science.gov (United States)

    Eom, Tae-Yeon; Bayazitov, Ildar T; Anderson, Kara; Yu, Jing; Zakharenko, Stanislav S

    2017-05-23

    Individuals with 22q11.2 deletion syndrome (22q11DS) are at high risk of developing psychiatric diseases such as schizophrenia. Individuals with 22q11DS and schizophrenia are impaired in emotional memory, anticipating, recalling, and assigning a correct context to emotions. The neuronal circuits responsible for these emotional memory deficits are unknown. Here, we show that 22q11DS mouse models have disrupted synaptic transmission at thalamic inputs to the lateral amygdala (thalamo-LA projections). This synaptic deficit is caused by haploinsufficiency of the 22q11DS gene Dgcr8, which is involved in microRNA processing, and is mediated by the increased dopamine receptor Drd2 levels in the thalamus and by reduced probability of glutamate release from thalamic inputs. This deficit in thalamo-LA synaptic transmission is sufficient to cause fear memory deficits. Our results suggest that dysregulation of the Dgcr8-Drd2 mechanism at thalamic inputs to the amygdala underlies emotional memory deficits in 22q11DS. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  18. Circulating type 1 vaccine-derived poliovirus may evolve under the pressure of adenosine deaminases acting on RNA.

    Science.gov (United States)

    Liu, Yanhan; Ma, Tengfei; Liu, Jianzhu; Zhao, Xiaona; Cheng, Ziqiang; Guo, Huijun; Xu, Ruixue; Wang, Shujing

    2015-01-01

    Poliovirus, the causative agent of poliomyelitis, is a human enterovirus and member of the Picornaviridae family. An effective live-attenuated poliovirus vaccine strain (Sabin 1) has been developed and has protected humans from polio. However, a few cases of vaccine virulence reversion have been documented in several countries. For instance, circulating type 1 vaccine-derived poliovirus is a highly pathogenic poliovirus that evolved from an avirulent strain, but the mechanism by which vaccine strains undergo reversion remains unclear. In this study, vaccine strains exhibited A to G/U to C and G to A/C to U hypermutations in the reversed evolution of Sabin 1. Furthermore, the mutation ratios of U to C and C to U were higher than those of other mutation types. Dinucleotide editing context was then analyzed. Results showed that A to G and U to C mutations exhibited preferences similar to adenosine deaminases acting on RNA (ADAR). Hence, ADARs may participate in poliovirus vaccine evolution.

  19. An intronic ncRNA-dependent regulation of SORL1 expression affecting Aβ formation is upregulated in post-mortem Alzheimer's disease brain samples

    Directory of Open Access Journals (Sweden)

    Eleonora Ciarlo

    2013-03-01

    Recent studies indicated that sortilin-related receptor 1 (SORL1 is a risk gene for late-onset Alzheimer's disease (AD, although its role in the aetiology and/or progression of this disorder is not fully understood. Here, we report the finding of a non-coding (nc RNA (hereafter referred to as 51A that maps in antisense configuration to intron 1 of the SORL1 gene. 51A expression drives a splicing shift of SORL1 from the synthesis of the canonical long protein variant A to an alternatively spliced protein form. This process, resulting in a decreased synthesis of SORL1 variant A, is associated with impaired processing of amyloid precursor protein (APP, leading to increased Aβ formation. Interestingly, we found that 51A is expressed in human brains, being frequently upregulated in cerebral cortices from individuals with Alzheimer's disease. Altogether, these findings document a novel ncRNA-dependent regulatory pathway that might have relevant implications in neurodegeneration.

  20. NSs protein of rift valley fever virus induces the specific degradation of the double-stranded RNA-dependent protein kinase.

    Science.gov (United States)

    Habjan, Matthias; Pichlmair, Andreas; Elliott, Richard M; Overby, Anna K; Glatter, Timo; Gstaiger, Matthias; Superti-Furga, Giulio; Unger, Hermann; Weber, Friedemann

    2009-05-01

    Rift Valley fever virus (RVFV) continues to cause large outbreaks of acute febrile and often fatal illness among humans and domesticated animals in Africa, Saudi Arabia, and Yemen. The high pathogenicity of this bunyavirus is mainly due to the viral protein NSs, which was shown to prevent transcriptional induction of the antivirally active type I interferons (alpha/beta interferon [IFN-alpha/beta]). Viruses lacking the NSs gene induce synthesis of IFNs and are therefore attenuated, whereas the noninducing wild-type RVFV strains can only be inhibited by pretreatment with IFN. We demonstrate here in vitro and in vivo that a substantial part of the antiviral activity of IFN against RVFV is due to a double-stranded RNA-dependent protein kinase (PKR). PKR-mediated virus inhibition, however, was much more pronounced for the strain Clone 13 with NSs deleted than for the NSs-expressing strain ZH548. In vivo, Clone 13 was nonpathogenic for wild-type (wt) mice but could regain pathogenicity if mice lacked the PKR gene. ZH548, in contrast, killed both wt and PKR knockout mice indiscriminately. ZH548 was largely resistant to the antiviral properties of PKR because RVFV NSs triggered the specific degradation of PKR via the proteasome. The NSs proteins of the related but less virulent sandfly fever Sicilian virus and La Crosse virus, in contrast, had no such anti-PKR activity despite being efficient suppressors of IFN induction. Our data suggest that RVFV NSs has gained an additional anti-IFN function that may explain the extraordinary pathogenicity of this virus.

  1. Generation of Recombinant Polioviruses Harboring RNA Affinity Tags in the 5′ and 3′ Noncoding Regions of Genomic RNAs

    Science.gov (United States)

    Flather, Dylan; Cathcart, Andrea L.; Cruz, Casey; Baggs, Eric; Ngo, Tuan; Gershon, Paul D.; Semler, Bert L.

    2016-01-01

    Despite being intensely studied for more than 50 years, a complete understanding of the enterovirus replication cycle remains elusive. Specifically, only a handful of cellular proteins have been shown to be involved in the RNA replication cycle of these viruses. In an effort to isolate and identify additional cellular proteins that function in enteroviral RNA replication, we have generated multiple recombinant polioviruses containing RNA affinity tags within the 3′ or 5′ noncoding region of the genome. These recombinant viruses retained RNA affinity sequences within the genome while remaining viable and infectious over multiple passages in cell culture. Further characterization of these viruses demonstrated that viral protein production and growth kinetics were unchanged or only slightly altered relative to wild type poliovirus. However, attempts to isolate these genetically-tagged viral genomes from infected cells have been hindered by high levels of co-purification of nonspecific proteins and the limited matrix-binding efficiency of RNA affinity sequences. Regardless, these recombinant viruses represent a step toward more thorough characterization of enterovirus ribonucleoprotein complexes involved in RNA replication. PMID:26861382

  2. The TAL effector PthA4 interacts with nuclear factors involved in RNA-dependent processes including a HMG protein that selectively binds poly(U RNA.

    Directory of Open Access Journals (Sweden)

    Tiago Antonio de Souza

    Full Text Available Plant pathogenic bacteria utilize an array of effector proteins to cause disease. Among them, transcriptional activator-like (TAL effectors are unusual in the sense that they modulate transcription in the host. Although target genes and DNA specificity of TAL effectors have been elucidated, how TAL proteins control host transcription is poorly understood. Previously, we showed that the Xanthomonas citri TAL effectors, PthAs 2 and 3, preferentially targeted a citrus protein complex associated with transcription control and DNA repair. To extend our knowledge on the mode of action of PthAs, we have identified new protein targets of the PthA4 variant, required to elicit canker on citrus. Here we show that all the PthA4-interacting proteins are DNA and/or RNA-binding factors implicated in chromatin remodeling and repair, gene regulation and mRNA stabilization/modification. The majority of these proteins, including a structural maintenance of chromosomes protein (CsSMC, a translin-associated factor X (CsTRAX, a VirE2-interacting protein (CsVIP2, a high mobility group (CsHMG and two poly(A-binding proteins (CsPABP1 and 2, interacted with each other, suggesting that they assemble into a multiprotein complex. CsHMG was shown to bind DNA and to interact with the invariable leucine-rich repeat region of PthAs. Surprisingly, both CsHMG and PthA4 interacted with PABP1 and 2 and showed selective binding to poly(U RNA, a property that is novel among HMGs and TAL effectors. Given that homologs of CsHMG, CsPABP1, CsPABP2, CsSMC and CsTRAX in other organisms assemble into protein complexes to regulate mRNA stability and translation, we suggest a novel role of TAL effectors in mRNA processing and translational control.

  3. Structural organization of poliovirus RNA replication is mediated by viral proteins of the P2 genomic region

    International Nuclear Information System (INIS)

    Bienz, K.; Egger, D.; Troxler, M.; Pasamontes, L.

    1990-01-01

    Transcriptionally active replication complexes bound to smooth membrane vesicles were isolated from poliovirus-infected cells. In electron microscopic, negatively stained preparations, the replication complex appeared as an irregularly shaped, oblong structure attached to several virus-induced vesicles of a rosettelike arrangement. Electron microscopic immunocytochemistry of such preparations demonstrated that the poliovirus replication complex contains the proteins coded by the P2 genomic region (P2 proteins) in a membrane-associated form. In addition, the P2 proteins are also associated with viral RNA, and they can be cross-linked to viral RNA by UV irradiation. Guanidine hydrochloride prevented the P2 proteins from becoming membrane bound but did not change their association with viral RNA. The findings allow the conclusion that the protein 2C or 2C-containing precursor(s) is responsible for the attachment of the viral RNA to the vesicular membrane and for the spatial organization of the replication complex necessary for its proper functioning in viral transcription. A model for the structure of the viral replication complex and for the function of the 2C-containing P2 protein(s) and the vesicular membranes is proposed

  4. Characterization of product RNAs synthesized in vitro by poliovirus RNA polymerase purified by chromatography on hydroxylapatite or poly(U) Sepharose.

    OpenAIRE

    Young, D C; Tobin, G J; Flanegan, J B

    1987-01-01

    The size of the product RNA synthesized by the poliovirus RNA polymerase and host factor was significantly affected by the type of column chromatography used to purify the polymerase. Dimer length product RNA was synthesized by the polymerase purified by chromatography on hydroxylapatite. This contrasted with the monomer length product RNA synthesized by the polymerase purified by chromatography on poly(U) Sepharose. The poly(U) Sepharose-purified polymerase was shown to contain oligo(U) that...

  5. Relationship between synthesis and cleavage of poliovirus-specific proteins.

    OpenAIRE

    Thomas, A A; Voorma, H O; Boeye, A

    1983-01-01

    Poliovirus proteinase was studied in vitro in lysates from poliovirus-infected HeLa cells. Preincubation of these lysates caused (i) a reduction in poliovirus proteinase activity and (ii) a partial dependence on exogenous mRNA for optimal translation. Proteins translated from endogenous poliovirus RNA in preincubated extracts from virus-infected HeLa cells are poorly cleaved. This cleavage deficiency is alleviated by adding fresh poliovirus RNA to the translation system, thus, allowing re-ini...

  6. 3-(imidazo[1,2-a:5,4-b']dipyridin-2-yl)aniline inhibits pestivirus replication by targeting a hot spot drug binding pocket in the RNA-dependent RNA polymerase.

    Science.gov (United States)

    Musiu, Simone; Leyssen, Pieter; Froeyen, Mathy; Chezal, Jean-Michel; Neyts, Johan; Paeshuyse, Jan

    2016-05-01

    The compound 3-(imidazo[1,2-a:5,4-b']dipyridin-2-yl)aniline (CF02334) was identified as a selective inhibitor of the cytopathic effect (CPE) caused by bovine viral diarrhea virus (BVDV) in a virus-cell-based assay. The EC50-values for inhibition of CPE, viral RNA synthesis and the production of infectious virus progeny were 13.0 ± 0.6 μM, 2.6 ± 0.9 μM and 17.8 ± 0.6 μM, respectively. CF02334 was found to be inactive in the hepatitis C subgenomic replicon system. CF02334-resistant BVDV was obtained and was found to carry the N264D mutation in the viral RNA-dependent RNA polymerase (RdRp). Molecular modeling revealed that N264D is located in a small cavity near the fingertip domain of the pestivirus polymerase. CF02334-resistant BVDV was proven to be cross-resistant to BPIP, AG110 and LZ37, inhibitors that have previously been described to target the same region of the BVDV RdRp. CF02334 did not inhibit the in vitro activity of recombinant BVDV RdRp, but did inhibit the activity of BVDV replication complexes. Taken together, these observations indicate that CF02334 likely interacts with the fingertip of the pestivirus RdRp at the same position as BPIP, AG110 and LZ37, which marks this region of the viral polymerase as a "hot spot" for inhibition of pestivirus replication. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Human Immunodeficiency Virus Tat-Activated Expression of Poliovirus Protein 2A Inhibits mRNA Translation

    Science.gov (United States)

    Sun, Xiao-Hong; Baltimore, David

    1989-04-01

    To study the effect of poliovirus protein 2A on cellular RNA translation, the tat control system of human immunodeficiency virus (HIV) was used. Protein 2A was expressed from a plasmid construct (pHIV/2A) incorporating the HIV long terminal repeat. Protein synthesis was measured by using chloramphenicol acetyltransferase as a reporter gene driven by the Rous sarcoma virus long terminal repeat. When HIV/2A was contransfected with the reporter, addition of a tat-producing plasmid caused at least a 50-fold drop in chloramphenicol acetyltransferase synthesis. A HeLa cell line carrying HIV/2A was established. In it, tat expression caused more than a 10-fold drop in chloramphenicol acetyltransferase synthesis from the reporter plasmid. Furthermore, 2A induction by tat caused cleavage of the cellular translation factor P220, a part of eukaryotic translation initiation factor 4F. Thus protein 2A can, by itself, carry out the inhibition of cellular protein synthesis characteristic of a poliovirus infection. Also, the HIV tat activation provides a very effective method to control gene expression in mammalian cells.

  8. Nuclear export of human hepatitis B virus core protein and pregenomic RNA depends on the cellular NXF1-p15 machinery.

    Science.gov (United States)

    Yang, Ching-Chun; Huang, Er-Yi; Li, Hung-Cheng; Su, Pei-Yi; Shih, Chiaho

    2014-01-01

    Hepatitis B virus (HBV) core protein (HBc) can shuttle between nucleus and cytoplasm. Cytoplasm-predominant HBc is clinically associated with severe liver inflammation. Previously, we found that HBc arginine-rich domain (ARD) can associate with a host factor NXF1 (TAP) by coimmunoprecipitation. It is well known that NXF1-p15 heterodimer can serve as a major export receptor of nuclear mRNA as a ribonucleoprotein complex (RNP). In the NXF1-p15 pathway, TREX (transcription/export) complex plays an important role in coupling nuclear pre-mRNA processing with mRNA export in mammalian cells. Here, we tested the hypothesis whether HBc and HBV specific RNA can be exported via the TREX and NXF1-p15 mediated pathway. We demonstrated here that HBc can physically and specifically associate with TREX components, and the NXF1-p15 export receptor by coimmunoprecipitation. Accumulation of HBc protein in the nucleus can be induced by the interference with TREX and NXF1-p15 mediated RNA export machinery. HBV transcripts encodes a non-spliced 3.5 kb pregenomic RNA (pgRNA) which can serve as a template for reverse transcription. Cytoplasmic HBV pgRNA appeared to be reduced by siRNA treatment specific for the NXF1-p15 complex by quantitative RT-qPCR and Northern blot analyses. This result suggests that the pgRNA was also exported via the NXF1-p15 machinery. We entertain the hypothesis that HBc protein can be exported as an RNP cargo via the mRNA export pathway by hijacking the TREX and NXF1-p15 complex. In our current and previous studies, HBc is not required for pgRNA accumulation in the cytoplasm. Furthermore, HBc ARD can mediate nuclear export of a chimeric protein containing HBc ARD in a pgRNA-independent manner. Taken together, it suggests that while both pgRNA and HBc protein exports are dependent on NXF1-p15, they are using the same export machinery in a manner independent of each other.

  9. Packaging of Mason-Pfizer monkey virus (MPMV) genomic RNA depends upon conserved long-range interactions (LRIs) between U5 and gag sequences.

    Science.gov (United States)

    Kalloush, Rawan M; Vivet-Boudou, Valérie; Ali, Lizna M; Mustafa, Farah; Marquet, Roland; Rizvi, Tahir A

    2016-06-01

    MPMV has great potential for development as a vector for gene therapy. In this respect, precisely defining the sequences and structural motifs that are important for dimerization and packaging of its genomic RNA (gRNA) are of utmost importance. A distinguishing feature of the MPMV gRNA packaging signal is two phylogenetically conserved long-range interactions (LRIs) between U5 and gag complementary sequences, LRI-I and LRI-II. To test their biological significance in the MPMV life cycle, we introduced mutations into these structural motifs and tested their effects on MPMV gRNA packaging and propagation. Furthermore, we probed the structure of key mutants using SHAPE (selective 2'hydroxyl acylation analyzed by primer extension). Disrupting base-pairing of the LRIs affected gRNA packaging and propagation, demonstrating their significance to the MPMV life cycle. A double mutant restoring a heterologous LRI-I was fully functional, whereas a similar LRI-II mutant failed to restore gRNA packaging and propagation. These results demonstrate that while LRI-I acts at the structural level, maintaining base-pairing is not sufficient for LRI-II function. In addition, in vitro RNA dimerization assays indicated that the loss of RNA packaging in LRI mutants could not be attributed to the defects in dimerization. Our findings suggest that U5-gag LRIs play an important architectural role in maintaining the structure of the 5' region of the MPMV gRNA, expanding the crucial role of LRIs to the nonlentiviral group of retroviruses. © 2016 Kalloush et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  10. Substituted 2,6-bis(benzimidazol-2-yl)pyridines: a novel chemical class of pestivirus inhibitors that targets a hot spot for inhibition of pestivirus replication in the RNA-dependent RNA polymerase.

    Science.gov (United States)

    Musiu, Simone; Pürstinger, Gerhard; Stallinger, Sylvia; Vrancken, Robert; Haegeman, Andy; Koenen, Frank; Leyssen, Pieter; Froeyen, Mathy; Neyts, Johan; Paeshuyse, Jan

    2014-06-01

    2,6-Bis(benzimidazol-2-yl)pyridine (BBP/CSFA-0) was identified in a CPE-based screening as a selective inhibitor of the in vitro bovine viral diarrhea virus (BVDV) replication. The EC50-values for the inhibition of BVDV-induced cytopathic (CPE) effect, viral RNA synthesis and the production of infectious virus were 0.3±0.1μM, 0.05±0.01μM and 0.3±0.04μM, respectively. Furthermore, BBP/CSFA-0 inhibits the in vitro replication of the classical swine fever virus (CSFV) with an EC50 of 0.33±0.25μM. BBP/CSFA-0 proved in vitro inactive against the hepatitis C virus, that belongs like BVDV and CSFV to the family of Flaviviridae. Modification of the substituents on the two 1H-benzimidazole groups of BBP resulted in analogues equipotent in anti-BVDV activity (EC50=0.7±0.1μM), devoid of cytotoxicity (S.I.=142). BBP resistant BVDV was selected for and was found to carry the I261M mutation in the viral RNA-dependent RNA polymerase (RdRp). Likewise, BBP-resistant CSFV was selected for; this variant carries either an I261N or a P262A mutation in NS5B. Molecular modeling revealed that I261 and P262 are located in a small cavity near the fingertip domain of the pestivirus polymerase. BBP-resistant BVDV and CSFV proved to be cross-resistant to earlier reported pestivirus inhibitors (BPIP, AG110 and LZ37) that are known to target the same region of the RdRp. BBP did not inhibit the in vitro activity of recombinant BVDV RdRp but inhibited the activity of BVDV replication complexes (RCs). BBP interacts likely with the fingertip of the pestivirus RdRp at the same position as BPIP, AG110 and LZ37. This indicates that this region is a "hot spot" for inhibition of pestivirus replication. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. UV-induced modifications in the peptidyl transferase loop of 23S rRNA dependent on binding of the streptogramin B antibiotic, pristinamycin IA

    DEFF Research Database (Denmark)

    Porse, B T; Kirillov, S V; Awayez, M J

    1999-01-01

    The naturally occurring streptogramin B antibiotic, pristinamycin IA, which inhibits peptide elongation, can produce two modifications in 23S rRNA when bound to the Escherichia coli 70S ribosome and irradiated at 365 nm. Both drug-induced effects map to highly conserved nucleotides within...... in the latter modification to A2062/C2063. Pristinamycin IA can also produce a modification on binding to deproteinized, mature 23S rRNA, at position U2500/C2501. The same modification occurs on an approximately 37-nt fragment, encompassing positions approximately 2496-2532 of the peptidyl transferase loop...... the sequence Cm-C-U-C-G-m2A-psi-G2505 are important for pristinamycin IA binding and/or the antibiotic-dependent modification of 23S rRNA....

  12. The RNA-Binding Site of Poliovirus 3C Protein Doubles as a Phosphoinositide-Binding Domain.

    Science.gov (United States)

    Shengjuler, Djoshkun; Chan, Yan Mei; Sun, Simou; Moustafa, Ibrahim M; Li, Zhen-Lu; Gohara, David W; Buck, Matthias; Cremer, Paul S; Boehr, David D; Cameron, Craig E

    2017-12-05

    Some viruses use phosphatidylinositol phosphate (PIP) to mark membranes used for genome replication or virion assembly. PIP-binding motifs of cellular proteins do not exist in viral proteins. Molecular-docking simulations revealed a putative site of PIP binding to poliovirus (PV) 3C protein that was validated using nuclear magnetic resonance spectroscopy. The PIP-binding site was located on a highly dynamic α helix, which also functions in RNA binding. Broad PIP-binding activity was observed in solution using a fluorescence polarization assay or in the context of a lipid bilayer using an on-chip, fluorescence assay. All-atom molecular dynamics simulations of the 3C protein-membrane interface revealed PIP clustering and perhaps PIP-dependent conformations. PIP clustering was mediated by interaction with residues that interact with the RNA phosphodiester backbone. We conclude that 3C binding to membranes will be determined by PIP abundance. We suggest that the duality of function observed for 3C may extend to RNA-binding proteins of other viruses. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. Poliovirus 2A protease triggers a selective nucleo-cytoplasmic redistribution of splicing factors to regulate alternative pre-mRNA splicing.

    Directory of Open Access Journals (Sweden)

    Enrique Álvarez

    Full Text Available Poliovirus protease 2A (2A(pro obstructs host gene expression by reprogramming transcriptional and post-transcriptional regulatory events during infection. Here we demonstrate that expression of 2A(pro induces a selective nucleo-cytoplasm translocation of several important RNA binding proteins and splicing factors. Subcellular fractionation studies, together with immunofluorescence microscopy revealed an asymmetric distribution of HuR and TIA1/TIAR in 2A(pro expressing cells, which modulates splicing of the human Fas exon 6. Consistent with this result, knockdown of HuR or overexpression of TIA1/TIAR, leads to Fas exon 6 inclusion in 2A(pro-expressing cells. Therefore, poliovirus 2A(pro can target alternative pre-mRNA splicing by regulating protein shuttling between the nucleus and the cytoplasm.

  14. Stimulation of poliovirus RNA synthesis and virus maturation in a HeLa cell-free in vitro translation-RNA replication system by viral protein 3CDpro

    Directory of Open Access Journals (Sweden)

    Wimmer Eckard

    2005-11-01

    Full Text Available Abstract Poliovirus protein 3CDpro possesses both proteinase and RNA binding activities, which are located in the 3Cpro domain of the protein. The RNA polymerase (3Dpol domain of 3CDpro modulates these activities of the protein. We have recently shown that the level of 3CDpro in HeLa cell-free in vitro translation-RNA replication reactions is suboptimal for efficient virus production. However, the addition of either 3CDpro mRNA or of purified 3CDpro protein to in vitro reactions, programmed with viral RNA, results in a 100-fold increase in virus yield. Mutational analyses of 3CDpro indicated that RNA binding by the 3Cpro domain and the integrity of interface I in the 3Dpol domain of the protein are both required for function. The aim of these studies was to determine the exact step or steps at which 3CDpro enhances virus yield and to determine the mechanism by which this occurs. Our results suggest that the addition of extra 3CDpro to in vitro translation RNA-replication reactions results in a mild enhancement of both minus and plus strand RNA synthesis. By examining the viral particles formed in the in vitro reactions on sucrose gradients we determined that 3CDpro has only a slight stimulating effect on the synthesis of capsid precursors but it strikingly enhances the maturation of virus particles. Both the stimulation of RNA synthesis and the maturation of the virus particles are dependent on the presence of an intact RNA binding site within the 3Cpro domain of 3CDpro. In addition, the integrity of interface I in the 3Dpol domain of 3CDpro is required for efficient production of mature virus. Surprisingly, plus strand RNA synthesis and virus production in in vitro reactions, programmed with full-length transcript RNA, are not enhanced by the addition of extra 3CDpro. Our results indicate that the stimulation of RNA synthesis and virus maturation by 3CDpro in vitro is dependent on the presence of a VPg-linked RNA template.

  15. The postpolio syndrome: no evidence for poliovirus persistence

    NARCIS (Netherlands)

    Melchers, W.; de Visser, M.; Jongen, P.; van Loon, A.; Nibbeling, R.; Oostvogel, P.; Willemse, D.; Galama, J.

    1992-01-01

    To investigate the possibility of poliovirus persistence in patients with the postpolio syndrome, we examined skeletal muscle biopsy specimens, cerebrospinal fluid specimens, and sera for the presence of poliovirus RNA by the polymerase chain reaction, and for IgM antibodies by a poliovirus

  16. Detection of negative and positive RNA strand of poliovirus Sabin 1 and echovirus E19 by a stem-loop reverse transcription PCR.

    Science.gov (United States)

    Fikatas, A; Dimitriou, T G; Kyriakopoulou, Z; Moschonas, G D; Amoutzias, G D; Mossialos, D; Gartzonika, C; Levidiotou-Stefanou, S; Markoulatos, P

    2017-09-01

    In this report a strand specific RT-PCR was established for the detection of the replicative negative RNA strand of poliovirus sabin 1 (Sabin1) and Echovirus 19 (E19) strains. The key for the successful conduction of the assay was the use of a specific reverse transcription primer targeting the 5'-UTR of enteroviruses that consisted of a stem-loop structure at the 5'-end and an enteroviral-specific sequence at the 3'-end. The stem loop RT-PCR was found to be an accurate and sensitive method, detecting even 10 -2 CCID 50 of poliovirus sabin 1 (Sabin1) and E19 strains 6 h postinfection (p.i.), while CPE appeared 3 days later. This assay was also validated in SiHa and Caski cell lines that are not used for the detection of enteroviruses. The negative RNA strand was detected 6 h and 12 h p.i. in SiHa and Caski cells, when these cell lines were inoculated with 10 5 and 1 CCID 50 respectively, whereas CPE was observed 5 days p.i for SiHa cells and 8 days p.i for Caski cells and that only at 10 5 CCID 50 . The results show that this approach may be used for replacing the time-consuming cell cultures in order to detect the active replication of enteroviruses. Enteroviruses are positive stranded RNA viruses that may cause severe diseases. The conventional method for detection of active viral replication involves virus isolation in sensitive cell cultures followed by titration and seroneutralization. In this report, we describe the use of a stem-loop secondary structured oligonucleotide in RT-PCR assay for the detection of the replicative negative strand of the positive-stranded RNA of poliovirus sabin 1 and E19 strains. This approach proved to be a useful tool that may be used for replacing the time-consuming cell culture assays in order to detect the active replication of enteroviruses. © 2017 The Society for Applied Microbiology.

  17. Prevention of the β-amyloid peptide-induced inflammatory process by inhibition of double-stranded RNA-dependent protein kinase in primary murine mixed co-cultures

    Directory of Open Access Journals (Sweden)

    Terro F

    2011-06-01

    Full Text Available Abstract Background Inflammation may be involved in the pathogenesis of Alzheimer's disease (AD. There has been little success with anti-inflammatory drugs in AD, while the promise of anti-inflammatory treatment is more evident in experimental models. A new anti-inflammatory strategy requires a better understanding of molecular mechanisms. Among the plethora of signaling pathways activated by β-amyloid (Aβ peptides, the nuclear factor-kappa B (NF-κB pathway could be an interesting target. In virus-infected cells, double-stranded RNA-dependent protein kinase (PKR controls the NF-κB signaling pathway. It is well-known that PKR is activated in AD. This led us to study the effect of a specific inhibitor of PKR on the Aβ42-induced inflammatory response in primary mixed murine co-cultures, allowing interactions between neurons, astrocytes and microglia. Methods Primary mixed murine co-cultures were prepared in three steps: a primary culture of astrocytes and microglia for 14 days, then a primary culture of neurons and astrocytes which were cultured with microglia purified from the first culture. Before exposure to Aβ neurotoxicity (72 h, co-cultures were treated with compound C16, a specific inhibitor of PKR. Levels of tumor necrosis factor-α (TNFα, interleukin (IL-1β, and IL-6 were assessed by ELISA. Levels of PT451-PKR and activation of IκB, NF-κB and caspase-3 were assessed by western blotting. Apoptosis was also followed using annexin V-FITC immunostaining kit. Subcellular distribution of PT451-PKR was assessed by confocal immunofluorescence and morphological structure of cells by scanning electron microscopy. Data were analysed using one-way ANOVA followed by a Newman-Keuls' post hoc test Results In these co-cultures, PKR inhibition prevented Aβ42-induced activation of IκB and NF-κB, strongly decreased production and release of tumor necrosis factor (TNFα and interleukin (IL-1β, and limited apoptosis. Conclusion In spite of the

  18. [Poliovirus vaccine].

    Science.gov (United States)

    Shimizu, Hiroyuki

    2012-06-01

    To avoid the risk of vaccine-associated paralytic poliomyelitis (VAPP) and polio outbreaks due to circulating vaccine-derived polioviruses, an inactivated poliovirus vaccine (IPV) was introduced for routine immunization in a number of countries with a low risk of polio outbreaks. Currently, production and marketing of a standalone conventional IPV and two diphtheria-pertussis-tetanus-IPV (Sabin-derived IPV; sIPV) products have been submitted, and it is expected that the IPV products will be introduced in Japan in the autumn of 2012. At the same time, a decline in the OPV immunization rate became apparent in Japan due to serious public concerns about a remaining risk of VAPP and introduction of IPV in the near future. Therefore, the recent development of polio immunity gaps should be carefully monitored, and surveillance of suspected polio cases and laboratory diagnosis of polioviruses have to be intensified for the transition period from OPV to IPV in Japan. The development of sIPV is one of the most realistic options to introduce affordable IPV to developing countries. In this regard, further clinical studies on its efficacy, safety, and interchangeability of sIPV will be needed after the introduction of the sIPV products, which will be licensed in Japan for the first time in the world.

  19. Cell-Free, De Nova Synthesis of Poliovirus

    Science.gov (United States)

    Molla, Akhteruzzaman; Paul, Aniko V.; Wimmer, Eckard

    1991-12-01

    Cell-free translation of poliovirus RNA in an extract of uninfected human (HeLa) cells yielded viral proteins through proteolysis of the polyprotein. In the extract, newly synthesized proteins catalyzed poliovirus-specific RNA synthesis, and formed infectious poliovirus de novo. Newly formed virions were neutralized by type-specific antiserum, and infection of human cells with them was prevented by poliovirus receptor-specific antibodies. Poliovirus synthesis was increased nearly 70-fold when nucleoside triphosphates were added, but it was abolished in the presence of inhibitors of translation or viral genome replication. The ability to conduct cell-free synthesis of poliovirus will aid in the study of picornavirus proliferation and in the search for the control of picornaviral disease.

  20. Conformational Ensemble of the Poliovirus 3CD Precursor Observed by MD Simulations and Confirmed by SAXS: A Strategy to Expand the Viral Proteome?

    Science.gov (United States)

    Moustafa, Ibrahim M; Gohara, David W; Uchida, Akira; Yennawar, Neela; Cameron, Craig E

    2015-11-23

    The genomes of RNA viruses are relatively small. To overcome the small-size limitation, RNA viruses assign distinct functions to the processed viral proteins and their precursors. This is exemplified by poliovirus 3CD protein. 3C protein is a protease and RNA-binding protein. 3D protein is an RNA-dependent RNA polymerase (RdRp). 3CD exhibits unique protease and RNA-binding activities relative to 3C and is devoid of RdRp activity. The origin of these differences is unclear, since crystal structure of 3CD revealed "beads-on-a-string" structure with no significant structural differences compared to the fully processed proteins. We performed molecular dynamics (MD) simulations on 3CD to investigate its conformational dynamics. A compact conformation of 3CD was observed that was substantially different from that shown crystallographically. This new conformation explained the unique properties of 3CD relative to the individual proteins. Interestingly, simulations of mutant 3CD showed altered interface. Additionally, accelerated MD simulations uncovered a conformational ensemble of 3CD. When we elucidated the 3CD conformations in solution using small-angle X-ray scattering (SAXS) experiments a range of conformations from extended to compact was revealed, validating the MD simulations. The existence of conformational ensemble of 3CD could be viewed as a way to expand the poliovirus proteome, an observation that may extend to other viruses.

  1. Mucosal immunity to poliovirus.

    Science.gov (United States)

    Ogra, Pearay L; Okayasu, Hiromasa; Czerkinsky, Cecil; Sutter, Roland W

    2011-10-01

    The Global Polio Eradication Initiative (GPEI) currently based on use of oral poliovirus vaccine (OPV) has identified suboptimal immunogenicity of this vaccine as a major impediment to eradication, with a failure to induce protection against paralytic poliomyelitis in certain population segments in some parts of the world. The Mucosal Immunity and Poliovirus Vaccines: Impact on Wild Poliovirus Infection, Transmission and Vaccine Failure conference was organized to obtain a better understanding of the current status of global control of poliomyelitis and identify approaches to improve the immune responsiveness and effectiveness of the orally administered poliovirus vaccines in order to accelerate the global eradication of paralytic poliomyelitis.

  2. microRNA-4516 Contributes to Different Functions of Epithelial Permeability Barrier by Targeting Poliovirus Receptor Related Protein 1 in Enterovirus 71 and Coxsackievirus A16 Infections

    Directory of Open Access Journals (Sweden)

    Yajie Hu

    2018-04-01

    Full Text Available Enterovirus 71 (EV-A71 and coxsackievirus A16 (CV-A16 remain the predominant etiological agents of hand, foot, and mouth disease (HFMD. The clinical manifestations caused by the two viruses are obviously different. CV-A16 usually triggers a repeated infection, and airway epithelial integrity is often the potential causative factor of respiratory repeated infections. Our previous studies have demonstrated that there were some differentially expressed miRNAs involved in the regulation of adhesion function of epithelial barrier in EV-A71 and CV-A16 infections. In this study, we compared the differences between EV-A71 and CV-A16 infections on the airway epithelial barrier function in human bronchial epithelial (16HBE cells and further screened the key miRNA which leaded to the formation of these differences. Our results showed that more rapid proliferation, more serious destruction of 16HBE cells permeability, more apoptosis and disruption of intercellular adhesion-associated molecules were found in CV-A16 infection as compared to EV-A71 infection. Furthermore, we also identified that microRNA-4516 (miR-4516, which presented down-regulation in EV-A71 infection and up-regulation in CV-A16 infection was an important regulator of intercellular junctions by targeting Poliovirus receptor related protein 1(PVRL1. The expressions of PVRL1, claudin4, ZO-1 and E-cadherin in CV-A16-infected cells were significantly less than those in EV-A71-infected cells, while the expressions of these proteins were subverted when pre-treated with miR-4516-overexpression plasmid in EV-A71 infected and miR-4516-knockdown plasmid in CV-A16 infected 16HBE cells. Thus, these data suggested that the opposite expression of miR-4516 in EV-A71 and CV-A16 infections might be the initial steps leading to different epithelial impairments of 16HBE cells by destroying intercellular adhesion, which finally resulted in different outcomes of EV-A71 and CV-A16 infections.

  3. Construction and characterization of poliovirus subgenomic replicons.

    Science.gov (United States)

    Kaplan, G; Racaniello, V R

    1988-05-01

    Poliovirus RNAs containing in-frame deletions within the capsid-coding region were produced by in vitro transcription of altered poliovirus type 1 cDNA by using bacteriophage T7 RNA polymerase. Three RNAs were transcribed that contained deletions of 2,317 nucleotides (bases 747 to 3064), 1,781 nucleotides (bases 1,175 to 2,956), and 1,295 nucleotides (bases 1,175 to 2,470). All three subgenomic RNAs replicated after transfection into HeLa cells, demonstrating that sequences encoding the capsid polypeptides are not essential for viral RNA replication in vivo. Viral RNA containing the largest deletion (R1) replicated approximately three times better than full-length RNA produced in vitro. Northern blot (RNA blot) hybridization analysis of total cellular RNA from HeLa cells at different times after transfection with R1 demonstrated the presence of increasing amounts of the expected 5.1-kilobase subgenomic RNA. Analysis by immunoprecipitation of viral proteins induced after transfection of R1 RNA into HeLa cells revealed the presence of proteins 2Apro, 2C, and 3Dpol and its precursors, suggesting that the polyprotein cleavages are similar to those occurring in virus-infected cells. Replication of P2/Lansing virion RNA was inhibited by cotransfection with the R1 replicon, as demonstrated by hybridization analysis with a serotype-specific oligonucleotide probe. A higher level of inhibition of RNA replication was observed when P2/Lansing RNA was cotransfected into HeLa cells with truncated R1 transcripts (R1-PvuII) that were missing 395 3' nucleotides and a poly(A) tail. These internally and terminally deleted RNAs inhibited the replication of subgenomic replicons R1, R2, and R3 and caused a reduction in plaque size when cotransfected with P1/Mahoney or P2/Lansing viral RNA, suggesting that individual cells had received both RNAs. No inhibition of plaque size was observed when replicon RNAs were used that were missing 1,384 or 1,839 3' nucleotides or contained plasmid

  4. Construction and characterization of poliovirus subgenomic replicons

    International Nuclear Information System (INIS)

    Kaplan, G.; Racaniello, V.R.

    1988-01-01

    Poliovirus RNAs containing in-frame deletions within the capsid-coding region were produced by in vitro transcription of altered poliovirus type 1 cDNA by using bacteriophage T7 RNA polymerase. Three RNAs were transcribed that contained deletions of 2,317 nucleotides (bases 747 to 3,064), 1,781 nucleotides (bases 1,175 to 2,956), and 1,295 nucleotides (bases 1,175 to 2,470). All three subgenomic RNAs replicated after transfection into HeLa cells, demonstrating that sequences encoding the capsid polypeptides are not essential for viral RNA replication in vivo. Viral RNA containing the largest deletion (R1) replicated approximately three times better than full-length RNA produced in vitro. Northern blot (RNA blot) hybridization analysis of total cellular RNA from HeLa cells at different times after transfection with R1 demonstrated the presence of increasing amounts of the expected 5.1-kilobase subgenomic RNA. Analysis by immunoprecipitation of [ 35 S-labeled] viral proteins induced after transfection of R1 RNA into HeLa cells revealed the presence of proteins 2A pro , 2C, and 3D pol and its precursors, suggesting that the polyprotein cleavages are similar to those occurring in virus-infected cells. These internally and terminally deleted RNAs inhibited the replication of subgenomic replicons R1, R2, and R3 and caused a reduction in plaque size when cotransfected with P1/Mahoney or P2/Lansing viral RNA, suggesting that individual cells had received both RNAs

  5. Cyclophilin B stimulates RNA synthesis by the HCV RNA dependent RNA polymerase

    OpenAIRE

    Heck, Julie A.; Meng, Xiao; Frick, David N.

    2009-01-01

    Cyclophilins are cellular peptidyl isomerases that have been implicated in regulating hepatitis C virus (HCV) replication. Cyclophilin B (CypB) is a target of cyclosporin A (CsA), an immunosuppressive drug recently shown to suppress HCV replication in cell culture. Watashi et al. recently demonstrated that CypB is important for efficient HCV replication, and proposed that it mediates the anti-HCV effects of CsA through an interaction with NS5B (Mol. Cell 19:111). We examined the effects of pu...

  6. Sensitivity of C6 Glioma Cells Carrying the Human Poliovirus Receptor to Oncolytic Polioviruses.

    Science.gov (United States)

    Sosnovtseva, A O; Lipatova, A V; Grinenko, N F; Baklaushev, V P; Chumakov, P M; Chekhonin, V P

    2016-10-01

    A humanized line of rat C6 glioma cells expressing human poliovirus receptor was obtained and tested for the sensitivity to oncolytic effects of vaccine strains of type 1, 2, and 3 polioviruses. Presentation of the poliovirus receptor on the surface of C6 glioma cells was shown to be a necessary condition for the interaction of cells with polioviruses, but insufficient for complete poliovirus oncolysis.

  7. Poliovirus intrahost evolution is required to overcome tissue-specific innate immune responses.

    Science.gov (United States)

    Xiao, Yinghong; Dolan, Patrick Timothy; Goldstein, Elizabeth Faul; Li, Min; Farkov, Mikhail; Brodsky, Leonid; Andino, Raul

    2017-08-29

    RNA viruses, such as poliovirus, have a great evolutionary capacity, allowing them to quickly adapt and overcome challenges encountered during infection. Here we show that poliovirus infection in immune-competent mice requires adaptation to tissue-specific innate immune microenvironments. The ability of the virus to establish robust infection and virulence correlates with its evolutionary capacity. We further identify a region in the multi-functional poliovirus protein 2B as a hotspot for the accumulation of minor alleles that facilitate a more effective suppression of the interferon response. We propose that population genetic dynamics enables poliovirus spread between tissues through optimization of the genetic composition of low frequency variants, which together cooperate to circumvent tissue-specific challenges. Thus, intrahost virus evolution determines pathogenesis, allowing a dynamic regulation of viral functions required to overcome barriers to infection.RNA viruses, such as polioviruses, have a great evolutionary capacity and can adapt quickly during infection. Here, the authors show that poliovirus infection in mice requires adaptation to innate immune microenvironments encountered in different tissues.

  8. Changes in secondary structure of poliovirus ribonucleic acid

    International Nuclear Information System (INIS)

    Koza, J.

    1975-01-01

    Infectious single-stranded RNA isolated from mature purified poliovirus was separated into three fractions by means of chromatography on an ''evaporated'' calcium phosphate column. RNA molecules with a higher degree of secondary structure were detected in two of the fractions as a result of the chromatography. These RNA molecules (1) were resistant to hydrolysis by pancreatic ribonuclease A, (2) retained unchanged the original infectivity for actinomycin D-pretreated cells, (3) were resistant to ultraviolet-light inactivation and (4) were partially resistant to formaldehyde inactivation

  9. Adenosine triphosphate analogs can efficiently inhibit the Zika virus RNA-dependent RNA polymerase

    Czech Academy of Sciences Publication Activity Database

    Hercík, Kamil; Kozák, Jaroslav; Šála, Michal; Dejmek, Milan; Hřebabecký, Hubert; Zborníková, Eva; Smola, Miroslav; Růžek, Daniel; Nencka, Radim; Bouřa, Evžen

    2017-01-01

    Roč. 137, Jan (2017), s. 131-133 ISSN 0166-3542 R&D Projects: GA ČR GA15-09310S Institutional support: RVO:61388963 ; RVO:60077344 Keywords : hepatitis C virus * borne encephalitis virus * crystal structure Subject RIV: CC - Organic Chemistry OBOR OECD: Organic chemistry Impact factor: 4.271, year: 2016

  10. The Dicer-like, Argonaute and RNA-dependent RNA polymerase ...

    Indian Academy of Sciences (India)

    15 days after cal ind. 15 days after callus induction. 4.796359765 5.345679052 2.367724275 2.373588326 8.296583735 8.470631911 10.40219234. 10.4819069. 3 days after shoot ind 3 days after the start of shoot induction 3.976837223 4.475576805 2.414958455 2.390819138 7.834091661 8.554726998 10.07637957.

  11. High-Throughput Next-Generation Sequencing of Polioviruses

    Science.gov (United States)

    Montmayeur, Anna M.; Schmidt, Alexander; Zhao, Kun; Magaña, Laura; Iber, Jane; Castro, Christina J.; Chen, Qi; Henderson, Elizabeth; Ramos, Edward; Shaw, Jing; Tatusov, Roman L.; Dybdahl-Sissoko, Naomi; Endegue-Zanga, Marie Claire; Adeniji, Johnson A.; Oberste, M. Steven; Burns, Cara C.

    2016-01-01

    ABSTRACT The poliovirus (PV) is currently targeted for worldwide eradication and containment. Sanger-based sequencing of the viral protein 1 (VP1) capsid region is currently the standard method for PV surveillance. However, the whole-genome sequence is sometimes needed for higher resolution global surveillance. In this study, we optimized whole-genome sequencing protocols for poliovirus isolates and FTA cards using next-generation sequencing (NGS), aiming for high sequence coverage, efficiency, and throughput. We found that DNase treatment of poliovirus RNA followed by random reverse transcription (RT), amplification, and the use of the Nextera XT DNA library preparation kit produced significantly better results than other preparations. The average viral reads per total reads, a measurement of efficiency, was as high as 84.2% ± 15.6%. PV genomes covering >99 to 100% of the reference length were obtained and validated with Sanger sequencing. A total of 52 PV genomes were generated, multiplexing as many as 64 samples in a single Illumina MiSeq run. This high-throughput, sequence-independent NGS approach facilitated the detection of a diverse range of PVs, especially for those in vaccine-derived polioviruses (VDPV), circulating VDPV, or immunodeficiency-related VDPV. In contrast to results from previous studies on other viruses, our results showed that filtration and nuclease treatment did not discernibly increase the sequencing efficiency of PV isolates. However, DNase treatment after nucleic acid extraction to remove host DNA significantly improved the sequencing results. This NGS method has been successfully implemented to generate PV genomes for molecular epidemiology of the most recent PV isolates. Additionally, the ability to obtain full PV genomes from FTA cards will aid in facilitating global poliovirus surveillance. PMID:27927929

  12. Exploring the fitness landscape of poliovirus

    Science.gov (United States)

    Bianco, Simone; Acevedo, Ashely; Andino, Raul; Tang, Chao

    2012-02-01

    RNA viruses are known to display extraordinary adaptation capabilities to different environments, due to high mutation rates. Their very dynamical evolution is captured by the quasispecies concept, according to which the viral population forms a swarm of genetic variants linked through mutation, which cooperatively interact at a functional level and collectively contribute to the characteristics of the population. The description of the viral fitness landscape becomes paramount towards a more thorough understanding of the virus evolution and spread. The high mutation rate, together with the cooperative nature of the quasispecies, makes it particularly challenging to explore its fitness landscape. I will present an investigation of the dynamical properties of poliovirus fitness landscape, through both the adoption of new experimental techniques and theoretical models.

  13. Standardized Methods for Detection of Poliovirus Antibodies.

    Science.gov (United States)

    Weldon, William C; Oberste, M Steven; Pallansch, Mark A

    2016-01-01

    Testing for neutralizing antibodies against polioviruses has been an established gold standard for assessing individual protection from disease, population immunity, vaccine efficacy studies, and other vaccine clinical trials. Detecting poliovirus specific IgM and IgA in sera and mucosal specimens has been proposed for evaluating the status of population mucosal immunity. More recently, there has been a renewed interest in using dried blood spot cards as a medium for sample collection to enhance surveillance of poliovirus immunity. Here, we describe the modified poliovirus microneutralization assay, poliovirus capture IgM and IgA ELISA assays, and dried blood spot polio serology procedures for the detection of antibodies against poliovirus serotypes 1, 2, and 3.

  14. Generation of Infectious Poliovirus with Altered Genetic Information from Cloned cDNA.

    Science.gov (United States)

    Bujaki, Erika

    2016-01-01

    The effect of specific genetic alterations on virus biology and phenotype can be studied by a great number of available assays. The following method describes the basic protocol to generate infectious poliovirus with altered genetic information from cloned cDNA in cultured cells.The example explained here involves generation of a recombinant poliovirus genome by simply replacing a portion of the 5' noncoding region with a synthetic gene by restriction cloning. The vector containing the full length poliovirus genome and the insert DNA with the known mutation(s) are cleaved for directional cloning, then ligated and transformed into competent bacteria. The recombinant plasmid DNA is then propagated in bacteria and transcribed to RNA in vitro before RNA transfection of cultured cells is performed. Finally, viral particles are recovered from the cell culture.

  15. The linker domain of poly(rC) binding protein 2 is a major determinant in poliovirus cap-independent translation.

    Science.gov (United States)

    Sean, Polen; Nguyen, Joseph H C; Semler, Bert L

    2008-09-01

    Poliovirus, a member of the enterovirus genus in the family Picornaviridae, is the causative agent of poliomyelitis. Translation of the viral genome is mediated through an internal ribosomal entry site (IRES) encoded within the 5' noncoding region (5' NCR). IRES elements are highly structured RNA sequences that facilitate the recruitment of ribosomes for translation. Previous studies have shown that binding of a cellular protein, poly(rC) binding protein 2 (PCBP2), to a major stem-loop structure in the genomic 5' NCR is necessary for the translation of picornaviruses containing type I IRES elements, including poliovirus, coxsackievirus, and human rhinovirus. PCBP1, an isoform that shares approximately 90% amino acid identity to PCBP2, cannot efficiently stimulate poliovirus IRES-mediated translation, most likely due to its reduced binding affinity to stem-loop IV within the poliovirus IRES. The primary differences between PCBP1 and PCBP2 are found in the so-called linker domain between the second and third K-homology (KH) domains of these proteins. We hypothesize that the linker region of PCBP2 augments binding to poliovirus stem-loop IV RNA. To test this hypothesis, we generated six PCBP1/PCBP2 chimeric proteins. The recombinant PCBP1/PCBP2 chimeric proteins were able to interact with poliovirus stem-loop I RNA and participate in protein-protein interactions. We demonstrated that the PCBP1/PCBP2 chimeric proteins with the PCBP2 linker, but not with the PCBP1 linker, were able to interact with poliovirus stem-loop IV RNA, and could subsequently stimulate poliovirus IRES-mediated translation. In addition, using a monoclonal anti-PCBP2 antibody (directed against the PCBP2 linker domain) in mobility shift assays, we showed that the PCBP2 linker domain modulates binding to poliovirus stem-loop IV RNA via a mechanism that is not inhibited by the antibody.

  16. Intracellular vesicle acidification promotes maturation of infectious poliovirus particles.

    Directory of Open Access Journals (Sweden)

    Alexsia L Richards

    Full Text Available The autophagic pathway acts as part of the immune response against a variety of pathogens. However, several pathogens subvert autophagic signaling to promote their own replication. In many cases it has been demonstrated that these pathogens inhibit or delay the degradative aspect of autophagy. Here, using poliovirus as a model virus, we report for the first time bona fide autophagic degradation occurring during infection with a virus whose replication is promoted by autophagy. We found that this degradation is not required to promote poliovirus replication. However, vesicular acidification, which in the case of autophagy precedes delivery of cargo to lysosomes, is required for normal levels of virus production. We show that blocking autophagosome formation inhibits viral RNA synthesis and subsequent steps in the virus cycle, while inhibiting vesicle acidification only inhibits the final maturation cleavage of virus particles. We suggest that particle assembly, genome encapsidation, and virion maturation may occur in a cellular compartment, and we propose the acidic mature autophagosome as a candidate vesicle. We discuss the implications of our findings in understanding the late stages of poliovirus replication, including the formation and maturation of virions and egress of infectious virus from cells.

  17. Strain differentiation of polioviruses with monoclonal antibodies.

    NARCIS (Netherlands)

    A.D.M.E. Osterhaus (Albert); A.L. van Wezel; A.J.H. Stegmann; J.A.A.M. van Asten (Jack)

    1984-01-01

    textabstractPanels of monoclonal antibodies raised against different poliovirus type 1, 2 and 3 strains, were tested in a micro-neutralization test and in a micro-enzyme linked immunosorbent assay against a large number of poliovirus strains. The results were compared with those obtained with the

  18. Immunity to poliovirus after infection and vaccination

    NARCIS (Netherlands)

    Herremans, Martina Maria Petronella Theresia

    1999-01-01

    The aim of this thesis was defined as the study of the contribution of IPV vaccination to the induction of a) protection against poliovirus infection and b) mucosal immunity.We have described the development of new immunological tools for the rapid detection of poliovirus-specific antibodies and

  19. Re-localization of Cellular Protein SRp20 during Poliovirus Infection: Bridging a Viral IRES to the Host Cell Translation Apparatus

    Science.gov (United States)

    Fitzgerald, Kerry D.; Semler, Bert L.

    2011-01-01

    Poliovirus IRES-mediated translation requires the functions of certain canonical as well as non-canonical factors for the recruitment of ribosomes to the viral RNA. The interaction of cellular proteins PCBP2 and SRp20 in extracts from poliovirus-infected cells has been previously described, and these two proteins were shown to function synergistically in viral translation. To further define the mechanism of ribosome recruitment for the initiation of poliovirus IRES-dependent translation, we focused on the role of the interaction between cellular proteins PCBP2 and SRp20. Work described here demonstrates that SRp20 dramatically re-localizes from the nucleus to the cytoplasm of poliovirus-infected neuroblastoma cells during the course of infection. Importantly, SRp20 partially co-localizes with PCBP2 in the cytoplasm of infected cells, corroborating our previous in vitro interaction data. In addition, the data presented implicate the presence of these two proteins in viral translation initiation complexes. We show that in extracts from poliovirus-infected cells, SRp20 is associated with PCBP2 bound to poliovirus RNA, indicating that this interaction occurs on the viral RNA. Finally, we generated a mutated version of SRp20 lacking the RNA recognition motif (SRp20ΔRRM) and found that this protein is localized similar to the full length SRp20, and also partially co-localizes with PCBP2 during poliovirus infection. Expression of this mutated version of SRp20 results in a ∼100 fold decrease in virus yield for poliovirus when compared to expression of wild type SRp20, possibly via a dominant negative effect. Taken together, these results are consistent with a model in which SRp20 interacts with PCBP2 bound to the viral RNA, and this interaction functions to recruit ribosomes to the viral RNA in a direct or indirect manner, with the participation of additional protein-protein or protein-RNA interactions. PMID:21779168

  20. Differential induction of Toll-like receptors & type 1 interferons by Sabin attenuated & wild type 1 polioviruses in human neuronal cells.

    Science.gov (United States)

    Mohanty, Madhu C; Deshpande, Jagadish M

    2013-01-01

    Polioviruses are the causative agent of paralytic poliomyelitis. Attenuated polioviruses (Sabin oral poliovirus vaccine strains) do not replicate efficiently in neurons as compared to the wild type polioviruses and therefore do not cause disease. This study was aimed to investigate the differential host immune response to wild type 1 poliovirus (wild PV) and Sabin attenuated type 1 poliovirus (Sabin PV) in cultured human neuronal cells. By using flow cytometry and real time PCR methods we examined host innate immune responses and compared the role of toll like receptors (TLRs) and cytoplasmic RNA helicases in cultured human neuronal cells (SK-N-SH) infected with Sabin PV and wild PV. Human neuronal cells expressed very low levels of TLRs constitutively. Sabin PV infection induced significantly higher expression of TLR3, TLR7 and melanoma differentiation-associated protein-5 (MDA-5) m-RNA in neuronal cells at the beginning of infection (up to 4 h) as compared to wild PV. Further, Sabin PV also induced the expression of interferon α/β at early time point of infection. The induced expression of IFN α/β gene by Sabin PV in neuronal cells could be suppressed by inhibiting TLR7. Neuronal cell innate immune response to Sabin and wild polioviruses differ significantly for TLR3, TLR7, MDA5 and type 1 interferons. Effects of TLR7 activation and interferon production and Sabin virus replication in neuronal cells need to be actively investigated in future studies.

  1. Differential induction of Toll-like receptors & type 1 interferons by Sabin attenuated & wild type 1 polioviruses in human neuronal cells

    Directory of Open Access Journals (Sweden)

    Madhu C Mohanty

    2013-01-01

    Full Text Available Background & objectives: Polioviruses are the causative agent of paralytic poliomyelitis. Attenuated polioviruses (Sabin oral poliovirus vaccine strains do not replicate efficiently in neurons as compared to the wild type polioviruses and therefore do not cause disease. This study was aimed to investigate the differential host immune response to wild type 1 poliovirus (wild PV and Sabin attenuated type 1 poliovirus (Sabin PV in cultured human neuronal cells. Methods: By using flow cytometry and real time PCR methods we examined host innate immune responses and compared the role of toll like receptors (TLRs and cytoplasmic RNA helicases in cultured human neuronal cells (SK-N-SH infected with Sabin PV and wild PV. Results: Human neuronal cells expressed very low levels of TLRs constitutively. Sabin PV infection induced significantly higher expression of TLR3, TLR7 and melanoma differentiation-associated protein-5 (MDA-5 m-RNA in neuronal cells at the beginning of infection (up to 4 h as compared to wild PV. Further, Sabin PV also induced the expression of interferon α/β at early time point of infection. The induced expression of IFN α/β gene by Sabin PV in neuronal cells could be suppressed by inhibiting TLR7. Interpretation & conclusions: Neuronal cell innate immune response to Sabin and wild polioviruses differ significantly for TLR3, TLR7, MDA5 and type 1 interferons. Effects of TLR7 activation and interferon production and Sabin virus replication in neuronal cells need to be actively investigated in future studies.

  2. Complex Dynamic Development of Poliovirus Membranous Replication Complexes

    Science.gov (United States)

    Nair, Vinod; Hansen, Bryan T.; Hoyt, Forrest H.; Fischer, Elizabeth R.; Ehrenfeld, Ellie

    2012-01-01

    Replication of all positive-strand RNA viruses is intimately associated with membranes. Here we utilize electron tomography and other methods to investigate the remodeling of membranes in poliovirus-infected cells. We found that the viral replication structures previously described as “vesicles” are in fact convoluted, branching chambers with complex and dynamic morphology. They are likely to originate from cis-Golgi membranes and are represented during the early stages of infection by single-walled connecting and branching tubular compartments. These early viral organelles gradually transform into double-membrane structures by extension of membranous walls and/or collapsing of the luminal cavity of the single-membrane structures. As the double-membrane regions develop, they enclose cytoplasmic material. At this stage, a continuous membranous structure may have double- and single-walled membrane morphology at adjacent cross-sections. In the late stages of the replication cycle, the structures are represented mostly by double-membrane vesicles. Viral replication proteins, double-stranded RNA species, and actively replicating RNA are associated with both double- and single-membrane structures. However, the exponential phase of viral RNA synthesis occurs when single-membrane formations are predominant in the cell. It has been shown previously that replication complexes of some other positive-strand RNA viruses form on membrane invaginations, which result from negative membrane curvature. Our data show that the remodeling of cellular membranes in poliovirus-infected cells produces structures with positive curvature of membranes. Thus, it is likely that there is a fundamental divergence in the requirements for the supporting cellular membrane-shaping machinery among different groups of positive-strand RNA viruses. PMID:22072780

  3. Studies towards the potential of poliovirus as a vector for the expression of HPV 16 virus-like-particles.

    Science.gov (United States)

    van Kuppeveld, Frank J M; de Jong, Arjan; Dijkman, Henri B P M; Andino, Raul; Melchers, Willem J G

    2002-11-15

    Development of human cervical carcinomas is associated with infection by certain human papillomavirus (HPV) types. Thus, protection against HPV infection through vaccination may prevent development of cervical cancer. The purpose of this study was to investigate the possibility of using a poliovirus recombinant vector to induce immunity against HPV. A poliovirus recombinant was constructed which contained the complete coding sequence of the HPV 16 major capsid protein L1, between the P1 and P2 region of the poliovirus polyprotein. A replication-competent virus was obtained after transfection of the recombinant RNA into tissue culture cells. Electron microscopically examination of cells infected with the poliovirus-HPV L1 recombinant indicated that HPV 16 L1 self-assembles into virus-like particles. To investigate the immunological response in vivo, susceptible transgenic mice carrying the poliovirus receptor were infected with the recombinant poliovirus. In all mice a modest but consistent immune response against HPV 16 was observed. Based on these results, the potential for picornavirus-derived vectors in vaccine development against HPV infection is discussed.

  4. Expression analysis of argonaute, Dicer-like, and RNA-dependent ...

    Indian Academy of Sciences (India)

    DEFANG GAN

    2Modern Agricultural Science and Technology Cooperative Extension Service Center of Mingguang City, Mingguang,. 239400, People's .... The quality and purity of the prepara- .... CsActin was used as an internal control to normalize the data.

  5. Current status of poliovirus infections.

    Science.gov (United States)

    Melnick, J L

    1996-07-01

    Two scientists who played leading roles in the conquest of poliomyelitis died recently. In 1954, Jonas Salk provided the first licensed polio vaccine, the formalin (and heat)-inactivated virus. Albert Sabin gave us the attenuated live virus vaccine, which was licensed in 1962. This paper takes the reader through the history of the disease, including its pathogenesis, epidemiology, vaccines, and future directions. The emphasis is on vaccines, for it seems that with proper vaccination the number of new cases is falling dramatically. It is hoped that by the year 2000, we will accomplish the goal of the World Health Organization of "a world without polio." Then, because there is no animal reservoir, we can seriously discuss when and how to eliminate the need for vaccination and ultimately destroy our stocks of poliovirus.

  6. The potential benefits of a new poliovirus vaccine for long-term poliovirus risk management.

    Science.gov (United States)

    Duintjer Tebbens, Radboud J; Thompson, Kimberly M

    2016-12-01

    To estimate the incremental net benefits (INBs) of a hypothetical ideal vaccine with all of the advantages and no disadvantages of existing oral and inactivated poliovirus vaccines compared with current vaccines available for future outbreak response. INB estimates based on expected costs and polio cases from an existing global model of long-term poliovirus risk management. Excluding the development costs, an ideal poliovirus vaccine could offer expected INBs of US$1.6 billion. The ideal vaccine yields small benefits in most realizations of long-term risks, but great benefits in low-probability-high-consequence realizations. New poliovirus vaccines may offer valuable insurance against long-term poliovirus risks and new vaccine development efforts should continue as the world gathers more evidence about polio endgame risks.

  7. Restricted fragmentation of poliovirus type 1, 2, and 3 RNAs by ribonuclease III

    Energy Technology Data Exchange (ETDEWEB)

    Nomoto, A. (State Univ. of New York, Stony Brook); Lee, Y.F.; Babich, A.; Jacobson, A.; Dunn, J.J.; Wimmer, E.

    1979-01-01

    Cleavage of the genome RNAs of poliovirus type 1, 2, and 3 with the ribonuclease III of Escherichia coli has been investigated with the following results: (1) at or above physiological salt concentration, the RNAs are completely resistant to the action of the enzyme, an observation suggesting that the RNAs lack primary cleavage sites; (2) lowering the salt concentration to 0.1 M or below allows RNase III to cleave the RNAs at secondary sites. Both large and small fragments can be obtained in a reproducible manner depending on salt conditions chosen for cleavage. Fingerprints of three large fragments of poliovirus type 2 RNA show that they originate from unique segments and represent most if not all sequences of the genome. Based upon binding to poly(U) filters of poly(A)-linked fragments, a physical map of the large fragments of poliovirus type 2 RNA was constructed. The data suggest that RNase III cleavage of single-stranded RNA provides a useful method to fragment the RNA for further studies.

  8. Gene expression in the muscle and central nervous system following intramuscular inoculation of encapsidated or naked poliovirus replicons

    International Nuclear Information System (INIS)

    Jackson, Cheryl A.; Messinger, Jeff; Palmer, Matthew T.; Peduzzi, Jean D.; Morrow, Casey D.

    2003-01-01

    The spread of intramuscularly inoculated poliovirus to the central nervous system (CNS) has been documented in humans, monkeys, and mice transgenic for the human poliovirus receptor. Poliovirus spread is thought to be due to infection of the peripheral nerve and retrograde transport of poliovirus through the axon to the neuron cell body, where final virus uncoating occurs and translation/replication ensues. In previous studies, we have shown that polio-based vectors (replicons) can be used for gene delivery to motor neurons of the CNS. Using a replicon that encodes green fluorescent protein (GFP), we found that following intrathecal inoculation, GFP expression was confined to motorneurons of the spinal cord. To further characterize the gene expression of poliovirus in the periphery and CNS, we have intramuscularly inoculated transgenic mice with poliovirus replicons encoding GFP. Expression of GFP was demonstrated in the muscle, sciatic nerve, dorsal root ganglion, and the ventral horn motorneurons following intramuscular inoculation. There was no evidence of paralysis or behavioral abnormalities in the mice following intramuscular inoculation of the replicon encoding GFP. Injection of replicon RNA alone (naked RNA) into the muscle of transgenic mice or rats, which do not express the poliovirus receptor, also resulted in expression of GFP in the muscle, sciatic nerve, dorsal root ganglion, and ventral horn motorneurons, indicating that transport of the replicon RNA from the periphery to CNS had occurred. GFP expression was found in the muscles and sciatic nerve as early as 6 h after injection of replicons or replicon RNA, even after sciatic nerve section. Analysis at longer times postinjection revealed GFP expression similar to 6 h levels in the cut sciatic nerves and robust expression in the nerves of uncut animals. The infection and expression of GFP in the CNS following intramuscular inoculation of encapsidated replicons encoding GFP occurred in juvenile or

  9. RNA.

    Science.gov (United States)

    Darnell, James E., Jr.

    1985-01-01

    Ribonucleic acid (RNA) converts genetic information into protein and usually must be processed to serve its function. RNA types, chemical structure, protein synthesis, translation, manufacture, and processing are discussed. Concludes that the first genes might have been spliced RNA and that humans might be closer than bacteria to primitive…

  10. Sabin and wild type polioviruses from children who presented with ...

    African Journals Online (AJOL)

    Conclusion: This study further confirms the presence of Sabin and wild-type poliovirus among children in Nigeria. The isolation of Sabin strain of poliovirus is advantageous to the polio eradication program as it is capable of inducing natural immunity in susceptible hosts. Transmission of wild-type poliovirus among children ...

  11. 21 CFR 866.3405 - Poliovirus serological reagents.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Poliovirus serological reagents. 866.3405 Section... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3405 Poliovirus serological reagents. (a) Identification. Poliovirus serological reagents are devices that consist of antigens...

  12. Alternative splicing, a new target to block cellular gene expression by poliovirus 2A protease

    International Nuclear Information System (INIS)

    Alvarez, Enrique; Castello, Alfredo; Carrasco, Luis; Izquierdo, Jose M.

    2011-01-01

    Highlights: → Novel role for poliovirus 2A protease as splicing modulator. → Poliovirus 2A protease inhibits the alternative splicing of pre-mRNAs. → Poliovirus 2A protease blocks the second catalytic step of splicing. -- Abstract: Viruses have developed multiple strategies to interfere with the gene expression of host cells at different stages to ensure their own survival. Here we report a new role for poliovirus 2A pro modulating the alternative splicing of pre-mRNAs. Expression of 2A pro potently inhibits splicing of reporter genes in HeLa cells. Low amounts of 2A pro abrogate Fas exon 6 skipping, whereas higher levels of protease fully abolish Fas and FGFR2 splicing. In vitro splicing of MINX mRNA using nuclear extracts is also strongly inhibited by 2A pro , leading to accumulation of the first exon and the lariat product containing the unspliced second exon. These findings reveal that the mechanism of action of 2A pro on splicing is to selectively block the second catalytic step.

  13. Alternative splicing, a new target to block cellular gene expression by poliovirus 2A protease

    Energy Technology Data Exchange (ETDEWEB)

    Alvarez, Enrique, E-mail: ealvarez@cbm.uam.es [Centro de Biologia Molecular Severo Ochoa (CSIC-UAM), Nicolas Cabrera, 1 Universidad Autonoma de Madrid, Cantoblanco, 28049 Madrid (Spain); Castello, Alfredo; Carrasco, Luis; Izquierdo, Jose M. [Centro de Biologia Molecular Severo Ochoa (CSIC-UAM), Nicolas Cabrera, 1 Universidad Autonoma de Madrid, Cantoblanco, 28049 Madrid (Spain)

    2011-10-14

    Highlights: {yields} Novel role for poliovirus 2A protease as splicing modulator. {yields} Poliovirus 2A protease inhibits the alternative splicing of pre-mRNAs. {yields} Poliovirus 2A protease blocks the second catalytic step of splicing. -- Abstract: Viruses have developed multiple strategies to interfere with the gene expression of host cells at different stages to ensure their own survival. Here we report a new role for poliovirus 2A{sup pro} modulating the alternative splicing of pre-mRNAs. Expression of 2A{sup pro} potently inhibits splicing of reporter genes in HeLa cells. Low amounts of 2A{sup pro} abrogate Fas exon 6 skipping, whereas higher levels of protease fully abolish Fas and FGFR2 splicing. In vitro splicing of MINX mRNA using nuclear extracts is also strongly inhibited by 2A{sup pro}, leading to accumulation of the first exon and the lariat product containing the unspliced second exon. These findings reveal that the mechanism of action of 2A{sup pro} on splicing is to selectively block the second catalytic step.

  14. Cold-Adapted Viral Attenuation (CAVA): Highly Temperature Sensitive Polioviruses as Novel Vaccine Strains for a Next Generation Inactivated Poliovirus Vaccine.

    Science.gov (United States)

    Sanders, Barbara P; de Los Rios Oakes, Isabel; van Hoek, Vladimir; Bockstal, Viki; Kamphuis, Tobias; Uil, Taco G; Song, Yutong; Cooper, Gillian; Crawt, Laura E; Martín, Javier; Zahn, Roland; Lewis, John; Wimmer, Eckard; Custers, Jerome H H V; Schuitemaker, Hanneke; Cello, Jeronimo; Edo-Matas, Diana

    2016-03-01

    The poliovirus vaccine field is moving towards novel vaccination strategies. Withdrawal of the Oral Poliovirus Vaccine and implementation of the conventional Inactivated Poliovirus Vaccine (cIPV) is imminent. Moreover, replacement of the virulent poliovirus strains currently used for cIPV with attenuated strains is preferred. We generated Cold-Adapted Viral Attenuation (CAVA) poliovirus strains by serial passage at low temperature and subsequent genetic engineering, which contain the capsid sequences of cIPV strains combined with a set of mutations identified during cold-adaptation. These viruses displayed a highly temperature sensitive phenotype with no signs of productive infection at 37°C as visualized by electron microscopy. Furthermore, decreases in infectious titers, viral RNA, and protein levels were measured during infection at 37°C, suggesting a block in the viral replication cycle at RNA replication, protein translation, or earlier. However, at 30°C, they could be propagated to high titers (9.4-9.9 Log10TCID50/ml) on the PER.C6 cell culture platform. We identified 14 mutations in the IRES and non-structural regions, which in combination induced the temperature sensitive phenotype, also when transferred to the genomes of other wild-type and attenuated polioviruses. The temperature sensitivity translated to complete absence of neurovirulence in CD155 transgenic mice. Attenuation was also confirmed after extended in vitro passage at small scale using conditions (MOI, cell density, temperature) anticipated for vaccine production. The inability of CAVA strains to replicate at 37°C makes reversion to a neurovirulent phenotype in vivo highly unlikely, therefore, these strains can be considered safe for the manufacture of IPV. The CAVA strains were immunogenic in the Wistar rat potency model for cIPV, inducing high neutralizing antibody titers in a dose-dependent manner in response to D-antigen doses used for cIPV. In combination with the highly productive

  15. Decay of Sabin inactivated poliovirus vaccine (IPV)-boosted poliovirus antibodies

    OpenAIRE

    Resik, Sonia; Tejeda, Alina; Fonseca, Magile; Sein, Carolyn; Hung, Lai Heng; Martinez, Yenisleidys; Diaz, Manuel; Okayasu, Hiromasa; Sutter, Roland W.

    2015-01-01

    Introduction: We conducted a follow-on study to a phase I randomized, controlled trial conducted in Cuba, 2012, to assess the persistence of poliovirus antibodies at 21–22 months following booster dose of Sabin-IPV compared to Salk-IPV in adults who had received multiple doses of oral poliovirus vaccine (OPV) during childhood. Methods: In 2012, 60 healthy adult males aged 19–23 were randomized to receive one booster dose, of either Sabin-inactivated poliovirus vaccine (Sabin-IPV), adjuvant...

  16. Mechanisms of poliovirus inactivation by the direct and indirect effects of ionizing radiation

    International Nuclear Information System (INIS)

    Ward, R.L.

    1980-01-01

    This study was designed to measure the effects of ionizing radiation on poliovirus particles when given under conditions where either direct (in broth) or indirect (in water) effects were predominant. Under direct conditions, inactivation of poliovirus was found to be due primarily to RNA damage, although capsid damage could account for about one-third of the viral inactivation. RNA damage did not appear to be due to strand breakage and therefore was probably caused primarily by base damage or crosslink formation. Capsid damage under direct irradiation conditions did not result in significant alterations of either the sedimentation coefficients or the isoelectric points of the poliovirus particles or detectable modification of the sizes of the viral proteins. It did, however, cause loss of availability to bind to host cells. Under indirect conditions no more than 25% of viral inactivation appeared to be due to RNA damage. However, the sedimentation coefficients and isoelectric points of the viral particles were greatly altered, and their abilities to bind to cells were lost at about three-fourths the rate of loss of infectivity. Capsid damage in this case did result in changes in the sizes of capsid proteins. Therefore, the majority of the radiation inactivation under indirect conditions appeared to be due to protein damage

  17. Proteolysis of MDA5 and IPS-1 is not required for inhibition of the type I IFN response by poliovirus.

    Science.gov (United States)

    Kotla, Swathi; Gustin, Kurt E

    2015-10-06

    The type I interferon (IFN) response is a critical component of the innate immune response to infection by RNA viruses and is initiated via recognition of viral nucleic acids by RIG-like receptors (RLR). Engagement of these receptors in the cytoplasm initiates a signal transduction pathway leading to activation of the transcription factors NF-κB, ATF-2 and IRF-3 that coordinately upregulate transcription of type I IFN genes, such as that encoding IFN-β. In this study the impact of poliovirus infection on the type I interferon response has been examined. The type I IFN response was assessed by measuring IFN-β mRNA levels using qRT-PCR and normalizing to levels of β-actin mRNA. The status of host factors involved in activation of the type I IFN response was examined by immunoblot, immunofluorescence microcopy and qRT-PCR. The results show that poliovirus infection results in induction of very low levels of IFN-β mRNA despite clear activation of NF-κB and ATF-2. In contrast, analysis of IRF-3 revealed no transcriptional induction of an IRF-3-responsive promoter or homodimerization of IRF-3 indicating it is not activated in poliovirus-infected cells. Exposure of poliovirus-infected cells to poly(I:C) results in lower levels of IFN-β mRNA synthesis and IRF-3 activation compared to mock-infected cells. Analysis of MDA-5 and IPS-1 revealed that these components of the RLR pathway were largely intact at times when the type I IFN response was suppressed. Collectively, these results demonstrate that poliovirus infection actively suppresses the host type I interferon response by blocking activation of IRF-3 and suggests that this is not mediated by cleavage of MDA-5 or IPS-1.

  18. Negative charge and membrane tethered viral 3B cooperate to recruit viral RNA dependent RNA polymerase 3Dpol

    Czech Academy of Sciences Publication Activity Database

    Dubánková, Anna; Humpolíčková, Jana; Šilhán, Jan; Bäumlová, Adriana; Chalupská, Dominika; Klíma, Martin; Bouřa, Evžen

    2017-01-01

    Roč. 284, Suppl 1 (2017), s. 190 ISSN 1742-464X. [FEBS Congress /42./ From Molecules to Cells and Back. 10.09.2017-14.09.2017, Jerusalem] Institutional support: RVO:61388963 Keywords : RdRp * ACBD3 Subject RIV: CE - Biochemistry

  19. Foot-and-mouth disease virus type O specific mutations determine RNA-dependent RNA polymerase fidelity and virus attenuation.

    Science.gov (United States)

    Li, Chen; Wang, Haiwei; Yuan, Tiangang; Woodman, Andrew; Yang, Decheng; Zhou, Guohui; Cameron, Craig E; Yu, Li

    2018-05-01

    Previous studies have shown that the FMDV Asia1/YS/CHA/05 high-fidelity mutagen-resistant variants are attenuated (Zeng et al., 2014). Here, we introduced the same single or multiple-amino-acid substitutions responsible for increased 3D pol fidelity of type Asia1 FMDV into the type O FMDV O/YS/CHA/05 infectious clone. The rescued viruses O-DA and O-DAMM are lower replication fidelity mutants and showed an attenuated phenotype. These results demonstrated that the same amino acid substitution of 3D pol in different serotypes of FMDV strains had different effects on viral fidelity. In addition, nucleoside analogues were used to select high-fidelity mutagen-resistant type O FMDV variants. The rescued mutagen-resistant type O FMDV high-fidelity variants exhibited significantly attenuated fitness and a reduced virulence phenotype. These results have important implications for understanding the molecular mechanism of FMDV evolution and pathogenicity, especially in developing a safer modified live-attenuated vaccine against FMDV. Copyright © 2018 Elsevier Inc. All rights reserved.

  20. Heat-accelerated radioinactivation of attenuated poliovirus

    International Nuclear Information System (INIS)

    Dugan, V.L.; Trujillo, R.

    1975-01-01

    Attenuated poliovirus is inactivated in a synergistic manner when exposed simultaneously to heat and ionizing radiation. The synergistic response is observed in both the thermally labile and stable forms of the virus. A three-term kinetic model may be used to describe the inactivation response of the virus in a thermal and/or ionizing radiation environment. (orig.) [de

  1. Poliovirus Laboratory Based Surveillance: An Overview.

    Science.gov (United States)

    Zaidi, Syed Sohail Zahoor; Asghar, Humayun; Sharif, Salmaan; Alam, Muhammad Masroor

    2016-01-01

    World Health Assembly (WHA) in 1988 encouraged the member states to launch Global Polio Eradication Initiative (GPEI) (resolution WHA41.28) against "the Crippler" called poliovirus, through strong routine immunization program and intensified surveillance systems. Since its launch, global incidence of poliomyelitis has been reduced by more than 99 % and the disease squeezed to only three endemic countries (Afghanistan, Pakistan, and Nigeria) out of 125. Today, poliomyelitis is on the verge of eradication, and their etiological agents, the three poliovirus serotypes, are on the brink of extinction from the natural environment. The last case of poliomyelitis due to wild type 2 strain occurred in 1999 in Uttar Pradesh, India whereas the last paralytic case due to wild poliovirus type 3 (WPV3) was seen in November, 2012 in Yobe, Nigeria. Despite this progress, undetected circulation cannot fully rule out the eradication as most of the poliovirus infections are entirely subclinical; hence sophisticated environmental surveillance is needed to ensure the complete eradication of virus. Moreover, the vaccine virus in under-immunized communities can sometimes revert and attain wild type characteristics posing a big challenge to the program.

  2. 3′-Terminal Sequence in Poliovirus Negative-Strand Templates Is the Primary cis-Acting Element Required for VPgpUpU-Primed Positive-Strand Initiation

    OpenAIRE

    Sharma, Nidhi; O'Donnell, Brian J.; Flanegan, James B.

    2005-01-01

    The 5′ cloverleaf in poliovirus RNA has a direct role in regulating the stability, translation, and replication of viral RNA. In this study, we investigated the role of stem a in the 5′ cloverleaf in regulating the stability and replication of poliovirus RNA in HeLa S10 translation-replication reactions. Our results showed that disrupting the duplex structure of stem a destabilized viral RNA and inhibited efficient negative-strand synthesis. Surprisingly, the duplex structure of stem a was no...

  3. Decay of Sabin inactivated poliovirus vaccine (IPV)-boosted poliovirus antibodies.

    Science.gov (United States)

    Resik, Sonia; Tejeda, Alina; Fonseca, Magile; Sein, Carolyn; Hung, Lai Heng; Martinez, Yenisleidys; Diaz, Manuel; Okayasu, Hiromasa; Sutter, Roland W

    We conducted a follow-on study to a phase I randomized, controlled trial conducted in Cuba, 2012, to assess the persistence of poliovirus antibodies at 21-22 months following booster dose of Sabin-IPV compared to Salk-IPV in adults who had received multiple doses of oral poliovirus vaccine (OPV) during childhood. In 2012, 60 healthy adult males aged 19-23 were randomized to receive one booster dose, of either Sabin-inactivated poliovirus vaccine (Sabin-IPV), adjuvanted Sabin-IPV (aSabin-IPV), or conventional Salk-IPV. In the original study, blood was collected at days 0 (before) and 28 (after vaccination), respectively. In this study, an additional blood sample was collected 21-22 months after vaccination, and tested for neutralizing antibodies to Sabin poliovirus types 1, 2 and 3. We collected sera from 59/60 (98.3%) subjects; 59/59 (100%) remained seropositive to all poliovirus types, 21-22 months after vaccination. The decay curves were very similar among the study groups. Between day 28 and 21-22 months, there was a reduction of ⩾87.4% in median antibody levels for all poliovirus types in all study groups, with no significant differences between the study groups. The decay of poliovirus antibodies over a 21-22-month period was similar regardless of the type of booster vaccine used, suggesting the scientific data of Salk IPV long-term persistence and decay may be broadly applicable to Sabin IPV.

  4. Progress Toward Containment of Poliovirus Type 2 - Worldwide, 2017.

    Science.gov (United States)

    Previsani, Nicoletta; Singh, Harpal; St Pierre, Jeanette; Boualam, Liliane; Fournier-Caruana, Jacqueline; Sutter, Roland W; Zaffran, Michel

    2017-06-23

    The Global Polio Eradication Initiative (GPEI) continues to make progress toward the eradication target. Only one of the three serotypes, wild poliovirus (WPV) type 1 (WPV1), is still circulating, and the numbers of cases and countries with endemic transmission are at record lows. With the certification of wild poliovirus type 2 (WPV2) eradication in 2015 and the global replacement of trivalent oral poliovirus vaccine (tOPV) containing Sabin poliovirus types 1, 2, and 3 with bivalent OPV containing only Sabin poliovirus types 1 and 3 during April-May 2016, poliovirus type 2 (PV2) is now an eradicated pathogen. However, in eight countries (Cameroon, Chad, Democratic Republic of Congo, Mozambique, Niger, Nigeria, Pakistan, and Syria), monovalent type 2 OPV (mOPV2) was authorized for large-scale outbreak control after tOPV withdrawal (1). Poliovirus containment, an evolving area of work that affects every country, aims to ensure that all PV2 specimens are safely contained to minimize the risk for reintroducing the virus into communities. This report summarizes the current status of poliovirus containment and progress since the last report (2), and outlines remaining challenges. Within 30 countries, 86 facilities have been designated by the relevant national authorities (usually the Ministry of Health) to become poliovirus-essential facilities for the continued storage or handling of PV2 materials; each country is responsible for ensuring that these facilities meet all biorisk management requirements.

  5. Nectin-like interactions between poliovirus and its receptor trigger conformational changes associated with cell entry.

    Science.gov (United States)

    Strauss, Mike; Filman, David J; Belnap, David M; Cheng, Naiqian; Noel, Roane T; Hogle, James M

    2015-04-01

    family, by involving the burying of otherwise-exposed hydrophobic groups. Importantly, poliovirus expansion is regulated by the binding of a lipid molecule within the viral capsid. We show that receptor binding either causes this molecule to be expelled or requires it, but that its loss is not sufficient to trigger irreversible expansion. Based on our model, we propose testable hypotheses to explain how the viral shell becomes destabilized, leading to RNA uncoating. These findings give us a better understanding of how poliovirus has evolved to exploit a natural process of its host to penetrate the membrane barrier. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  6. Limits of variation, specific infectivity, and genome packaging of massively recoded poliovirus genomes.

    Science.gov (United States)

    Song, Yutong; Gorbatsevych, Oleksandr; Liu, Ying; Mugavero, JoAnn; Shen, Sam H; Ward, Charles B; Asare, Emmanuel; Jiang, Ping; Paul, Aniko V; Mueller, Steffen; Wimmer, Eckard

    2017-10-10

    Computer design and chemical synthesis generated viable variants of poliovirus type 1 (PV1), whose ORF (6,189 nucleotides) carried up to 1,297 "Max" mutations (excess of overrepresented synonymous codon pairs) or up to 2,104 "SD" mutations (randomly scrambled synonymous codons). "Min" variants (excess of underrepresented synonymous codon pairs) are nonviable except for P2 Min , a variant temperature-sensitive at 33 and 39.5 °C. Compared with WT PV1, P2 Min displayed a vastly reduced specific infectivity (si) (WT, 1 PFU/118 particles vs. P2 Min , 1 PFU/35,000 particles), a phenotype that will be discussed broadly. Si of haploid PV presents cellular infectivity of a single genotype. We performed a comprehensive analysis of sequence and structures of the PV genome to determine if evolutionary conserved cis-acting packaging signal(s) were preserved after recoding. We showed that conserved synonymous sites and/or local secondary structures that might play a role in determining packaging specificity do not survive codon pair recoding. This makes it unlikely that numerous "cryptic, sequence-degenerate, dispersed RNA packaging signals mapping along the entire viral genome" [Patel N, et al. (2017) Nat Microbiol 2:17098] play the critical role in poliovirus packaging specificity. Considering all available evidence, we propose a two-step assembly strategy for +ssRNA viruses: step I, acquisition of packaging specificity, either ( a ) by specific recognition between capsid protein(s) and replication proteins (poliovirus), or ( b ) by the high affinity interaction of a single RNA packaging signal (PS) with capsid protein(s) (most +ssRNA viruses so far studied); step II, cocondensation of genome/capsid precursors in which an array of hairpin structures plays a role in virion formation.

  7. Poliovirus infection induces the co-localization of cellular protein SRp20 with TIA-1, a cytoplasmic stress granule protein.

    Science.gov (United States)

    Fitzgerald, Kerry D; Semler, Bert L

    2013-09-01

    Different types of environmental stress cause mammalian cells to form cytoplasmic foci, termed stress granules, which contain mRNPs that are translationally silenced. These foci are transient and dynamic, and contain components of the cellular translation machinery as well as certain mRNAs and RNA binding proteins. Stress granules are known to be induced by conditions such as hypoxia, nutrient deprivation, and oxidative stress, and a number of cellular factors have been identified that are commonly associated with these foci. More recently it was discovered that poliovirus infection also induces the formation of stress granules, although these cytoplasmic foci appear to be somewhat compositionally unique. Work described here examined the punctate pattern of SRp20 (a host cell mRNA splicing protein) localization in the cytoplasm of poliovirus-infected cells, demonstrating the partial co-localization of SRp20 with the stress granule marker protein TIA-1. We determined that SRp20 does not co-localize with TIA-1, however, under conditions of oxidative stress, indicating that the close association of these two proteins during poliovirus infection is not representative of a general response to cellular stress. We confirmed that the expression of a dominant negative version of TIA-1 (TIA-1-PRD) results in the dissociation of stress granules. Finally, we demonstrated that expression of wild type TIA-1 or dominant negative TIA-1-PRD in cells during poliovirus infection does not dramatically affect viral translation. Taken together, these studies provide a new example of the unique cytoplasmic foci that form during poliovirus infection. Copyright © 2013 Elsevier B.V. All rights reserved.

  8. Poliovirus infection induces the co-localization of cellular protein SRp20 with TIA-1, a cytoplasmic stress granule protein

    Science.gov (United States)

    Fitzgerald, Kerry D.; Semler, Bert L.

    2013-01-01

    Different types of environmental stress cause mammalian cells to form cytoplasmic foci, termed stress granules, which contain mRNPs that are translationally silenced. These foci are transient and dynamic, and contain components of the cellular translation machinery as well as certain mRNAs and RNA binding proteins. Stress granules are known to be induced by conditions such as hypoxia, nutrient deprivation, and oxidative stress, and a number of cellular factors have been identified that are commonly associated with these foci. More recently it was discovered that poliovirus infection also induces the formation of stress granules, although these cytoplasmic foci appear to be somewhat compositionally unique. Work described here examined the punctate pattern of SRp20 (a host cell mRNA splicing protein) localization in the cytoplasm of poliovirus-infected cells, demonstrating the partial co-localization of SRp20 with the stress granule marker protein TIA-1. We determined that SRp20 does not co-localize with TIA-1, however, under conditions of oxidative stress, indicating that the close association of these two proteins during poliovirus infection is not representative of a general response to cellular stress. We confirmed that the expression of a dominant negative version of TIA-1 (TIA-1-PRD) results in the dissociation of stress granules. Finally, we demonstrated that expression of wild type TIA-1 or dominant negative TIA-1-PRD in cells during poliovirus infection does not dramatically affect viral translation. Taken together, these studies provide a new example of the unique cytoplasmic foci that form during poliovirus infection. PMID:23830997

  9. Cleavage sites in the polypeptide precursors of poliovirus protein P2-X

    International Nuclear Information System (INIS)

    Selmer, B.L.; Hanecak, R.; Anderson, C.W.; Wimmer, E.

    1981-01-01

    Partial amino-terminal sequence analysis has been performed on the three major polypeptide products (P2-3b, P2-5b, and P2-X) from the central region (P2) of the poliovirus polyprotein, and this analysis precisely locates the amino termini of these products with respect to the nucleotide sequence of the poliovirus RNA genome. Like most of the products of the replicase region (P3), the amino termini of P2-5b and P2-X are generated by cleavage between glutamine and glycine residues. Thus, P2-5b and P2-X are probably both produced by the action of a singly (virus-encoded.) proteinase. The amino terminus of P2-3b, on the other hand, is produced by a cleavage between the carboxy-terminal tyrosine of VP1 and the glycine encoded by nucleotides 3381-3383. This result may suggest that more than one proteolytic activity is required for the complete processing of the poliovirus polyprotein

  10. Functional conservation of the hydrophobic domain of polypeptide 3AB between human rhinovirus and poliovirus

    International Nuclear Information System (INIS)

    Towner, Jonathan S.; Brown, David M.; Nguyen, Joseph H.C.; Semler, Bert L.

    2003-01-01

    In this study we exchanged portions of the poliovirus type 1 (PV1) hydrophobic domain within the membrane-associated polypeptide 3AB for the analogous sequences from human rhinovirus 14 (HRV14). The sequence exchanges were based upon a previous report in which the 22 amino acid hydrophobic region was subdivided into two domains, I and II, the latter of which was shown to be required for membrane association (J. Biol. Chem. 271 (1996), 26810). Using these divisions, the HRV14 sequences were cloned into the complete poliovirus type 1 cDNA sequence. RNAs transcribed from these cDNAs were transfected into HeLa cell monolayers and used in HeLa cell-free translation/replication assays. The data indicated that 3AB sequences from PV1 and HRV14 are interchangeable; however, the substitutions cause a range of significant RNA replication defects, and in some cases, protein processing defects. Following transfection of RNAs encoding the domain substitutions into HeLa cell monolayers, virus isolates were harvested, and the corresponding viral RNAs were sequenced. The sequence data revealed that for the carboxy-terminal domain substitutions (domain II), multiple nucleotide changes were identified in the first, second, and third positions of different codons. In addition, the data indicated that for one of the PV1/HRV14 chimeras to replicate, compensatory mutations within poliovirus protein 2B may be required

  11. The Role of Electron Microscopy in Studying the Continuum of Changes in Membranous Structures during Poliovirus Infection

    Science.gov (United States)

    Rossignol, Evan D.; Yang, Jie E.; Bullitt, Esther

    2015-01-01

    Replication of the poliovirus genome is localized to cytoplasmic replication factories that are fashioned out of a mixture of viral proteins, scavenged cellular components, and new components that are synthesized within the cell due to viral manipulation/up-regulation of protein and phospholipid synthesis. These membranous replication factories are quite complex, and include markers from multiple cytoplasmic cellular organelles. This review focuses on the role of electron microscopy in advancing our understanding of poliovirus RNA replication factories. Structural data from the literature provide the basis for interpreting a wide range of biochemical studies that have been published on virus-induced lipid biosynthesis. In combination, structural and biochemical experiments elucidate the dramatic membrane remodeling that is a hallmark of poliovirus infection. Temporal and spatial membrane modifications throughout the infection cycle are discussed. Early electron microscopy studies of morphological changes following viral infection are re-considered in light of more recent data on viral manipulation of lipid and protein biosynthesis. These data suggest the existence of distinct subcellular vesicle populations, each of which serves specialized roles in poliovirus replication processes. PMID:26473912

  12. Environmental Surveillance System To Track Wild Poliovirus Transmission

    Science.gov (United States)

    Deshpande, Jagadish M.; Shetty, Sushmitha J.; Siddiqui, Zaeem A.

    2003-01-01

    Eradication of poliomyelitis from large metropolis cities in India has been difficult due to high population density and the presence of large urban slums. Three paralytic poliomyelitis cases were reported in Mumbai, India, in 1999 and 2000 in spite of high immunization coverage and good-quality supplementary immunization activities. We therefore established a systematic environmental surveillance study by weekly screening of sewage samples from three high-risk slum areas to detect the silent transmission of wild poliovirus. In 2001, from among the 137 sewage samples tested, wild poliovirus type 1 was isolated from 35 and wild poliovirus type 3 was isolated from 1. Acute flaccid paralysis (AFP) surveillance indicated one case of paralytic poliomyelitis from the city. Phylogenetic analysis with complete VP1 sequences revealed that the isolates from environmental samples belonged to four lineages of wild polioviruses recently isolated from poliomyelitis cases in Uttar Pradesh and not to those previously isolated from AFP cases in Mumbai. Wild poliovirus thus introduced caused one case of paralytic poliomyelitis. The virus was detected in environmental samples 3 months before. It was found that wild polioviruses introduced several times during the year circulated in Mumbai for a limited period before being eliminated. Environmental surveillance was found to be sensitive for the detection of wild poliovirus silent transmission. Nucleotide sequence analysis helped identify wild poliovirus reservoir areas. PMID:12732567

  13. Evaluation of immunity against poliovirus serotypes among children ...

    African Journals Online (AJOL)

    Eight (4%) of the children had no detectable antibody, 178 (89%), 180 (90%) and 181 (90.5%) were positive for antibodies to poliovirus types 1, 2 and 3, respectively. Overall, 162 (81%) of the children had antibodies to the three poliovirus serotypes at a titre of at least 1:8. The study shows the need for proper monitoring of ...

  14. Decay of Sabin inactivated poliovirus vaccine (IPV-boosted poliovirus antibodies

    Directory of Open Access Journals (Sweden)

    Sonia Resik

    2015-01-01

    Conclusion: The decay of poliovirus antibodies over a 21–22-month period was similar regardless of the type of booster vaccine used, suggesting the scientific data of Salk IPV long-term persistence and decay may be broadly applicable to Sabin IPV.

  15. Environmental Isolation of Circulating Vaccine-Derived Poliovirus After Interruption of Wild Poliovirus Transmission - Nigeria, 2016.

    Science.gov (United States)

    Etsano, Andrew; Damisa, Eunice; Shuaib, Faisal; Nganda, Gatei Wa; Enemaku, Ogu; Usman, Samuel; Adeniji, Adekunle; Jorba, Jaume; Iber, Jane; Ohuabunwo, Chima; Nnadi, Chimeremma; Wiesen, Eric

    2016-08-05

    In September 2015, more than 1 year after reporting its last wild poliovirus (WPV) case in July 2014 (1), Nigeria was removed from the list of countries with endemic poliovirus transmission,* leaving Afghanistan and Pakistan as the only remaining countries with endemic WPV. However, on April 29, 2016, a laboratory-confirmed, circulating vaccine-derived poliovirus type 2 (cVDPV2) isolate was reported from an environmental sample collected in March from a sewage effluent site in Maiduguri Municipal Council, Borno State, a security-compromised area in northeastern Nigeria. VDPVs are genetic variants of the vaccine viruses with the potential to cause paralysis and can circulate in areas with low population immunity. The Nigeria National Polio Emergency Operations Center initiated emergency response activities, including administration of at least 2 doses of oral poliovirus vaccine (OPV) to all children aged <5 years through mass campaigns; retroactive searches for missed cases of acute flaccid paralysis (AFP), and enhanced environmental surveillance. Approximately 1 million children were vaccinated in the first OPV round. Thirteen previously unreported AFP cases were identified. Enhanced environmental surveillance has not resulted in detection of additional VDPV isolates. The detection of persistent circulation of VDPV2 in Borno State highlights the low population immunity, surveillance limitations, and risk for international spread of cVDPVs associated with insurgency-related insecurity. Increasing vaccination coverage with additional targeted supplemental immunization activities and reestablishment of effective routine immunization activities in newly secured and difficult-to-reach areas in Borno is urgently needed.

  16. A novel multiplex poliovirus binding inhibition assay applicable for large serosurveillance and vaccine studies, without the use of live poliovirus.

    NARCIS (Netherlands)

    Schepp, Rutger M; Berbers, Guy A M; Ferreira, José A; Reimerink, Johan H; van der Klis, Fiona R

    2017-01-01

    Large-scale serosurveillance or vaccine studies for poliovirus using the "gold standard" WHO neutralisation test (NT) are very laborious and time consuming. With the polio eradication at hand and with the removal of live attenuated Sabin strains from the oral poliovirus vaccine (OPV), starting with

  17. Bioinformatics analysis and genetic diversity of the poliovirus.

    Science.gov (United States)

    Liu, Yanhan; Ma, Tengfei; Liu, Jianzhu; Zhao, Xiaona; Cheng, Ziqiang; Guo, Huijun; Wang, Shujing; Xu, Ruixue

    2014-12-01

    Poliomyelitis, a disease which can manifest as muscle paralysis, is caused by the poliovirus, which is a human enterovirus and member of the family Picornaviridae that usually transmits by the faecal-oral route. The viruses of the OPV (oral poliovirus attenuated-live vaccine) strains can mutate in the human intestine during replication and some of these mutations can lead to the recovery of serious neurovirulence. Informatics research of the poliovirus genome can be used to explain further the characteristics of this virus. In this study, sequences from 100 poliovirus isolates were acquired from GenBank. To determine the evolutionary relationship between the strains, we compared and analysed the sequences of the complete poliovirus genome and the VP1 region. The reconstructed phylogenetic trees for the complete sequences and the VP1 sequences were both divided into two branches, indicating that the genetic relationships of the whole poliovirus genome and the VP1 sequences are very similar. This branching indicates that the virulence and pathogenicity of poliomyelitis may be associated with the VP1 region. Sequence alignment of the VP1 region revealed numerous mutation sites in which mutation rates of >30 % were detected. In a group of strains recorded in the USA, mutation sites and mutation types were the same and this may be associated with their distribution in the evolutionary tree and their genetic relationship. In conclusion, the genetic evolutionary relationships of poliovirus isolate sequences are determined to a great extent by the VP1 protein, and poliovirus strains located on the same branch of the phylogenetic tree contain the same mutation spots and mutation types. Hence, the genetic characteristics of the VP1 region in the poliovirus genome should be analysed to identify the transmission route of poliovirus and provide the basis of viral immunity development. © 2014 The Authors.

  18. New approaches to poliovirus diagnosis using laboratory techniques: memorandum from a WHO meeting.

    OpenAIRE

    1992-01-01

    Laboratory diagnosis of poliomyelitis is an important part of the WHO initiative for global eradication of poliomyelitis. During the last year, new methods have been developed for the detection of poliovirus in clinical specimens, for intratypic differentiation, for the analysis of poliovirus neurovirulence, and for the detection of poliovirus antibodies. Progress in laboratory techniques for detection of poliovirus antibodies and for characterization of poliovirus isolates has suggested seve...

  19. All-atom molecular dynamics calculation study of entire poliovirus empty capsids in solution

    Energy Technology Data Exchange (ETDEWEB)

    Andoh, Y.; Yoshii, N.; Yamada, A.; Kojima, H.; Mizutani, K.; Okazaki, S., E-mail: okazaki@apchem.nagoya-u.ac.jp [Department of Applied Chemistry, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603 (Japan); Fujimoto, K. [Department of Pharmacy, College of Pharmaceutical Sciences, Ritsumeikan University, Nojihigashi, Kusatsu, Shiga 525-8577 (Japan); Nakagawa, A. [Institute for Protein Research, Osaka University, Yamadaoka, Suita, Osaka 565-0871 (Japan); Nomoto, A. [Institute of Microbial Chemistry, Kamiosaki, Shinagawa-ku, Tokyo 141-0021 (Japan)

    2014-10-28

    Small viruses that belong, for example, to the Picornaviridae, such as poliovirus and foot-and-mouth disease virus, consist simply of capsid proteins and a single-stranded RNA (ssRNA) genome. The capsids are quite stable in solution to protect the genome from the environment. Here, based on long-time and large-scale 6.5 × 10{sup 6} all-atom molecular dynamics calculations for the Mahoney strain of poliovirus, we show microscopic properties of the viral capsids at a molecular level. First, we found equilibrium rapid exchange of water molecules across the capsid. The exchange rate is so high that all water molecules inside the capsid (about 200 000) can leave the capsid and be replaced by water molecules from the outside in about 25 μs. This explains the capsid's tolerance to high pressures and deactivation by exsiccation. In contrast, the capsid did not exchange ions, at least within the present simulation time of 200 ns. This implies that the capsid can function, in principle, as a semipermeable membrane. We also found that, similar to the xylem of trees, the pressure of the solution inside the capsid without the genome was negative. This is caused by coulombic interaction of the solution inside the capsid with the capsid excess charges. The negative pressure may be compensated by positive osmotic pressure by the solution-soluble ssRNA and the counter ions introduced into it.

  20. All-atom molecular dynamics calculation study of entire poliovirus empty capsids in solution

    Science.gov (United States)

    Andoh, Y.; Yoshii, N.; Yamada, A.; Fujimoto, K.; Kojima, H.; Mizutani, K.; Nakagawa, A.; Nomoto, A.; Okazaki, S.

    2014-10-01

    Small viruses that belong, for example, to the Picornaviridae, such as poliovirus and foot-and-mouth disease virus, consist simply of capsid proteins and a single-stranded RNA (ssRNA) genome. The capsids are quite stable in solution to protect the genome from the environment. Here, based on long-time and large-scale 6.5 × 106 all-atom molecular dynamics calculations for the Mahoney strain of poliovirus, we show microscopic properties of the viral capsids at a molecular level. First, we found equilibrium rapid exchange of water molecules across the capsid. The exchange rate is so high that all water molecules inside the capsid (about 200 000) can leave the capsid and be replaced by water molecules from the outside in about 25 μs. This explains the capsid's tolerance to high pressures and deactivation by exsiccation. In contrast, the capsid did not exchange ions, at least within the present simulation time of 200 ns. This implies that the capsid can function, in principle, as a semipermeable membrane. We also found that, similar to the xylem of trees, the pressure of the solution inside the capsid without the genome was negative. This is caused by coulombic interaction of the solution inside the capsid with the capsid excess charges. The negative pressure may be compensated by positive osmotic pressure by the solution-soluble ssRNA and the counter ions introduced into it.

  1. All-atom molecular dynamics calculation study of entire poliovirus empty capsids in solution

    International Nuclear Information System (INIS)

    Andoh, Y.; Yoshii, N.; Yamada, A.; Kojima, H.; Mizutani, K.; Okazaki, S.; Fujimoto, K.; Nakagawa, A.; Nomoto, A.

    2014-01-01

    Small viruses that belong, for example, to the Picornaviridae, such as poliovirus and foot-and-mouth disease virus, consist simply of capsid proteins and a single-stranded RNA (ssRNA) genome. The capsids are quite stable in solution to protect the genome from the environment. Here, based on long-time and large-scale 6.5 × 10 6 all-atom molecular dynamics calculations for the Mahoney strain of poliovirus, we show microscopic properties of the viral capsids at a molecular level. First, we found equilibrium rapid exchange of water molecules across the capsid. The exchange rate is so high that all water molecules inside the capsid (about 200 000) can leave the capsid and be replaced by water molecules from the outside in about 25 μs. This explains the capsid's tolerance to high pressures and deactivation by exsiccation. In contrast, the capsid did not exchange ions, at least within the present simulation time of 200 ns. This implies that the capsid can function, in principle, as a semipermeable membrane. We also found that, similar to the xylem of trees, the pressure of the solution inside the capsid without the genome was negative. This is caused by coulombic interaction of the solution inside the capsid with the capsid excess charges. The negative pressure may be compensated by positive osmotic pressure by the solution-soluble ssRNA and the counter ions introduced into it

  2. Immune Serum From Sabin Inactivated Poliovirus Vaccine Immunization Neutralizes Multiple Individual Wild and Vaccine-Derived Polioviruses.

    Science.gov (United States)

    Sun, Mingbo; Li, Changgui; Xu, Wenbo; Liao, Guoyang; Li, Rongcheng; Zhou, Jian; Li, Yanping; Cai, Wei; Yan, Dongmei; Che, Yanchun; Ying, Zhifang; Wang, Jianfeng; Yang, Huijuan; Ma, Yan; Ma, Lei; Ji, Guang; Shi, Li; Jiang, Shude; Li, Qihan

    2017-05-15

    A Sabin strain-based inactivated poliomyelitis vaccine (Sabin-IPV) is the rational option for completely eradicating poliovirus transmission. The neutralizing capacity of Sabin-IPV immune serum to different strains of poliovirus is a key indicator of the clinical protective efficacy of this vaccine. Sera collected from 500 infants enrolled in a randomized, blinded, positive control, phase 2 clinical trial were randomly divided into 5 groups: Groups A, B, and C received high, medium, and low doses, respectively, of Sabin-IPV, while groups D and E received trivalent oral polio vaccine and Salk strain-based IPV, respectively, all on the same schedule. Immune sera were collected after the third dose of primary immunization, and tested in cross-neutralization assays against 19 poliovirus strains of all 3 types. All immune sera from all 5 groups interacted with the 19 poliovirus strains with various titers and in a dose-dependent manner. One type 2 immunodeficiency-associated vaccine-derived poliovirus strain was not recognized by these immune sera. Sabin-IPV vaccine can induce protective antibodies against currently circulating and reference wild poliovirus strains and most vaccine-derived poliovirus strains, with rare exceptions. NCT01056705. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

  3. Identification and Analysis of Antiviral Compounds Against Poliovirus.

    Science.gov (United States)

    Leyssen, Pieter; Franco, David; Tijsma, Aloys; Lacroix, Céline; De Palma, Armando; Neyts, Johan

    2016-01-01

    The Global Polio Eradication Initiative, launched in 1988, had as its goal the eradication of polio worldwide by the year 2000 through large-scale vaccinations campaigns with the live attenuated oral PV vaccine (OPV) (Griffiths et al., Biologicals 34:73-74, 2006). Despite substantial progress, polio remains endemic in several countries and new imported cases are reported on a regular basis ( http://www.polioeradication.org/casecount.asp ).It was recognized by the poliovirus research community that developing antivirals against poliovirus would be invaluable in the post-OPV era. Here, we describe three methods essential for the identification of selective inhibitors of poliovirus replication and for determining their mode of action by time-of-drug-addition studies as well as by the isolation of compound-resistant poliovirus variants.

  4. Intradermal Inactivated Poliovirus Vaccine: A Preclinical Dose-Finding Study

    OpenAIRE

    Kouiavskaia, Diana; Mirochnitchenko, Olga; Dragunsky, Eugenia; Kochba, Efrat; Levin, Yotam; Troy, Stephanie; Chumakov, Konstantin

    2014-01-01

    Intradermal delivery of vaccines has been shown to result in dose sparing. We tested the ability of fractional doses of inactivated poliovirus vaccine (IPV) delivered intradermally to induce levels of serum poliovirus-neutralizing antibodies similar to immunization through the intramuscular route. Immunogenicity of fractional doses of IPV was studied by comparing intramuscular and intradermal immunization of Wistar rats using NanoPass MicronJet600 microneedles. Intradermal delivery of partial...

  5. Plant RNA binding proteins for control of RNA virus infection

    Directory of Open Access Journals (Sweden)

    Sung Un eHuh

    2013-12-01

    Full Text Available Plant RNA viruses have effective strategies to infect host plants through either direct or indirect interactions with various host proteins, thus suppressing the host immune system. When plant RNA viruses enter host cells exposed RNAs of viruses are recognized by the host immune system through processes such as siRNA-dependent silencing. Interestingly, some host RNA binding proteins have been involved in the inhibition of RNA virus replication, movement, and translation through RNA-specific binding. Host plants intensively use RNA binding proteins for defense against viral infections in nature. In this mini review, we will summarize the function of some host RNA binding proteins which act in a sequence-specific binding manner to the infecting virus RNA. It is important to understand how plants effectively suppresses RNA virus infections via RNA binding proteins, and this defense system can be potentially developed as a synthetic virus defense strategy for use in crop engineering.

  6. Rare adverse events associated with oral poliovirus vaccine in Brazil

    Directory of Open Access Journals (Sweden)

    Friedrich F.

    1997-01-01

    Full Text Available Oral poliovirus vaccine (OPV developed by A. Sabin has been effectively used to control poliomyelitis in Brazil, and the last case with the isolation of a wild poliovirus strain occurred in March 1989. Although the vaccine controlled the circulation of wild strains and poliomyelitis cases associated with these strains were not detected during the last eight years, rare cases classified as vaccine-associated paralytic poliomyelitis (VAPP have been detected. Molecular characterization studies of poliovirus strains isolated from VAPP cases and from healthy contacts have confirmed that the isolates are derived from the Sabin vaccine strains and also detected genomic modifications known or suspected to increase neurovirulence such as mutations and recombination. The molecular characterization of polioviruses isolated during the last eight years from paralysis cases classified as Guillain-Barré (GBS syndrome and transverse myelitits (TM, and from facial paralysis (FP cases also confirmed the vaccine origin of the strains and demonstrated mutations known to increase neurovirulence. Analysis of the epidemiologic data of these GBS, TM and FP cases demonstrated that in most of them the last OPV dose was given months or years before the onset of the disease and the isolation of the polioviruses. The temporal association between the isolation of these strains and the GBS, TM and FP suggested that the Sabin vaccine-derived poliovirus strains could also rarely trigger the diseases.

  7. Detection of Emerging Vaccine-Related Polioviruses by Deep Sequencing.

    Science.gov (United States)

    Sahoo, Malaya K; Holubar, Marisa; Huang, ChunHong; Mohamed-Hadley, Alisha; Liu, Yuanyuan; Waggoner, Jesse J; Troy, Stephanie B; Garcia-Garcia, Lourdes; Ferreyra-Reyes, Leticia; Maldonado, Yvonne; Pinsky, Benjamin A

    2017-07-01

    Oral poliovirus vaccine can mutate to regain neurovirulence. To date, evaluation of these mutations has been performed primarily on culture-enriched isolates by using conventional Sanger sequencing. We therefore developed a culture-independent, deep-sequencing method targeting the 5' untranslated region (UTR) and P1 genomic region to characterize vaccine-related poliovirus variants. Error analysis of the deep-sequencing method demonstrated reliable detection of poliovirus mutations at levels of vaccinated, asymptomatic children and their close contacts collected during a prospective cohort study in Veracruz, Mexico, revealed no vaccine-derived polioviruses. This was expected given that the longest duration between sequenced sample collection and the end of the most recent national immunization week was 66 days. However, we identified many low-level variants (Sabin serotypes, as well as vaccine-related viruses with multiple canonical mutations associated with phenotypic reversion present at high levels (>90%). These results suggest that monitoring emerging vaccine-related poliovirus variants by deep sequencing may aid in the poliovirus endgame and efforts to ensure global polio eradication. Copyright © 2017 Sahoo et al.

  8. Environmental surveillance for polioviruses in the Global Polio Eradication Initiative.

    Science.gov (United States)

    Asghar, Humayun; Diop, Ousmane M; Weldegebriel, Goitom; Malik, Farzana; Shetty, Sushmitha; El Bassioni, Laila; Akande, Adefunke O; Al Maamoun, Eman; Zaidi, Sohail; Adeniji, Adekunle J; Burns, Cara C; Deshpande, Jagadish; Oberste, M Steve; Lowther, Sara A

    2014-11-01

    This article summarizes the status of environmental surveillance (ES) used by the Global Polio Eradication Initiative, provides the rationale for ES, gives examples of ES methods and findings, and summarizes how these data are used to achieve poliovirus eradication. ES complements clinical acute flaccid paralysis (AFP) surveillance for possible polio cases. ES detects poliovirus circulation in environmental sewage and is used to monitor transmission in communities. If detected, the genetic sequences of polioviruses isolated from ES are compared with those of isolates from clinical cases to evaluate the relationships among viruses. To evaluate poliovirus transmission, ES programs must be developed in a manner that is sensitive, with sufficiently frequent sampling, appropriate isolation methods, and specifically targeted sampling sites in locations at highest risk for poliovirus transmission. After poliovirus ceased to be detected in human cases, ES documented the absence of endemic WPV transmission and detected imported WPV. ES provides valuable information, particularly in high-density populations where AFP surveillance is of poor quality, persistent virus circulation is suspected, or frequent virus reintroduction is perceived. Given the benefits of ES, GPEI plans to continue and expand ES as part of its strategic plan and as a supplement to AFP surveillance. Published by Oxford University Press on behalf of the Infectious Diseases Society of America 2014. This work is written by (a) US Government employee(s) and is in the public domain in the US.

  9. Poliovirus seroprevalence before and after interruption of poliovirus transmission in Kano State, Nigeria.

    Science.gov (United States)

    Iliyasu, Zubairu; Verma, Harish; Craig, Kehinde T; Nwaze, Eric; Ahmad-Shehu, Amina; Jibir, Binta Wudil; Gwarzo, Garba Dayyabu; Gajida, Auwalu U; Weldon, William C; Steven Oberste, M; Takane, Marina; Mkanda, Pascal; Muhammad, Ado J G; Sutter, Roland W

    2016-09-30

    In September 2015, Nigeria was removed from the list of polio-endemic countries after more than 12months had passed since the detection of last wild poliovirus case in the country on 24 July 2014. We are presenting here a report of two polio seroprevalence surveys conducted in September 2013 and October 2014, respectively, in the Kano state of northern Nigeria. Health facility based seroprevalence surveys were undertaken at Murtala Mohammad Specialist Hospital, Kano. Parents or guardians of children aged 6-9months, 36-47months, 5-9years and 10-14years in 2013 and 6-9months and 19-22months (corresponding to 6-9months range at the time of 2013 survey) in 2014 presenting to the outpatient department, were approached for participation, screened for eligibility and asked to provide informed consent. A questionnaire was administered and a blood sample collected for polio neutralization assay. Among subjects aged 6-9months in the 2013 survey, seroprevalence was 58% (95% confidence interval [CI] 51-66%) to poliovirus type 1, 42% (95% CI 34-50%) to poliovirus type 2, and 52% (95% CI 44-60%) to poliovirus type 3. Among children 36-47months and older, seroprevalence was 85% or higher for all three serotypes. In 2014, seroprevalence in 6-9month infants was 72% (95% CI 65-79%) for type 1, 59% (95% CI 52-66%) for type 2, and 65% (95% CI 57-72%) for type 3 and in 19-22months, 80% (95% CI 74-85%), 57% (49-63%) and 78% (71-83%) respectively. Seroprevalence was positively associated with history of increasing oral poliovirus vaccine doses. There was significant improvement in seroprevalence in 2014 over the 2013 levels indicating a positive impact of recent programmatic interventions. However the continued low seroprevalence in 6-9month age is a concern and calls for improved immunization efforts to sustain the polio-free Nigeria. Copyright © 2016. Published by Elsevier Ltd.

  10. Sabin and wild type polioviruses from children who presented with acute flaccid paralysis in Nigeria.

    Science.gov (United States)

    Adedeji, A O; Okonko, I O; Adu, F D

    2012-09-01

    Sensitive poliovirus surveillance to detect vaccine-derived-polioviruses will continue to increase in importance. Isolating and identifying poliovirus strains from children of pediatrics age in Nigeria. A total of 120 fecal samples were randomly collected from children under the age of five who presented with acute flaccid paralysis. Samples were tested by tissue culture technique and further characterized by intratypic differentiation testing using ELISA and PCR methods. The study confirmed the presence of 22(18.3%) enteroviral isolates comprising 19(86.4%) polioviruses and 3(13.6%) non-polio enteroviruses. These 19 polioviruses include: Sabin-type poliovirus-1 (15.8%), poliovirus-2 (10.5%), poliovirus-3 (10.5%) and wild-type poliovirus-1 (63.2%) isolates. It showed that poliovirus infection was higher in children ages 6-11 months (18.9%), females (18.4%), northern states (91.0%) with no vaccination record (75.0%). Wild-type poliovirus-1 was isolated from the stool samples of 12(54.6%) children from northern states and in all age groups except 18-23 months. No significant differences (P >0.05) between poliovirus infection and age (18.9% vs. 17.7%; 81.9% vs. 18.2%) and sex (18.3% vs. 18.4%). There was significant differences (Pvaccination (75.0% vs. 0.0%). No wild-type poliovirus was found in those with complete vaccination. This study further confirms the presence of Sabin and wild-type poliovirus among children in Nigeria. The isolation of Sabin strain of poliovirus is advantageous to the polio eradication program as it is capable of inducing natural immunity in susceptible hosts. Transmission of wild-type poliovirus among children with incomplete vaccination poses a serious threat to polio eradication program in Nigeria. Environmental and serological surveillance with larger sample size are important for monitoring poliovirus circulation in Nigeria.

  11. A novel multiplex poliovirus binding inhibition assay applicable for large serosurveillance and vaccine studies, without the use of live poliovirus.

    Science.gov (United States)

    Schepp, Rutger M; Berbers, Guy A M; Ferreira, José A; Reimerink, Johan H; van der Klis, Fiona R

    2017-03-01

    Large-scale serosurveillance or vaccine studies for poliovirus using the "gold standard" WHO neutralisation test (NT) are very laborious and time consuming. With the polio eradication at hand and with the removal of live attenuated Sabin strains from the oral poliovirus vaccine (OPV), starting with type 2 (as of April 2016), laboratories will need to conform to much more stringent laboratory biosafety regulations when handling live poliovirus strains. In this study, a poliovirus binding inhibition multiplex immunoassay (polio MIA) using inactivated poliovirus vaccine (IPV-Salk) was developed for simultaneous quantification of serum antibodies directed to all three poliovirus types. Our assay shows a good correlation with the NT and an excellent correlation with the ELISA-based binding inhibition assay (POBI). The assay is highly type-specific and reproducible. Additionally, serum sample throughput increases about fivefold relative to NT and POBI and the amount of serum needed is reduced by more than 90%. In conclusion, the polio MIA can be used as a safe and high throughput application, especially for large-scale surveillance and vaccine studies, reducing laboratory time and serum amounts needed. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  12. Cell-Specific Establishment of Poliovirus Resistance to an Inhibitor Targeting a Cellular Protein

    Science.gov (United States)

    Viktorova, Ekaterina G.; Nchoutmboube, Jules; Ford-Siltz, Lauren A.

    2015-01-01

    ABSTRACT It is hypothesized that targeting stable cellular factors involved in viral replication instead of virus-specific proteins may raise the barrier for development of resistant mutants, which is especially important for highly adaptable small (+)RNA viruses. However, contrary to this assumption, the accumulated evidence shows that these viruses easily generate mutants resistant to the inhibitors of cellular proteins at least in some systems. We investigated here the development of poliovirus resistance to brefeldin A (BFA), an inhibitor of the cellular protein GBF1, a guanine nucleotide exchange factor for the small cellular GTPase Arf1. We found that while resistant viruses can be easily selected in HeLa cells, they do not emerge in Vero cells, in spite that in the absence of the drug both cultures support robust virus replication. Our data show that the viral replication is much more resilient to BFA than functioning of the cellular secretory pathway, suggesting that the role of GBF1 in the viral replication is independent of its Arf activating function. We demonstrate that the level of recruitment of GBF1 to the replication complexes limits the establishment and expression of a BFA resistance phenotype in both HeLa and Vero cells. Moreover, the BFA resistance phenotype of poliovirus mutants is also cell type dependent in different cells of human origin and results in a fitness loss in the form of reduced efficiency of RNA replication in the absence of the drug. Thus, a rational approach to the development of host-targeting antivirals may overcome the superior adaptability of (+)RNA viruses. IMPORTANCE Compared to the number of viral diseases, the number of available vaccines is miniscule. For some viruses vaccine development has not been successful after multiple attempts, and for many others vaccination is not a viable option. Antiviral drugs are needed for clinical practice and public health emergencies. However, viruses are highly adaptable and can

  13. Purification and properties of cowpea mosaic virus RNA replicase

    NARCIS (Netherlands)

    Zabel, P.

    1978-01-01

    This thesis concerns the partial purification and properties of an RNA-dependent RNA polymerase (RNA replicase) produced upon infection of Vigna unguiculata plants with Cowpea Mosaic Virus (CPMV). The enzyme is believed to be coded, at least in part, by the virus genome and to

  14. MicroRNA related polymorphisms and breast cancer risk

    NARCIS (Netherlands)

    S. Khan (Sofia); D. Greco (Dario); K. Michailidou (Kyriaki); R.L. Milne (Roger); T.A. Muranen (Taru); T. Heikkinen (Tuomas); K. Aaltonen (Kirsimari); J. Dennis (Joe); M.K. Bolla (Manjeet); J. Liu (Jianjun); P. Hall (Per); A. Irwanto (Astrid); M.K. Humphreys (Manjeet); J. Li (Jingmei); K. Czene (Kamila); J. Chang-Claude (Jenny); R. Hein (Rebecca); A. Rudolph (Anja); P. Seibold (Petra); D. Flesch-Janys (Dieter); O. Fletcher (Olivia); J. Peto (Julian); I. dos Santos Silva (Isabel); N. Johnson (Nichola); L.J. Gibson (Lorna); A. Aitken; J.L. Hopper (John); H. Tsimiklis (Helen); M. Bui (Minh); E. Makalic (Enes); D.F. Schmidt (Daniel); M.C. Southey (Melissa); C. Apicella (Carmel); J. Stone (Jennifer); Q. Waisfisz (Quinten); E.J. Meijers-Heijboer (Hanne); M.A. Adank (Muriel); R.B. van der Luijt (Rob); A. Meindl (Alfons); R.K. Schmutzler (Rita); B. Müller-Myhsok (B.); P. Lichtner (Peter); C. Turnbull (Clare); N. Rahman (Nazneen); S.J. Chanock (Stephen); D. Hunter (David); A. Cox (Angela); S.S. Cross (Simon); M.W.R. Reed (Malcolm); M.K. Schmidt (Marjanka); A. Broeks (Annegien); L.J. van 't Veer (Laura); F.B.L. Hogervorst (Frans); P.A. Fasching (Peter); A. Schrauder (André); A.B. Ekici (Arif); M.W. Beckmann (Matthias); S.E. Bojesen (Stig); B.G. Nordestgaard (Børge); S.F. Nielsen (Sune); H. Flyger (Henrik); J. Benítez (Javier); P.M. Zamora (Pilar M.); J.I.A. Perez (Jose Ignacio Arias); C.A. Haiman (Christopher); B.E. Henderson (Brian); F.R. Schumacher (Fredrick); L.L. March (Loic Le); P.D.P. Pharoah (Paul); A.M. Dunning (Alison); M. Shah (Mitul); R.N. Luben (Robert); J. Brown (Judith); F.J. Couch (Fergus); X. Wang (X.); C. Vachon (Celine); J.E. Olson (Janet); D. Lambrechts (Diether); M. Moisse (Matthieu); R. Paridaens (Robert); M.R. Christiaens (Marie Rose); P. Guénel (Pascal); T. Truong (Thérèse); P. Laurent-Puig (Pierre); C. Mulot (Claire); F. Marme (Frederick); B. Burwinkel (Barbara); A. Schneeweiss (Andreas); C. Sohn (Christof); E.J. Sawyer (Elinor); I.P. Tomlinson (Ian); M. Kerin (Michael); N. Miller (Nicola); I.L. Andrulis (Irene); J.A. Knight (Julia); S. Tchatchou (Srine); A.-M. Mulligan (Anna-Marie); T. Dörk (Thilo); N.V. Bogdanova (Natalia); N.N. Antonenkova (Natalia); H. Anton-Culver (Hoda); H. Darabi (Hatef); M. Eriksson (Mats); M. García-Closas (Montserrat); J.D. Figueroa (Jonine); J. Lissowska (Jolanta); L.A. Brinton (Louise); P. Devilee (Peter); R.A.E.M. Tollenaar (Rob); C.M. Seynaeve (Caroline); C.J. van Asperen (Christi); V. Kristensen (Vessela); S. Slager (Susan); A.E. Tol (Ama E.); C.B. Ambrosone (Christine); D. Yannoukakos (Drakoulis); A. Lindblom (Annika); S. Margolin (Sara); P. Radice (Paolo); P. Peterlongo (Paolo); M. Barile (Monica); P. Mariani (Paolo); M.J. Hooning (Maartje); J.W.M. Martens (John); J.M. Collée (Margriet); A. Jager (Agnes); A. Jakubowska (Anna); J. Lubinski (Jan); K. Jaworska-Bieniek (Katarzyna); K. Durda (Katarzyna); G.G. Giles (Graham); C.A. McLean (Catriona Ann); H. Brauch (Hiltrud); T. Brüning (Thomas); Y.-D. Ko (Yon-Dschun); H. Brenner (Hermann); A.K. Dieffenbach (Aida Karina); V. Arndt (Volker); C. Stegmaier (Christa); A.J. Swerdlow (Anthony ); A. Ashworth (Alan); N. Orr (Nick); M. Jones (Michael); J. Simard (Jacques); M.S. Goldberg (Mark); F. Labrèche (France); M. Dumont (Martine); R. Winqvist (Robert); K. Pykäs (Katri); A. Jukkola-Vuorinen (Arja); M. Grip (Mervi); V. Kataja (Vesa); V-M. Kosma (Veli-Matti); J.M. Hartikainen (J.); A. Mannermaa (Arto); U. Hamann (Ute); G. Chenevix-Trench (Georgia); C. Blomqvist (Carl); K. Aittomäki (Kristiina); D.F. Easton (Douglas); H. Nevanlinna (Heli)

    2014-01-01

    textabstractGenetic variations, such as single nucleotide polymorphisms (SNPs) in microRNAs (miRNA) or in the miRNA binding sites may affect the miRNA dependent gene expression regulation, which has been implicated in various cancers, including breast cancer, and may alter individual susceptibility

  15. Cryo-electron Microscopy Structures of Expanded Poliovirus with VHHs Sample the Conformational Repertoire of the Expanded State.

    Science.gov (United States)

    Strauss, Mike; Schotte, Lise; Karunatilaka, Krishanthi S; Filman, David J; Hogle, James M

    2017-02-01

    By using cryo-electron microscopy, expanded 80S-like poliovirus virions (poliovirions) were visualized in complexes with four 80S-specific camelid VHHs (Nanobodies). In all four complexes, the VHHs bind to a site on the top surface of the capsid protein VP3, which is hidden in the native virus. Interestingly, although the four VHHs bind to the same site, the structures of the expanded virus differ in detail in each complex, suggesting that each of the Nanobodies has sampled a range of low-energy structures available to the expanded virion. By stabilizing unique structures of expanded virions, VHH binding permitted a more detailed view of the virus structure than was previously possible, leading to a better understanding of the expansion process that is a critical step in infection. It is now clear which polypeptide chains become disordered and which become rearranged. The higher resolution of these structures also revealed well-ordered conformations for the EF loop of VP2, the GH loop of VP3, and the N-terminal extensions of VP1 and VP2, which, in retrospect, were present in lower-resolution structures but not recognized. These structural observations help to explain preexisting mutational data and provide insights into several other stages of the poliovirus life cycle, including the mechanism of receptor-triggered virus expansion. When poliovirus infects a cell, it undergoes a change in its structure in order to pass RNA through its protein coat, but this altered state is short-lived and thus poorly understood. The structures of poliovirus bound to single-domain antibodies presented here capture the altered virus in what appear to be intermediate states. A careful analysis of these structures lets us better understand the molecular mechanism of infection and how these changes in the virus lead to productive-infection events. Copyright © 2017 American Society for Microbiology.

  16. Membrane-associated precursor to poliovirus VPg identified by immunoprecipitation with antibodies directed against a synthetic heptapeptide

    International Nuclear Information System (INIS)

    Semelr, B.L.; Anderson, C.W.; Hanecak, R.; Dorner, L.F.; Wimmer, E.

    1982-01-01

    A synthetic heptapeptide corresponding to the C-terminal sequence of the poliovirus genome protein (VPg) has been linked to bovine serum albumin and used to raise antibodies in rabbits. These antibodies precipitate not only VPg but also at least two more virus-specific polypeptides. The smaller polypeptide, denoted P3-9 (12,000 daltons), has been mapped by Edman degradation and by fragmentation with cyanogen bromide and determined to be the N-terminal cleavage product of polypeptide P3-1b, a precursor to the RNA polymerase. P3-9 contains the sequence of the basic protein VPg (22 amino acids) at its C terminus. As predicted by the known RNA sequence of poliovirus, P3-9 also contains a hydrophobic region of 22 amino acids preceding VPg, an observation suggesting that P3-9 may be membrane-associated. This was confirmed by fractionation of infected cells in the presence or absence of detergent. We speculate that P3-9 may be the donor of VPg to RNA chains in the membrane-bound RNA replication complex

  17. Systematic review of mucosal immunity induced by oral and inactivated poliovirus vaccines against virus shedding following oral poliovirus challenge.

    Directory of Open Access Journals (Sweden)

    Thomas R Hird

    Full Text Available Inactivated poliovirus vaccine (IPV may be used in mass vaccination campaigns during the final stages of polio eradication. It is also likely to be adopted by many countries following the coordinated global cessation of vaccination with oral poliovirus vaccine (OPV after eradication. The success of IPV in the control of poliomyelitis outbreaks will depend on the degree of nasopharyngeal and intestinal mucosal immunity induced against poliovirus infection. We performed a systematic review of studies published through May 2011 that recorded the prevalence of poliovirus shedding in stool samples or nasopharyngeal secretions collected 5-30 days after a "challenge" dose of OPV. Studies were combined in a meta-analysis of the odds of shedding among children vaccinated according to IPV, OPV, and combination schedules. We identified 31 studies of shedding in stool and four in nasopharyngeal samples that met the inclusion criteria. Individuals vaccinated with OPV were protected against infection and shedding of poliovirus in stool samples collected after challenge compared with unvaccinated individuals (summary odds ratio [OR] for shedding 0.13 (95% confidence interval [CI] 0.08-0.24. In contrast, IPV provided no protection against shedding compared with unvaccinated individuals (summary OR 0.81 [95% CI 0.59-1.11] or when given in addition to OPV, compared with individuals given OPV alone (summary OR 1.14 [95% CI 0.82-1.58]. There were insufficient studies of nasopharyngeal shedding to draw a conclusion. IPV does not induce sufficient intestinal mucosal immunity to reduce the prevalence of fecal poliovirus shedding after challenge, although there was some evidence that it can reduce the quantity of virus shed. The impact of IPV on poliovirus transmission in countries where fecal-oral spread is common is unknown but is likely to be limited compared with OPV.

  18. Pressure for Pattern-Specific Intertypic Recombination between Sabin Polioviruses: Evolutionary Implications.

    Science.gov (United States)

    Korotkova, Ekaterina; Laassri, Majid; Zagorodnyaya, Tatiana; Petrovskaya, Svetlana; Rodionova, Elvira; Cherkasova, Elena; Gmyl, Anatoly; Ivanova, Olga E; Eremeeva, Tatyana P; Lipskaya, Galina Y; Agol, Vadim I; Chumakov, Konstantin

    2017-11-22

    Complete genomic sequences of a non-redundant set of 70 recombinants between three serotypes of attenuated Sabin polioviruses as well as location (based on partial sequencing) of crossover sites of 28 additional recombinants were determined and compared with the previously published data. It is demonstrated that the genomes of Sabin viruses contain distinct strain-specific segments that are eliminated by recombination. The presumed low fitness of these segments could be linked to mutations acquired upon derivation of the vaccine strains and/or may have been present in wild-type parents of Sabin viruses. These "weak" segments contribute to the propensity of these viruses to recombine with each other and with other enteroviruses as well as determine the choice of crossover sites. The knowledge of location of such segments opens additional possibilities for the design of more genetically stable and/or more attenuated variants, i.e., candidates for new oral polio vaccines. The results also suggest that the genome of wild polioviruses, and, by generalization, of other RNA viruses, may harbor hidden low-fitness segments that can be readily eliminated only by recombination.

  19. Phenotypic and genomic analysis of serotype 3 Sabin poliovirus vaccine produced in MRC-5 cell substrate.

    Science.gov (United States)

    Alirezaie, Behnam; Taqavian, Mohammad; Aghaiypour, Khosrow; Esna-Ashari, Fatemeh; Shafyi, Abbas

    2011-05-01

    The cell substrate has a pivotal role in live virus vaccines production. It is necessary to evaluate the effects of the cell substrate on the properties of the propagated viruses, especially in the case of viruses which are unstable genetically such as polioviruses, by monitoring the molecular and phenotypical characteristics of harvested viruses. To investigate the presence/absence of mutation(s), the near full-length genomic sequence of different harvests of the type 3 Sabin strain of poliovirus propagated in MRC-5 cells were determined. The sequences were compared with genomic sequences of different virus seeds, vaccines, and OPV-like isolates. Nearly complete genomic sequencing results, however, revealed no detectable mutations throughout the genome RNA-plaque purified (RSO)-derived monopool of type 3 OPVs manufactured in MRC-5. Thirty-six years of experience in OPV production, trend analysis, and vaccine surveillance also suggest that: (i) different monopools of serotype 3 OPV produced in MRC-5 retained their phenotypic characteristics (temperature sensitivity and neuroattenuation), (ii) MRC-5 cells support the production of acceptable virus yields, (iii) OPV replicated in the MRC-5 cell substrate is a highly efficient and safe vaccine. These results confirm previous reports that MRC-5 is a desirable cell substrate for the production of OPV. Copyright © 2011 Wiley-Liss, Inc.

  20. Pressure for Pattern-Specific Intertypic Recombination between Sabin Polioviruses: Evolutionary Implications

    Directory of Open Access Journals (Sweden)

    Ekaterina Korotkova

    2017-11-01

    Full Text Available Complete genomic sequences of a non-redundant set of 70 recombinants between three serotypes of attenuated Sabin polioviruses as well as location (based on partial sequencing of crossover sites of 28 additional recombinants were determined and compared with the previously published data. It is demonstrated that the genomes of Sabin viruses contain distinct strain-specific segments that are eliminated by recombination. The presumed low fitness of these segments could be linked to mutations acquired upon derivation of the vaccine strains and/or may have been present in wild-type parents of Sabin viruses. These “weak” segments contribute to the propensity of these viruses to recombine with each other and with other enteroviruses as well as determine the choice of crossover sites. The knowledge of location of such segments opens additional possibilities for the design of more genetically stable and/or more attenuated variants, i.e., candidates for new oral polio vaccines. The results also suggest that the genome of wild polioviruses, and, by generalization, of other RNA viruses, may harbor hidden low-fitness segments that can be readily eliminated only by recombination.

  1. [Inactivated poliovirus vaccines: an inevitable choice for eliminating poliomyelitis].

    Science.gov (United States)

    Vidor, J D; Jean-Denis, Shu

    2016-12-06

    The inactivated poliovirus vaccine (IPV) is a very old tool in the fight against poliomyelitis. Though supplanted by oral poliovirus vaccine (OPV) in the 1960s and 1970s, the IPV has now become an inevitable choice because of the increasingly recognized risks associated with continuous use of OPVs. Following the pioneering work of Jonas Salk, who established key principles for the IPV, considerable experience has accumulated over the years. This work has led to modern Salk IPV-containing vaccines, based on the use of inactivated wildtype polioviruses, which have been deployed for routine use in many countries. Very good protection against paralysis is achieved with IPV through the presence of circulating antibodies able to neutralize virus infectivity toward motor neurons. In addition, with IPV, a variable degree of protection against mucosal infection (and therefore transmission) through mucosal antibodies and immune cells is achieved, depending on previous exposure of subjects to wildtype or vaccine polioviruses. The use of an IPV-followed-by-OPV sequential immunization schedule has the potential advantage of eliminating the vaccine-associated paralytic poliomyelitis (VAPP) risk, while limiting the risks of vaccine-derived poliovirus (VDPVs). Sabin strain-derived IPVs are new tools, only recently beginning to be deployed, and data are being generated to document their performance. IPVs will play an irreplaceable role in global eradication of polio.

  2. Modeling the dynamics of oral poliovirus vaccine cessation.

    Science.gov (United States)

    Thompson, Kimberly M; Duintjer Tebbens, Radboud J

    2014-11-01

    Oral poliovirus vaccine (OPV) results in an ongoing burden of poliomyelitis due to vaccine-associated paralytic poliomyelitis and circulating vaccine-derived polioviruses (cVDPVs). This motivates globally coordinated OPV cessation after wild poliovirus eradication. We modeled poliovirus transmission and OPV evolution to characterize the interaction between population immunity, OPV-related virus prevalence, and the emergence of cVDPVs after OPV cessation. We explored strategies to prevent and manage cVDPVs for countries that currently use OPV for immunization and characterized cVDPV emergence risks and OPV use for outbreak response. Continued intense supplemental immunization activities until OPV cessation represent the best strategy to prevent cVDPV emergence after OPV cessation in areas with insufficient routine immunization coverage. Policy makers must actively manage population immunity before OPV cessation to prevent cVDPVs and aggressively respond if prevention fails. Sufficiently aggressive response with OPV to interrupt transmission of the cVDPV outbreak virus will lead to die-out of OPV-related viruses used for response in the outbreak population. Further analyses should consider the risk of exportation to other populations of the outbreak virus and any OPV used for outbreak response. OPV cessation can successfully eliminate all circulating live polioviruses in a population. The polio end game requires active risk management. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  3. The differential impact of oral poliovirus vaccine formulation choices on serotype-specific population immunity to poliovirus transmission.

    Science.gov (United States)

    Thompson, Kimberly M; Duintjer Tebbens, Radboud J

    2015-09-17

    Prior analyses demonstrated the need for some countries and the Global Polio Eradication Initiative (GPEI) to conduct additional supplemental immunization activities (SIAs) with trivalent oral poliovirus vaccine (tOPV) prior to globally-coordinated cessation of all serotype 2-containing OPV (OPV2 cessation) to prevent the creation of serotype 2 circulating vaccine-derived poliovirus (cVDPV2) outbreaks after OPV2 cessation. The GPEI continues to focus on achieving and ensuring interruption of wild poliovirus serotype 1 (WPV1) and making vaccine choices that prioritize bivalent OPV (bOPV) for SIAs, nominally to increase population immunity to serotype 1, despite an aggressive timeline for OPV2 cessation. We use an existing dynamic poliovirus transmission model of northwest Nigeria and an integrated global model for long-term poliovirus risk management to explore the impact of tOPV vs. bOPV vaccine choices on population immunity and cVDPV2 risks. Using tOPV instead of bOPV for SIAs leads to a minimal decrease in population immunity to transmission of serotypes 1 and 3 polioviruses, but a significantly higher population immunity to transmission of serotype 2 polioviruses. Failure to use tOPV in enough SIAs results in cVDPV2 emergence after OPV2 cessation in both the northwest Nigeria model and the global model. Despite perceptions to the contrary, prioritizing the use of bOPV over tOPV prior to OPV2 cessation does not significantly improve serotype 1 population immunity to transmission. Immunization leaders need to focus on all three poliovirus serotypes to appropriately manage the risks of OPV cessation in the polio endgame. Focusing on population immunity to transmission to interrupt WPV1 transmission and manage pre-OPV cessation risks of cVDPVs, all countries performing poliovirus SIAs should use tOPV up until the time of OPV2 cessation, after which time they should continue to use the OPV vaccine formulation with all remaining serotypes until coordinated global

  4. Immunogenicity to poliovirus type 2 following two doses of fractional intradermal inactivated poliovirus vaccine: A novel dose sparing immunization schedule.

    Science.gov (United States)

    Anand, Abhijeet; Molodecky, Natalie A; Pallansch, Mark A; Sutter, Roland W

    2017-05-19

    The polio eradication endgame strategic plan calls for the sequential removal of Sabin poliovirus serotypes from the trivalent oral poliovirus vaccine (tOPV), starting with type 2, and the introduction of ≥1 dose of inactivated poliovirus vaccine (IPV), to maintain an immunity base against poliovirus type 2. The global removal of oral poliovirus type 2 was successfully implemented in May 2016. However, IPV supply constraints has prevented introduction in 21 countries and led to complete stock-out in >20 countries. We conducted a literature review and contacted corresponding authors of recent studies with fractional-dose IPV (fIPV), one-fifth of intramuscular dose administered intradermally, to conduct additional type 2 immunogenicity analyses of two fIPV doses compared with one full-dose IPV. Four studies were identified that assessed immunogenicity of two fIPV doses compared to one full-dose IPV. Two fractional doses are more immunogenic than 1 full-dose, with type 2 seroconversion rates improving between absolute 19-42% (median: 37%, pvaccine compared to one full-dose IPV. In response to the current IPV shortage, a schedule of two fIPV doses at ages 6 and 14weekshas been endorsed by technical oversight committees and has been introduced in some affected countries. Copyright © 2017. Published by Elsevier Ltd.

  5. [Immunogenicity of sabin inactivated poliovirus vaccine induced by diphtheria-tetanus-acellular pertussis and Sabin inactivated poliovirus combined vaccine].

    Science.gov (United States)

    Ma, Yan; Qin, Min; Hu, Hui-Qiong; Ji, Guang; Feng, Ling; Gao, Na; Gu, Jie; Xie, Bing-Feng; He, Ji-Hong; Sun, Ming-Bo

    2011-06-01

    In order to search the preparation process and optimazing dosage ratio of adsorbed diphtheria-tetanus-acellular pertussis and sabin inactivated poliovirus combined vaccine (DTaP-sIPV), the neutralizing antibody titers of IPV induced by different concentration of DTaP-sIPV were investigated on rats. Two batches of DTaP-sLPV were produced using different concentration of sIPV and the quality control was carried. Together with sabin-IPV and DTaP-wIPV ( boostrix-polio, GSK, Belgium) as control group, the DTaP-sIPV were administrated on three-dose schedule at 0, 1, 2 month on rats. Serum sample were collected 30 days after each dose and neutralizing antibody titers against three types poliovirus were determined using micro-neutralization test. Two batches of prepared DTaP-sIPV and control sLPV were according to the requirement of Chinese Pharmacopoeia (Volume III, 2005 edition) and showed good stability. The seropositivity rates were 100% for sabin inactivated poliovirus antigen in all groups. The GMTs (Geometric mean titers) of neutralizing antibodies against three types poliovirus increased. The prepared DTaP-sIPV was safe, stable and effective and could induced high level neutralizing antibody against poliovirus on rats.

  6. Virologic Monitoring of Poliovirus Type 2 after Oral Poliovirus Vaccine Type 2 Withdrawal in April 2016 - Worldwide, 2016-2017.

    Science.gov (United States)

    Diop, Ousmane M; Asghar, Humayun; Gavrilin, Evgeniy; Moeletsi, Nicksy Gumede; Benito, Gloria Rey; Paladin, Fem; Pattamadilok, Sirima; Zhang, Yan; Goel, Ajay; Quddus, Arshad

    2017-05-26

    The Global Polio Eradication Initiative (GPEI) has made substantial progress since its launch in 1988; only 37 wild poliovirus type 1 (WPV1) cases were detected in 2016, the lowest annual count ever. Wild poliovirus type 3 has not been detected since November 2012, and wild poliovirus type 2 was officially declared eradicated in September 2015. This success is attributable to the wide use of live oral poliovirus vaccines (OPVs). Since 2001, numerous outbreaks were caused by the emergence of genetically divergent vaccine-derived polioviruses (VDPVs) whose genetic drift from the parental OPV strains indicates prolonged replication or circulation (1). In 2015, circulating VDPV type 2 (cVDPV2) outbreaks were detected in five countries worldwide (Nigeria, Pakistan, Guinea, Burma, and South Sudan), and VDPV2 single events were reported in 22 countries. These events prompted the GPEI to withdraw the type 2 component (Sabin2) of trivalent OPV (tOPV) in a globally coordinated, synchronized manner in April 2016 (2,3), at which time all OPV-using countries switched to using bivalent OPV (bOPV), containing Sabin types 1 and 3. This report details for the first time the virologic tracking of elimination of a live vaccine that has been withdrawn from routine and mass immunization systems worldwide (3). To secure elimination, further monitoring is warranted to detect any use of tOPV or monovalent OPV type 2 (mOPV2).

  7. Comprehensive screening for immunodeficiency-associated vaccine-derived poliovirus: an essential oral poliovirus vaccine cessation risk management strategy.

    Science.gov (United States)

    Duintjer Tebbens, R J; Thompson, K M

    2017-01-01

    If the world can successfully control all outbreaks of circulating vaccine-derived poliovirus that may occur soon after global oral poliovirus vaccine (OPV) cessation, then immunodeficiency-associated vaccine-derived polioviruses (iVDPVs) from rare and mostly asymptomatic long-term excretors (defined as ⩾6 months of excretion) will become the main source of potential poliovirus outbreaks for as long as iVDPV excretion continues. Using existing models of global iVDPV prevalence and global long-term poliovirus risk management, we explore the implications of uncertainties related to iVDPV risks, including the ability to identify asymptomatic iVDPV excretors to treat with polio antiviral drugs (PAVDs) and the transmissibility of iVDPVs. The expected benefits of expanded screening to identify and treat long-term iVDPV excretors with PAVDs range from US$0.7 to 1.5 billion with the identification of 25-90% of asymptomatic long-term iVDPV excretors, respectively. However, these estimates depend strongly on assumptions about the transmissibility of iVDPVs and model inputs affecting the global iVDPV prevalence. For example, the expected benefits may decrease to as low as US$260 million with the identification of 90% of asymptomatic iVDPV excretors if iVDPVs behave and transmit like partially reverted viruses instead of fully reverted viruses. Comprehensive screening for iVDPVs will reduce uncertainties and maximize the expected benefits of PAVD use.

  8. Nonhomologous recombination between defective poliovirus and coxsackievirus genomes suggests a new model of genetic plasticity for picornaviruses.

    Science.gov (United States)

    Holmblat, Barbara; Jégouic, Sophie; Muslin, Claire; Blondel, Bruno; Joffret, Marie-Line; Delpeyroux, Francis

    2014-08-05

    Most of the circulating vaccine-derived polioviruses (cVDPVs) implicated in poliomyelitis outbreaks in Madagascar have been shown to be recombinants between the type 2 poliovirus (PV) strain of the oral polio vaccine (Sabin 2) and another species C human enterovirus (HEV-C), such as type 17 coxsackie A virus (CA17) in particular. We studied intertypic genetic exchanges between PV and non-PV HEV-C by developing a recombination model, making it possible to rescue defective type 2 PV RNA genomes with a short deletion at the 3' end by the cotransfection of cells with defective or infectious CA17 RNAs. We isolated over 200 different PV/CA17 recombinants, using murine cells expressing the human PV receptor (PVR) and selecting viruses with PV capsids. We found some homologous (H) recombinants and, mostly, nonhomologous (NH) recombinants presenting duplications of parental sequences preferentially located in the regions encoding proteins 2A, 2B, and 3A. Short duplications appeared to be stable, whereas longer duplications were excised during passaging in cultured cells or after multiplication in PVR-transgenic mice, generating H recombinants with diverse sites of recombination. This suggests that NH recombination events may be a transient, intermediate step in the generation and selection of the fittest H recombinants. In addition to the classical copy-choice mechanism of recombination thought to generate mostly H recombinants, there may also be a modular mechanism of recombination, involving NH recombinant precursors, shaping the genomes of recombinant enteroviruses and other picornaviruses. Importance: The multiplication of circulating vaccine-derived polioviruses (cVDPVs) in poorly immunized human populations can render these viruses pathogenic, causing poliomyelitis outbreaks. Most cVDPVs are intertypic recombinants between a poliovirus (PV) strain and another human enterovirus, such as type 17 coxsackie A viruses (CA17). For further studies of the genetic exchanges

  9. 100 years poliovirus: from discovery to eradication. A meeting report.

    Science.gov (United States)

    Skern, Tim

    2010-09-01

    Just over hundred years ago, Karl Landsteiner and Erwin Popper identified a virus, later termed poliovirus, as the causative agent of poliomyelitis. This groundbreaking discovery simultaneously provided the basis for the measures that today prevent the outbreaks of the terrible epidemics caused by poliovirus. In 1988, the WHO started its eradication program to eliminate the virus from the planet. The symposium celebrated the discovery of poliovirus and discussed our current state of knowledge of poliovirus biology. Prospects for the eradication program were evaluated, with particular emphasis being placed on why certain countries still have not succeeding in interrupting wild-type transmission of poliovirus. Discussion also centred on the role of inactivated poliovirus vaccines in the eradication program and the maintenance of a poliovirus-free world, whenever this goal should be achieved.

  10. Health facility-based survey of poliovirus antibody prevalence ...

    African Journals Online (AJOL)

    Background: High level of Poliovirus protective antibodies, must at all times be sustained in a community if poliomyelitis eradication is to be achieved. For some time now children have been vaccinated against poliomyelitis through various means in Northern Nigeria without authorities taking steps to evaluate the ...

  11. Interrupting poliovirus transmission -- new solutions to an old problem.

    Science.gov (United States)

    Aylward, R Bruce; Maher, Chris

    2006-06-01

    Since the launch of the Global Polio Eradication Initiative (GPEI) in 1988, knowledge as to the nature of circulating polioviruses and the challenges to their interruption has increased tremendously, particularly during the period 2000-2005. By January 2006, however, the systematic application of the standard polio eradication strategies, combined with recent refinements, had reduced the number of countries with ongoing transmission of indigenous wild polioviruses to just four (Nigeria, India, Pakistan, and Afghanistan), the lowest ever in history. In addition, only 8 of the 22 areas that had been re-infected by wild poliovirus in 2003-2005 still required large-scale 'mop-up' activities and circulating vaccine-derived poliovirus (cVDPV) outbreaks were being readily addressed. This progress, despite new challenges late in the GPEI, was greatly facilitated by a range of solutions that included two new monovalent oral polio vaccines (mOPVs), new and robust international standards for polio outbreak response, and renewed political commitment across the remaining infected countries.

  12. Antigenic variation and resistance to neutralization in poliovirus type 1.

    NARCIS (Netherlands)

    D.C. Diamond; B.A. Jameson; J. Bonin; M. Kohara; S. Abe; H. Itoh; T. Komatsu; M. Arita; S. Kuge; A. Nomoto; A.D.M.E. Osterhaus (Albert); R. Crainic; E. Wimmer

    1985-01-01

    textabstractMutations have been identified in variants of poliovirus, type 1 (Mahoney) on the basis of their resistance to neutralization by individual monoclonal antibodies. The phenotypes of these variants were defined in terms of antibody binding; the pattern of epitopes expressed or able to be

  13. Isolation and Characterization of Poliovirus in Cell Culture Systems.

    Science.gov (United States)

    Thorley, Bruce R; Roberts, Jason A

    2016-01-01

    The isolation and characterization of enteroviruses by cell culture was accepted as the "gold standard" by clinical virology laboratories. Methods for the direct detection of all enteroviruses by reverse transcription polymerase chain reaction, targeting a conserved region of the genome, have largely supplanted cell culture as the principal diagnostic procedure. However, the World Health Organization's Global Polio Eradication Initiative continues to rely upon cell culture to isolate poliovirus due to the lack of a reliable sensitive genetic test for direct typing of enteroviruses from clinical specimens. Poliovirus is able to infect a wide range of mammalian cell lines, with CD155 identified as the primary human receptor for all three seroytpes, and virus replication leads to an observable cytopathic effect. Inoculation of cell lines with extracts of clinical specimens and subsequent passaging of the cells leads to an increased virus titre. Cultured isolates of poliovirus are suitable for testing by a variety of methods and remain viable for years when stored at low temperature.This chapter describes general procedures for establishing a cell bank and routine passaging of cell lines. While the sections on specimen preparation and virus isolation focus on poliovirus, the protocols are suitable for other enteroviruses.

  14. Development of novel inactivated poliovirus vaccines: Breaking away from convention

    NARCIS (Netherlands)

    Sanders, B.P.

    2015-01-01

    Infection with poliovirus (a small, non-enveloped Picornavirus) can result in poliomyelitis, hallmarked by symptoms of paralysis which is caused by viral destruction of motor neurons. Circulating humoral neutralizing antibodies can effectively protect against the disease, an immune response which

  15. Poliovirus Excretion in Children with Primary Immunodeficiency Disorders, India.

    Science.gov (United States)

    Mohanty, Madhu Chhanda; Madkaikar, Manisha Rajan; Desai, Mukesh; Taur, Prasad; Nalavade, Uma Prajwal; Sharma, Deepa Kailash; Gupta, Maya; Dalvi, Aparna; Shabrish, Snehal; Kulkarni, Manasi; Aluri, Jahnavi; Deshpande, Jagadish Mohanrao

    2017-10-01

    Prolonged excretion of poliovirus can occur in immunodeficient patients who receive oral polio vaccine, which may lead to propagation of highly divergent vaccine-derived polioviruses (VDPVs), posing a concern for global polio eradication. This study aimed to estimate the proportion of primary immunodeficient children with enterovirus infection and to identify the long-term polio/nonpolio enterovirus excreters in a tertiary care unit in Mumbai, India. During September 2014-April 2017, 151 patients received diagnoses of primary immunodeficiency (PID). We isolated 8 enteroviruses (3 polioviruses and 5 nonpolio enteroviruses) in cell culture of 105 fecal samples collected from 42 patients. Only 1 patient with severe combined immunodeficiency was identified as a long-term VDPV3 excreter (for 2 years after identification of infection). Our results show that the risk of enterovirus excretion among children in India with PID is low; however, systematic screening is necessary to identify long-term poliovirus excreters until the use of oral polio vaccine is stopped.

  16. Monoclonal antibodies to polioviruses; comparison of intratypic strain differentiation of poliovirus type 1 using monoclonal antibodies versus cross-absorbed antisera.

    NARCIS (Netherlands)

    A.D.M.E. Osterhaus (Albert); A.L. van Wezel; T.G. Hazendonk; F.G.C.M. Uytdehaag (Fons); J.A.A.M. van Asten (Jack); G. van Steenis (Bert)

    1983-01-01

    textabstractA panel of 10 monoclonal antibodies raised to 3 different poliovirus type 1 strains was tested in a micro-enzyme-linked immunosorbent assay and in a micro-neutralization test against 87 poliovirus type 1 strains. The results, evaluated in a newly developed system for intratypic strain

  17. Genomic characterization of severe acute respiratory syndrome-related coronavirus in European bats and classification of coronaviruses based on partial RNA-dependent RNA polymerase gene sequences

    Czech Academy of Sciences Publication Activity Database

    Drexler, J. F.; Gloza-Rausch, F.; Glende, J.; Corman, V. M.; Muth, D.; Goettsche, M.; Seebens, A.; Niedrig, M.; Pfefferle, S.; Yordanov, S.; Zhelyazkov, L.; Hermanns, U.; Vallo, Peter; Lukashev, A.; Müller, M. A.; Deng, H.; Herrler, G.; Drosten, C.

    2010-01-01

    Roč. 84, č. 21 (2010), s. 11336-11349 ISSN 0022-538X Institutional research plan: CEZ:AV0Z60930519 Keywords : cross-species transmission * SARS-like coronavirus es * reservoir hosts * horseshoe bats Subject RIV: EE - Microbiology, Virology Impact factor: 5.189, year: 2010

  18. Dense particles and slow sedimenting particles produced by ultraviolet irradiation of poliovirus

    International Nuclear Information System (INIS)

    Wetz, K.; Zeichhardt, H.; Willingmann, P.; Habermehl, K.-O.

    1983-01-01

    Low doses of u.v. radiation rapidly inactivate poliovirus, the virus progressively converting into dense particles (DPs) of buoyant density 1.44 g/ml in CsCl. The DPs are structurally and antigenically related to standard virus (N-antigen), i.e. indistinguishable from virus in their RNA and protein content and in sedimentation properties. Furthermore, there is no difference in reactivity of the structural proteins of virus and DPs with the monofunctional reagent [ 3 H]N-succinimidyl propionate ( 3 H-NSP). However, DPs differ from virus in that their capsids are permeable to several ions, and they can be degraded by RNase and protease. Increasing the radiation dose causes a successive transformation of DPs into 105S slow-sedimenting particles (SSPs), antigenically related to 76S artificial empty capsids (AECs) or H-antigen, but differing physically and structurally. The SSPs have a higher S value than AECs and contain all the capsid proteins, including VP4, and the RNA, both of these macromolecules being absent from AECs. It is concluded, therefore, that transformation from N- to H- antigenicity by u.v. radiation does not require release of RNA and VP4. Conversion of virus particles to SSPs correlates with altered reactivity of VP2 and to a lesser extent VP1 and VP3, with the monofunctional reagent 3 H-NSP. (author)

  19. Dense particles and slow sedimenting particles produced by ultraviolet irradiation of poliovirus

    Energy Technology Data Exchange (ETDEWEB)

    Wetz, K.; Zeichhardt, H.; Willingmann, P.; Habermehl, K.O. (Freie Univ. Berlin (Germany, F.R.))

    1983-06-01

    Low doses of u.v. radiation rapidly inactivate poliovirus, the virus progressively converting into dense particles (DPs) of buoyant density 1.44 g/ml in CsCl. The DPs are structurally and antigenically related to standard virus (N-antigen), i.e. indistinguishable from virus in their RNA and protein content and in sedimentation properties. Furthermore, there is no difference in reactivity of the structural proteins of virus and DPs with the monofunctional reagent (/sup 3/H)N-succinimidyl propionate (/sup 3/H-NSP). However, DPs differ from virus in that their capsids are permeable to several ions, and they can be degraded by RNase and protease. Increasing the radiation dose causes a successive transformation of DPs into 105S slow-sedimenting particles (SSPs), antigenically related to 76S artificial empty capsids (AECs) or H-antigen, but differing physically and structurally. The SSPs have a higher S value than AECs and contain all the capsid proteins, including VP4, and the RNA, both of these macromolecules being absent from AECs. It is concluded, therefore, that transformation from N- to H- antigenicity by u.v. radiation does not require release of RNA and VP4. Conversion of virus particles to SSPs correlates with altered reactivity of VP2 and to a lesser extent VP1 and VP3, with the monofunctional reagent /sup 3/H-NSP.

  20. Determinants of attenuation and temperature sensitivity in the type 1 poliovirus Sabin vaccine.

    OpenAIRE

    Bouchard, M J; Lam, D H; Racaniello, V R

    1995-01-01

    To identify determinants of attenuation in the poliovirus type 1 Sabin vaccine strain, a series of recombinant viruses were constructed by using infectious cDNA clones of the virulent type 1 poliovirus P1/Mahoney and the attenuated type 1 vaccine strain P1/Sabin. Intracerebral inoculation of these viruses into transgenic mice which express the human receptor for poliovirus identified regions of the genome that conferred reduced neurovirulence. Exchange of smaller restriction fragments and sit...

  1. Poliovirus trafficking toward central nervous system via human poliovirus receptor-dependent and -independent pathway.

    Directory of Open Access Journals (Sweden)

    Seii eOHKA

    2012-04-01

    Full Text Available In humans, paralytic poliomyelitis results from the invasion of the central nervous system by circulating poliovirus (PV via the blood-brain barrier (BBB. After the virus enters the central nervous system (CNS, it replicates in neurons, especially in motor neurons (MNs, inducing the cell death that causes paralytic poliomyelitis. Along with this route of dissemination, neural pathway has been reported in humans, monkeys, and PV-sensitive human PV receptor (hPVR/CD155-transgenic (Tg mice. We demonstrated that a fast retrograde axonal transport process is required for PV dissemination through the sciatic nerve of hPVR-Tg mice and that intramuscularly inoculated PV causes paralysis in a hPVR-dependent manner. We also showed that hPVR-independent axonal transport of PV exists in hPVR-Tg and non-Tg mice, indicating that several different pathways for PV axonal transport exist in these mice. Circulating PV after intravenous inoculation in mice cross the BBB at a high rate in a hPVR-independent manner. Recently, we identified transferrin receptor 1 (TfR1 of mouse brain capillary endothelial cells as a binding protein to PV, implicating that TfR1 is a possible receptor for PV to permeate the BBB.

  2. Initiation, elongation, and realignment during influenza virus mRNA synthesis

    NARCIS (Netherlands)

    Velthuis, te Aartjan J.W.; Oymans, Judith

    2018-01-01

    The RNA-dependent RNA polymerase (RdRp) of the influenza A virus replicates and transcribes the viral genome segments in the nucleus of the host cell. To transcribe these viral genome segments, the RdRp "snatches" capped RNA oligonucleotides from nascent host cell mRNAs and aligns these primers to

  3. Intradermal inactivated poliovirus vaccine: a preclinical dose-finding study.

    Science.gov (United States)

    Kouiavskaia, Diana; Mirochnitchenko, Olga; Dragunsky, Eugenia; Kochba, Efrat; Levin, Yotam; Troy, Stephanie; Chumakov, Konstantin

    2015-05-01

    Intradermal delivery of vaccines has been shown to result in dose sparing. We tested the ability of fractional doses of inactivated poliovirus vaccine (IPV) delivered intradermally to induce levels of serum poliovirus-neutralizing antibodies similar to immunization through the intramuscular route. Immunogenicity of fractional doses of IPV was studied by comparing intramuscular and intradermal immunization of Wistar rats using NanoPass MicronJet600 microneedles. Intradermal delivery of partial vaccine doses induced antibodies at titers comparable to those after immunization with full human dose delivered intramuscularly. The results suggest that intradermal delivery of IPV may lead to dose-sparing effect and reduction of the vaccination cost. Published by Oxford University Press on behalf of the Infectious Diseases Society of America 2014. This work is written by (a) US Government employee(s) and is in the public domain in the US.

  4. Inactivation of poliovirus by gamma irradiation of wastewater sludges

    International Nuclear Information System (INIS)

    Kaupert, Norma L.; Burgi, Elsa; Scolaro, L.

    1999-01-01

    The effect of gamma radiation on poliovirus infectivity seeded in sludge samples was investigated in order to determine the radiation dose required to inactivate 90% of viral infectivity (D 10 ). Sludges were obtained from anaerobic pretreated sewages produced by San Felipe, a wastewater treatment facility located at the Tucuman province, Argentina. A D 10 of 3.34 kGy was determined for poliovirus type III, Sabin strain, suspended in sludge samples. This value dropped to 1.92 kGy when the virus was suspended in water. A virucidal effect associated to sludges was also demonstrated. These results will be of interest when considering the dose of gamma radiation to be applied to wastewater sludges in order to preserve the environment from viral contamination. (author)

  5. Poliovirus Mutants Resistant to Neutralization with Soluble Cell Receptors

    Science.gov (United States)

    Kaplan, Gerardo; Peters, David; Racaniello, Vincent R.

    1990-12-01

    Poliovirus mutants resistant to neutralization with soluble cellular receptor were isolated. Replication of soluble receptor-resistant (srr) mutants was blocked by a monoclonal antibody directed against the HeLa cell receptor for poliovirus, indicating that the mutants use this receptor to enter cells. The srr mutants showed reduced binding to HeLa cells and cell membranes. However, the reduced binding phenotype did not have a major impact on viral replication, as judged by plaque size and one-step growth curves. These results suggest that the use of soluble receptors as antiviral agents could lead to the selection of neutralization-resistant mutants that are able to bind cell surface receptors, replicate, and cause disease.

  6. UV ability to destroy poliovirus end FRNA specific bacteriophages

    Energy Technology Data Exchange (ETDEWEB)

    Baron, J.; Joret, J.C.; Lesavre, J.; Perrot, J.Y.

    1996-01-01

    In France, the use of ultraviolet radiation to disinfect secondary effluents is only in its initial stage. The aim of this study was to examine the ability of UV to destroy Poliovirus Type 1 and FRNA specific bacteriophages (laboratory MS2 phages and indigenous phages). Concentrated viral solutions were mixed with secondary effluents artificially enriched with suspended solids and then irradiated at various UV dose in a collimated beam. Bacteriological analysis of Escherichia coli and enterococci were performed at the same time. UV were very efficient to kill Poliovirus : Inactivation of 3 and 5 log units were observed respectively at UV doses of 20 and 40 mW/cm{sup 2}. The Poliovirus disinfection rate was almost the same than Escherichia coli. Enterococci were more resistant than E. coli. Inactivation of MS2 bacteriophages was significantly correlated to UV dose following the relationship MS2 Inactivation = 0.047{sup *} Dose + 0,396. At UV dose of 20 mWs/cm{sup 2}, MS2 phages were 2.3 times more resistant to UV than Poliovirus, i.e. they need UV dose 2,3 times greater to be disinfected at the same level. A review of the literature has also shown that viruses more resistant to UV treatment have never been reported. All this would tend to confirm the interest of this group of virus as indicators of the disinfection efficiency of UV, which could indicate, on site, the inactivation of pathogenic viruses. Inactivation rates obtained for FRNA phages proved the good virucidal activity of UV. The inactivation of indigenous FRNA bacteriophages was not correlated with E. coli inactivation. On the other hand, it was correlated with enterococci inactivation. (Author). 23 refs., 7 figs., 4 tabs.

  7. Methods for the Quality Control of Inactivated Poliovirus Vaccines.

    Science.gov (United States)

    Wilton, Thomas

    2016-01-01

    Inactivated poliovirus vaccine (IPV) plays an instrumental role in the Global Poliovirus Eradication Initiative (GPEI). The quality of IPV is controlled by assessment of the potency of vaccine batches. The potency of IPV can be assessed by both in vivo and in vitro methods. In vitro potency assessment is based upon the assessment of the quantity of the D-Antigen (D-Ag) units in an IPV. The D-Ag unit is used as a measure of potency as it is largely expressed on native infectious virions and is the protective immunogen. The most commonly used in vitro test is the indirect ELISA which is used to ensure consistency throughout production.A range of in vivo assays have been developed in monkeys, chicks, guinea pigs, mice, and rats to assess the potency of IPV. All are based on assessment of the neutralizing antibody titer within the sera of the respective animal model. The rat potency test has become the favored in vivo potency test as it shows minimal variation between laboratories and the antibody patterns of rats and humans are similar. With the development of transgenic mice expressing the human poliovirus receptor, immunization-challenge tests have been developed to assess the potency of IPVs. This chapter describes in detail the methodology of these three laboratory tests to assess the quality of IPVs.

  8. Seroprevalence of poliovirus antibodies amongst children in Zaria, Northern Nigeria.

    Science.gov (United States)

    Giwa, F J; Olayinka, A T; Ogunshola, F T

    2012-11-06

    Poliomyelitis is endemic in Northern Nigeria where there is continuous transmission of wild poliovirus 1 and 3 (WPV1 and 3) and circulating vaccine derived poliovirus 2 (cVDPV2) resulting in a high number of cases of children with acute flaccid paralysis. The seroprevalence of antibodies to polio serotypes which can be used to assess the immune status of children and the effectiveness of the vaccine against poliomyelitis is unknown, despite its endemicity in this part of the world. This study aimed to determine the seroprevalence of poliovirus antibodies in children aged 1-10 years in Zaria, Northern Nigeria. A descriptive, cross sectional, community based study was undertaken in Zaria, North Western Nigeria between 2008 and 2009. Two hundred and sixty-four (264) children aged 1-10 years were enrolled from two local government in Zaria by multistage random sampling method. Demographic data and polio immunisation history were retrieved from parents and caregivers by an interviewer administered questionnaire. Neutralising antibody titres to polioserotypes 1, 2 and 3 were assayed according to the WHO Manual for the virological investigation of polio. Antibody titres ≥ 1:8 were considered positive. The mean age of the 264 children studied was 6.25 years. Fifty-five percent of the children were protected against the three polioserotypes, while 86.4%, 76.1% and 77.3% of children had neutralising antibodies to P1, P2 and P3 polioserotypes respectively. 5 (1.9%) of the children had no antibodies to all the three polioserotypes. Polio antibody seropositivity was significantly associated with higher socioeconomic status and immunisation was the single most important determinant of seropositivity to poliovirus serotypes. Seroprevalence to poliovirus serotypes, though higher than values found in previous studies done in Nigeria, was lower compared to findings in the developed world. The use of more immunogenic vaccines and the balanced use of OPV formulations in SIAs, with

  9. Sorveglianza della circolazione ambientale dei poliovirus nel Lazio

    Directory of Open Access Journals (Sweden)

    A.M. Patti

    2003-05-01

    Full Text Available Ancora oggi in tutto il mondo il vaccino antipolio più utilizzato è l’OPV costituito da virus viventi attenuati che vengono eliminati per un periodo di tempo variabile dal soggetto vaccinato. L’immissione di virus vaccinali nell’ambiente è stata in passato, e lo è tuttora nelle zone endemiche, estremamente importante per assicurare e la competizione con il poliovirus selvaggio e una immunità di gregge. Nei paesi polio-free, ed in futuro in tutto il mondo, la circolazione di virus vaccinali potrebbe viceversa diventare un punto critico in grado di inficiare i risultati dell’eradicazione. Infatti i virus vaccino derivati, replicando, retromutano verso la neurovirulenza e/o accumulano mutazioni che alla fine conferiscono loro caratteristiche del tutto diverse dai ceppi parentali; inoltre possono anche ricombinarsi con il selvaggio o con altri enterovirus assumendo caratteristiche di virulenza e di trasmissibilità interumana che emergono con lo scoppio di focolai epidemici. Obiettivo del presente progetto è stata la valutazione della circolazione dei poliovirus e degli eventuali virus vaccino derivati in matrici ambientali nella regione Lazio nel periodo 1996-2002. Metodo: sono stati analizzati 26 campioni di liquami e 36 campioni di acque superficiali contaminate da liquami. Le particelle virali sono state concentrate mediante ultra filtrazione tangenziale (10.000 NMWR – Millipore. I concentrati sono stati seminati su cellule BGM ed L20B. I virus isolati sono stati identificati con antisieri specifici (RIUM e sui poliovirus, presso l’ISS, sono stati effettuati la differenziazione intratipica, il sequenziamento della regione VPI/2A, il sequenziamento della regione 5’ NCR e la regione codificante la polimerasi virale. Risultati e conclusioni: sono stati isolati complessivamente 6 poliovirus di cui 4 da acque superficiali. I virus erano tutti Sabin-kike e retromutati ma non ricombinanti. I dati ottenuti sottolineano l

  10. Inhibition of host cell protein synthesis by UV-inactivated poliovirus

    International Nuclear Information System (INIS)

    Helentjaris, T.; Ehrenfeld, E.

    1977-01-01

    The ability of poliovirus that was irradiated with UV light at energies up to 2,160 ergs/mm 2 to subsequently inhibit host cell protein synthesis was measured. The inactivation of the host cell shutoff function followed one-hit kinetics. Increasing irradiation did not affect the rate of inhibition until the multiplicity of infection after irradiation was reduced to approximately 1 PFU/cell. At higher functional multiplicities, the rate was unchanged, but an increasing lag before the onset of inhibition was observed with increasing irradiation. The energy levels required to inactivate virus-induced inhibition of host cell protein synthesis suggest that damage to virus RNA rather than to virus capsid proteins is responsible for the loss of function. When the inactivation of host cell shutoff was compared with the inactivation of other viral functions by UV irradiation, it correlated exactly with the loss of infectivity but not with other viral functions measured. Guanidine treatment, which prevents detectable viral RNA and protein synthesis, completely inhibited host cell shutoff by low multiplicities of unirradiated virus infection but not higher multiplicities. When a high multiplicity of virus was first reduced to a low titer by irradiation, host cell shutoff was still evident in the presence of guanidine. The results demonstrate that the complete inhibition of host cell protein synthesis can be accomplished by one infectious viral genome per cell

  11. Investigation of an outbreak of type 3 wild poliovirus in Cote d'Ivoire ...

    African Journals Online (AJOL)

    adedamla

    three doses of polio vaccine compared to non-polio cases, OR: 16.9 [95% CI: 2.3 – 125.0]. More than 27% ... with only the poliovirus vaccine against the circulating poliovirus, people live in precarious socio-economic conditions ...... Sabin and.

  12. Poliovirus-specific memory immunity in seronegative elderly people does not protect against virus excretion.

    NARCIS (Netherlands)

    Abbink, Frithjofna; Buisman, Anne M; Doornbos, Gerda; Woldman, Jan; Kimman, Tjeerd G; Conyn- van Spaendonck, Marina A E

    2005-01-01

    BACKGROUND: Dutch people born between 1925 and 1945 were ineligible for vaccination with the inactivated poliovirus vaccine (IPV) introduced in 1957 and may have escaped natural infection because of reduced poliovirus circulation. We examined whether people with low or undetectable antibody levels

  13. STUDY OF IMMUNITY TO POLIOVIRUSES ON CERTAIN "SILENT" TERRITORIES OF RUSSIA

    Directory of Open Access Journals (Sweden)

    N. I. Romanenkova

    2011-01-01

    Full Text Available Abstract. The degree of immunity to polioviruses of three serotypes among children of different ages was analysed on certain "controlled" and "silent" territories of Russia in different periods of Polio Eradication Initiative. It was shown that the levels of immunity of children’s population to polioviruses on "controlled" and "silent" territories had no significant difference. It was stated that on the phase which preceded the certification for the absence of circulation of wild polioviruses, when the National Immunisation Days were conducted in the country, the percentage of eronegative children to polioviruses of different serotypes was low on all the territories of Russia. After Russia as a part of the WHO European region was certified as a polio free country and mass immunisation was stopped thepercentage of seronegative children increased, especially to poliovirus of serotype 3, both on the "controlled" and on the "silent" territories.

  14. NMR solution structure of poliovirus uridylyated peptide linked to the genome (VPgpU)

    Science.gov (United States)

    Schein, Catherine H.; Oezguen, Numan; van der Heden van Noort, Gerbrand J.; Filippov, Dmitri V.; Paul, Aniko; Kumar, Eric; Braun, Werner

    2010-01-01

    Picornaviruses have a 22–24 amino acid peptide, VPg, bound covalently at the 5’ end of their RNA, that is essential for replication. VPgs are uridylylated at a conserved Tyrosine to form VPgpU, the primer of RNA synthesis by the viral polymerase. This first complete structure for any uridylylated VPg, of poliovirus type 1 (PV1)-VPgpU, shows that conserved amino acids in VPg stabilize the bound UMP, with the uridine atoms involved in base pairing and chain elongation projected outward. Comparing this structure to PV1-VPg and partial structures of VPg/VPgpU from other picornaviruses suggests that enteroviral polymerases require a more stable VPg structure than does the distantly related aphthovirus, foot and mouth disease virus (FMDV). The glutamine residue at the C-terminus of PV1-VPgpU lies in back of the uridine base and may stabilize its position during chain elongation and/or contribute to base specificity. Under in vivo-like conditions with the authentic cre(2C) hairpin RNA and Mg++, 5-methylUTP cannot compete with UTP for VPg uridylyation in an in vitro uridylyation assay, but both nucleotides are equally incorporated by PV1-polymerase with Mn++ and a poly-A RNA template. This indicates the 5 position is recognized under in vivo conditions. The compact VPgpU structure docks within the active site cavity of the PV-polymerase, close to the position seen for the fragment of FMDV-VPgpU with its polymerase. This structure could aid in design of novel enterovirus inhibitors, and stabilization upon uridylylation may also be pertinent for post-translational uridylylation reactions that underlie other biological processes. PMID:20441784

  15. Picornavirus RNA is protected from cleavage by ribonuclease during virion uncoating and transfer across cellular and model membranes.

    Science.gov (United States)

    Groppelli, Elisabetta; Levy, Hazel C; Sun, Eileen; Strauss, Mike; Nicol, Clare; Gold, Sarah; Zhuang, Xiaowei; Tuthill, Tobias J; Hogle, James M; Rowlands, David J

    2017-02-01

    Picornaviruses are non-enveloped RNA viruses that enter cells via receptor-mediated endocytosis. Because they lack an envelope, picornaviruses face the challenge of delivering their RNA genomes across the membrane of the endocytic vesicle into the cytoplasm to initiate infection. Currently, the mechanism of genome release and translocation across membranes remains poorly understood. Within the enterovirus genus, poliovirus, rhinovirus 2, and rhinovirus 16 have been proposed to release their genomes across intact endosomal membranes through virally induced pores, whereas one study has proposed that rhinovirus 14 releases its RNA following disruption of endosomal membranes. For the more distantly related aphthovirus genus (e.g. foot-and-mouth disease viruses and equine rhinitis A virus) acidification of endosomes results in the disassembly of the virion into pentamers and in the release of the viral RNA into the lumen of the endosome, but no details have been elucidated as how the RNA crosses the vesicle membrane. However, more recent studies suggest aphthovirus RNA is released from intact particles and the dissociation to pentamers may be a late event. In this study we have investigated the RNase A sensitivity of genome translocation of poliovirus using a receptor-decorated-liposome model and the sensitivity of infection of poliovirus and equine-rhinitis A virus to co-internalized RNase A. We show that poliovirus genome translocation is insensitive to RNase A and results in little or no release into the medium in the liposome model. We also show that infectivity is not reduced by co-internalized RNase A for poliovirus and equine rhinitis A virus. Additionally, we show that all poliovirus genomes that are internalized into cells, not just those resulting in infection, are protected from RNase A. These results support a finely coordinated, directional model of viral RNA delivery that involves viral proteins and cellular membranes.

  16. Picornavirus RNA is protected from cleavage by ribonuclease during virion uncoating and transfer across cellular and model membranes.

    Directory of Open Access Journals (Sweden)

    Elisabetta Groppelli

    2017-02-01

    Full Text Available Picornaviruses are non-enveloped RNA viruses that enter cells via receptor-mediated endocytosis. Because they lack an envelope, picornaviruses face the challenge of delivering their RNA genomes across the membrane of the endocytic vesicle into the cytoplasm to initiate infection. Currently, the mechanism of genome release and translocation across membranes remains poorly understood. Within the enterovirus genus, poliovirus, rhinovirus 2, and rhinovirus 16 have been proposed to release their genomes across intact endosomal membranes through virally induced pores, whereas one study has proposed that rhinovirus 14 releases its RNA following disruption of endosomal membranes. For the more distantly related aphthovirus genus (e.g. foot-and-mouth disease viruses and equine rhinitis A virus acidification of endosomes results in the disassembly of the virion into pentamers and in the release of the viral RNA into the lumen of the endosome, but no details have been elucidated as how the RNA crosses the vesicle membrane. However, more recent studies suggest aphthovirus RNA is released from intact particles and the dissociation to pentamers may be a late event. In this study we have investigated the RNase A sensitivity of genome translocation of poliovirus using a receptor-decorated-liposome model and the sensitivity of infection of poliovirus and equine-rhinitis A virus to co-internalized RNase A. We show that poliovirus genome translocation is insensitive to RNase A and results in little or no release into the medium in the liposome model. We also show that infectivity is not reduced by co-internalized RNase A for poliovirus and equine rhinitis A virus. Additionally, we show that all poliovirus genomes that are internalized into cells, not just those resulting in infection, are protected from RNase A. These results support a finely coordinated, directional model of viral RNA delivery that involves viral proteins and cellular membranes.

  17. Patients with Primary Immunodeficiencies Are a Reservoir of Poliovirus and a Risk to Polio Eradication

    Directory of Open Access Journals (Sweden)

    Asghar Aghamohammadi

    2017-06-01

    Full Text Available Immunodeficiency-associated vaccine-derived polioviruses (iVDPVs have been isolated from primary immunodeficiency (PID patients exposed to oral poliovirus vaccine (OPV. Patients may excrete poliovirus strains for months or years; the excreted viruses are frequently highly divergent from the parental OPV and have been shown to be as neurovirulent as wild virus. Thus, these patients represent a potential reservoir for transmission of neurovirulent polioviruses in the post-eradication era. In support of WHO recommendations to better estimate the prevalence of poliovirus excreters among PIDs and characterize genetic evolution of these strains, 635 patients including 570 with primary antibody deficiencies and 65 combined immunodeficiencies were studied from 13 OPV-using countries. Two stool samples were collected over 4 days, tested for enterovirus, and the poliovirus positive samples were sequenced. Thirteen patients (2% excreted polioviruses, most for less than 2 months following identification of infection. Five (0.8% were classified as iVDPVs (only in combined immunodeficiencies and mostly poliovirus serotype 2. Non-polio enteroviruses were detected in 30 patients (4.7%. Patients with combined immunodeficiencies had increased risk of delayed poliovirus clearance compared to primary antibody deficiencies. Usually, iVDPV was detected in subjects with combined immunodeficiencies in a short period of time after OPV exposure, most for less than 6 months. Surveillance for poliovirus excretion among PID patients should be reinforced until polio eradication is certified and the use of OPV is stopped. Survival rates among PID patients are improving in lower and middle income countries, and iVDPV excreters are identified more frequently. Antivirals or enhanced immunotherapies presently in development represent the only potential means to manage the treatment of prolonged excreters and the risk they present to the polio endgame.

  18. Construction of a mutagenesis cartridge for poliovirus genome-linked viral protein: isolation and characterization of viable and nonviable mutants

    International Nuclear Information System (INIS)

    Kuhn, R.J.; Tada, H.; Ypma-Wong, M.F.; Dunn, J.J.; Semler, B.L.; Wimmer, E.

    1988-01-01

    By following a strategy of genetic analysis of poliovirus, the authors have constructed a synthetic mutagenesis cartridge spanning the genome-linked viral protein coding region and flanking cleavage sites in an infectious cDNA clone of the type I (Mahoney) genome. The insertion of new restriction sites within the infectious clone has allowed them to replace the wild-type sequences with short complementary pairs of synthetic oligonucleotides containing various mutations. A set of mutations have been made that create methionine codons within the genome-linked viral protein region. The resulting viruses have growth characteristics similar to wild type. Experiments that led to an alteration of the tyrosine residue responsible for the linkage to RNA have resulted in nonviable virus. In one mutant, proteolytic processing assayed in vitro appeared unimpaired by the mutation. They suggest that the position of the tyrosine residue is important for genome-linked viral protein function(s)

  19. A safe and reliable neutralization assay based on pseudovirus to measure neutralizing antibody titer against poliovirus.

    Science.gov (United States)

    Liu, Shaohua; Song, Dongmei; Bai, Han; Lu, Weiwei; Dai, Xinxian; Hao, Chunsheng; Zhang, Zhongyang; Guo, Huijie; Zhang, Yue; Li, Xiuling

    2017-12-01

    With the promotion of inactivated poliomyelitis vaccine (IPV) and live attenuated oral poliomyelitis vaccine (OPV), the global reported cases of poliomyelitis have reduced sharply from 0.35 million in 1988 to 74 in 2015. The Polio Eradication & Endgame Strategic Plan published by WHO in 2013 included the strategy of implementation of poliovirus safe handling and containment measures to minimize the risks of facility-associated reintroduction of virus into the polio-free community to prevent the re-import of poliovirus. Toward this strategy, we produced replication-incompetent pseudovirus of poliovirus type 1, 2, 3 attenuated strains by constructing poliovirus capsid expression vectors and poliovirus replicon then transfecting HEK293T cells and developed a pseudovirus-based neutralization assay (pNA) to determine neutralizing antibody titer which is more secure, time-saving and reliable than conventional neutralization assay (cNA). By using anti-poliovirus rat serum, we demonstrated excellent correlation between neutralizing antibody titers measured by cNA and pNA. It was concluded that pNA can be a potential alternative to replace cNA as a safe and time-saving system for titer determination after live poliovirus's safekeeping. © 2017 Wiley Periodicals, Inc.

  20. Seroprevalence of Poliovirus Antibodies in the United States Population, 2009-2010.

    Science.gov (United States)

    Wallace, Gregory S; Curns, Aaron T; Weldon, William C; Oberste, M Steven

    2016-08-05

    Polio is eliminated in the United States, with the last indigenous transmission occurring in 1979. However, global eradication of polio has not yet been completed, so importation of poliovirus into the U.S. is still possible. Specimens from the 2009-10 National Health and Nutrition Examination Survey (NHANES) were analyzed to evaluate population seroprevalence and assess overall risk from a poliovirus importation. We evaluated prevalence of serum antibodies to all three poliovirus types using the National Health and Nutrition Examination Survey during 2009-2010. The overall seroprevalence to poliovirus was 93.9 % for type 1, 97.0 % for type 2, and 83.1 % for type 3. Seroprevalence was higher for type 2 compared to the other types (p poliovirus in the US population during 2009-2010. While there were observed differences by serotype with type 2 having the highest seroprevalence and type 3 having the lowest, consistent with previous observations, no large immunity gaps to poliovirus suggesting an imminent substantial population risk from a poliovirus importation were observed at a population level.

  1. Seroprevalence of poliovirus antibodies in the Kansas City metropolitan area, 2012-2013.

    Science.gov (United States)

    Wallace, Gregory S; Pahud, Barbara A; Weldon, William C; Curns, Aaron T; Oberste, M Steven; Harrison, Christopher J

    2017-04-03

    No indigenous cases of poliomyelitis have occurred in the US since 1979; however the risk of importation persists until global eradication is achieved. The seropositivity rate for different age cohorts with exposures to different poliovirus vaccine types and wild virus in the US are not presently known. A convenience sample was conducted in the Kansas City metropolitan area during 2012-2103 with approximately 100 participants enrolled for each of 5 age cohorts categorized based on vaccine policy changes over time in the US. Immunization records for poliovirus vaccination were required for participants poliovirus serotypes. Seroprevalence was evaluated by demographics as well as between polio serotypes. The overall seroprevalence to poliovirus was 90.7%, 94.4%, and 83.3%, for types 1, 2, and 3, respectively. Seroprevalence was high (88.6%-96.2%) for all 3 types of poliovirus for the 6-10 y old age group that was likely to have received a complete schedule of IPV-only vaccination. Children 2-3 y of age, who have not yet completed their full IPV series, had lower seroprevalence compared with all older age groups for types 1 and 2 (p-value poliovirus in the population surveyed. Seroprevalence for subjects aged 2-3 y was lower than all other age groups for serotypes 1 and 2 highlighting the importance of completing the recommended poliovirus vaccine series with a booster dose at age 4-6 y.

  2. Implementation of coordinated global serotype 2 oral poliovirus vaccine cessation: risks of inadvertent trivalent oral poliovirus vaccine use.

    Science.gov (United States)

    Duintjer Tebbens, Radboud J; Hampton, Lee M; Thompson, Kimberly M

    2016-06-01

    The endgame for polio eradication includes coordinated global cessation of oral poliovirus vaccine (OPV), starting with the cessation of vaccine containing OPV serotype 2 (OPV2) by switching all trivalent OPV (tOPV) to bivalent OPV (bOPV). The logistics associated with this global switch represent a significant undertaking, with some possibility of inadvertent tOPV use after the switch. We used a previously developed poliovirus transmission and OPV evolution model to explore the relationships between the extent of inadvertent tOPV use, the time after the switch of the inadvertent tOPV use and corresponding population immunity to serotype 2 poliovirus transmission, and the ability of the inadvertently introduced viruses to cause a serotype 2 circulating vaccine-derived poliovirus (cVDPV2) outbreak in a hypothetical population. We then estimated the minimum time until inadvertent tOPV use in a supplemental immunization activity (SIA) or in routine immunization (RI) can lead to a cVDPV2 outbreak in realistic populations with properties like those of northern India, northern Pakistan and Afghanistan, northern Nigeria, and Ukraine. At low levels of inadvertent tOPV use, the minimum time after the switch for the inadvertent use to cause a cVDPV2 outbreak decreases sharply with increasing proportions of children inadvertently receiving tOPV. The minimum times until inadvertent tOPV use in an SIA or in RI can lead to a cVDPV2 outbreak varies widely among populations, with higher basic reproduction numbers, lower tOPV-induced population immunity to serotype 2 poliovirus transmission prior to the switch, and a lower proportion of transmission occurring via the oropharyngeal route all resulting in shorter times. In populations with the lowest expected immunity to serotype 2 poliovirus transmission after the switch, inadvertent tOPV use in an SIA leads to a cVDPV2 outbreak if it occurs as soon as 9 months after the switch with 0.5 % of children aged 0-4 years inadvertently

  3. Sabin and wild polioviruses from apparently healthy primary school children in northeastern Nigeria.

    Science.gov (United States)

    Baba, M M; Oderinde, B S; Patrick, P Z; Jarmai, M M

    2012-02-01

    Despite significant success of the Global Polio Eradication Initiative (GPEI) in Nigeria, Afghanistan, India, Pakistan, wild poliovirus still occurs due to persistently high proportions of under and unimmunized children. The study aimed at determining the type of poliovirus often excreted into the environment. Four hundred nine fecal samples collected from apparently healthy school children aged 5-16 years in Borno and Adamawa States, northeastern Nigeria, were tested for poliovirus by tissue culture technique. The isolates were characterized further by intratypic differentiation testing and genetic sequencing. Three wild poliovirus type, 11 Sabin type, combination of Sabin-types 1 + 2 and 2 + 3 poliovirus, and 22 non-polio enteroviruses were obtained. The continued excretion of wild-type poliovirus among children above 5 years old vaccinated with oral polio vaccine contributes to the persistent circulation of these viruses in the environment and may limit the population immunity. However, the excreted Sabin poliovirus is capable of immunizing the unvaccinated children and promotes herd immunity. Similarly, the excretion of combination of two polio serotypes indicates the child susceptibility to the missing serotype (s) and therefore indicates an immunity gap. The common unhygienic practices in the environment could aid the spread of these viruses through oral-fecal route. Asymptomatic transmission of wild poliovirus among older oral polio vaccine-vaccinated children poses a serious threat to polio eradication program in Nigeria and therefore, environmental and serological surveillance with larger sample size are important for monitoring poliovirus circulation in Nigeria. Copyright © 2011 Wiley Periodicals, Inc.

  4. Genetic relationships and epidemiological links between wild type 1 poliovirus isolates in Pakistan and Afghanistan.

    Science.gov (United States)

    Angez, Mehar; Shaukat, Shahzad; Alam, Muhammad M; Sharif, Salmaan; Khurshid, Adnan; Zaidi, Syed Sohail Zahoor

    2012-02-22

    Efforts have been made to eliminate wild poliovirus transmission since 1988 when the World Health Organization began its global eradication campaign. Since then, the incidence of polio has decreased significantly. However, serotype 1 and serotype 3 still circulate endemically in Pakistan and Afghanistan. Both countries constitute a single epidemiologic block representing one of the three remaining major global reservoirs of poliovirus transmission. In this study we used genetic sequence data to investigate transmission links among viruses from diverse locations during 2005-2007. In order to find the origins and routes of wild type 1 poliovirus circulation, polioviruses were isolated from faecal samples of Acute Flaccid Paralysis (AFP) patients. We used viral cultures, two intratypic differentiation methods PCR, ELISA to characterize as vaccine or wild type 1 and nucleic acid sequencing of entire VP1 region of poliovirus genome to determine the genetic relatedness. One hundred eleven wild type 1 poliovirus isolates were subjected to nucleotide sequencing for genetic variation study. Considering the 15% divergence of the sequences from Sabin 1, Phylogenetic analysis by MEGA software revealed that active inter and intra country transmission of many genetically distinct strains of wild poliovirus type 1 belonged to genotype SOAS which is indigenous in this region. By grouping wild type 1 polioviruses according to nucleotide sequence homology, three distinct clusters A, B and C were obtained with multiple chains of transmission together with some silent circulations represented by orphan lineages. Our results emphasize that there was a persistent transmission of wild type 1 polioviruses in Pakistan and Afghanistan during 2005-2007. The epidemiologic information provided by the sequence data can contribute to the formulation of better strategies for poliomyelitis control to those critical areas, associated with high risk population groups which include migrants

  5. High resolution identity testing of inactivated poliovirus vaccines.

    Science.gov (United States)

    Mee, Edward T; Minor, Philip D; Martin, Javier

    2015-07-09

    Definitive identification of poliovirus strains in vaccines is essential for quality control, particularly where multiple wild-type and Sabin strains are produced in the same facility. Sequence-based identification provides the ultimate in identity testing and would offer several advantages over serological methods. We employed random RT-PCR and high throughput sequencing to recover full-length genome sequences from monovalent and trivalent poliovirus vaccine products at various stages of the manufacturing process. All expected strains were detected in previously characterised products and the method permitted identification of strains comprising as little as 0.1% of sequence reads. Highly similar Mahoney and Sabin 1 strains were readily discriminated on the basis of specific variant positions. Analysis of a product known to contain incorrect strains demonstrated that the method correctly identified the contaminants. Random RT-PCR and shotgun sequencing provided high resolution identification of vaccine components. In addition to the recovery of full-length genome sequences, the method could also be easily adapted to the characterisation of minor variant frequencies and distinction of closely related products on the basis of distinguishing consensus and low frequency polymorphisms. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  6. Cleavage sites within the poliovirus capsid protein precursors

    International Nuclear Information System (INIS)

    Larsen, G.R.; Anderson, C.W.; Dorner, A.J.; Semler, B.L.; Wimmer, E.

    1982-01-01

    Partial amino-terminal sequence analysis was performed on radiolabeled poliovirus capsid proteins VP1, VP2, and VP3. A computer-assisted comparison of the amino acid sequences obtained with that predicted by the nucleotide sequence of the poliovirus genome allows assignment of the amino terminus of each capsid protein to a unique position within the virus polyprotein. Sequence analysis of trypsin-digested VP4, which has a blocked amino terminus, demonstrates that VP4 is encoded at or very near to the amino terminus of the polyprotein. The gene order of the capsid proteins is VP4-VP2-VP3-VP1. Cleavage of VP0 to VP4 and VP2 is shown to occur between asparagine and serine, whereas the cleavages that separate VP2/VP3 and VP3/VP1 occur between glutamine and glycine residues. This finding supports the hypothesis that the cleavage of VP0, which occurs during virion morphogenesis, is distinct from the cleavages that separate functional regions of the polyprotein

  7. Inactivation of poliovirus in wastewater sludge with radiation and thermoradiation

    International Nuclear Information System (INIS)

    Ward, R.L.

    1977-01-01

    The effect of sludge on the rate of viral inactivation by radiation and thermoradiation was determined. The virus used for the experiments was the poliovirus type 1 strain CHAT, which was grown in HeLa cells. Radiation, heat, and thermoradiation treatments were carried out in a chamber specifically designed to permit rapid heating and cooling of the samples at the beginning and completion of treatment, respectively. The treated samples were then assayed for plaque-forming units on HeLa cells after sonication in 0.1% sodium dodecylsulfate (SDS). For the radiation treatment virus was diluted 10-fold into PBS containing new sludge, irradiated at 20 0 C with 137 Cs at a dose rate of 30 krads/min, and assayed for infectious virus. The results show that raw sludge is protective of poliovirus against ionizing radiation but that small concentrations of sludge are nearly as protective as large concentrations. When heat and radiation are given simultaneously, however, the amount of protection afforded by sludge is less than the additive effects of the individual treatments. This result is especially evident at low concentrations of sludge. It appears, therefore, that thermoradiation treatment may be an effective way of inactivation viruses in waters containing low concentrations of suspended solids

  8. A RT-PCR method for selective amplification and phenotypic characterization of all three serotypes of Sabin-related polioviruses from viral mixtures

    Directory of Open Access Journals (Sweden)

    Eliane Veiga da Costa

    2012-08-01

    Full Text Available Outbreaks caused by vaccine-derived polioviruses are challenging the final eradication of paralytic poliomyelitis. Therefore, the surveillance of the acute flaccid paralysis cases based on poliovirus isolation and characterization remains an essential activity. Due to the use of trivalent oral poliovirus vaccine (OPV, mixtures containing more than one serotype of Sabin-related polioviruses are frequently isolated from clinical samples. Because each poliovirus isolate needs to be individually analyzed, we designed polymerase chain reaction primers that can selectively distinguish and amplify a genomic segment of the three Sabin-related poliovirus serotypes present in mixtures, thus, optimizing the diagnosis and providing prompt information to support epidemiologic actions.

  9. [The effect of aluminum adjuvant and immunization schedule on immunogenicity of Sabin inactivated poliovirus vaccine].

    Science.gov (United States)

    Wang, Fang; Zhang, Ming; Xie, Bing-Feng; Cao, Han; Tong, Shao-Yong; Wang, Jun-Rong; Yu, Xiao-Ping; Tang, Yang; Yang, Jing-Ran; Sun, Ming-Bo

    2013-04-01

    To study the effect of aluminume adjuvant and immunization schedule on immunogenicity of Sabin inactivated poliovirus vaccine. Four batches of Sabin IPV were produced by different concentrations of type 1, 2, and 3 poliovirus and administrated on three-dose schedule at 0, 1, 2 months and 0, 2, 4 months on rats. Serum samples were collected one month after each dose and neutralizing antibody titers against three types poliovirus were determined by micro-neutralization assay. The GMTs of neutralizing antibodies against three types poliovirus increased significantly and the seropositivity rates were 100% in all groups after 3 doses. There was no significant difference between two immunization schedules, and the 0, 2, 4 month schedule could induce higher level neutralizing antibody compared to the 0, 1, 2 month schedule. The groups with aluminum adjuvant could induce higher level neutralizing antibody compared to the groups without adjuvant. Aluminum djuvant and immunization schedule could improve the immunogenicity of Sabin IPV.

  10. Antibodies to poliovirus detected by immunoradiometric assay with a monoclonal antibody

    International Nuclear Information System (INIS)

    Spitz, M.; Fossati, C.A.; Schild, G.C.; Spitz, L.; Brasher, M.

    1982-01-01

    An immunoradiometric assay (IRMA) for the assay of antibodies to poliovirus antigens is described. Dilutions of the test sera or whole (finger prick) blood samples were incubated with the poliovirus antigen bound to a solid phase and the specific antibody was detected by the addition of a mouse anti-human IgG monoclonal antibody (McAb), which was itself revealed by iodinated sheep IgG antimouse F(ab). The authors have shown that this technique is suitable for the estimation of IgG anti-poliovirus antibodies induced in children following polio vaccine. The present study shows that SPRIA provides a simple and inexpensive method for serological studies with poliovirus particularly for use in large-scale surveys. (Auth.)

  11. Antibodies to poliovirus detected by immunoradiometric assay with a monoclonal antibody

    Energy Technology Data Exchange (ETDEWEB)

    Spitz, M.; Fossati, C.A.; Schild, G.C.; Spitz, L.; Brasher, M. (National Inst. for Biological Standards and Control, London (UK))

    1982-10-01

    An immunoradiometric assay (IRMA) for the assay of antibodies to poliovirus antigens is described. Dilutions of the test sera or whole (finger prick) blood samples were incubated with the poliovirus antigen bound to a solid phase and the specific antibody was detected by the addition of a mouse anti-human IgG monoclonal antibody (McAb), which was itself revealed by iodinated sheep IgG antimouse F(ab). The authors have shown that this technique is suitable for the estimation of IgG anti-poliovirus antibodies induced in children following polio vaccine. The present study shows that SPRIA provides a simple and inexpensive method for serological studies with poliovirus particularly for use in large-scale surveys.

  12. Poliovirus immunity in newly resettled adult refugees in Idaho, United States of America.

    Science.gov (United States)

    Roscoe, Clay; Gilles, Ryan; Reed, Alex J; Messerschmidt, Matt; Kinney, Rebecca

    2015-06-12

    In the United States, vaccines have eliminated wild poliovirus (WPV) infection, though resettling refugees may lack immunity and importation of WPV remains a concern. A cross-sectional survey was performed to determine the prevalence of poliovirus immunity in adult refugees resettling in Boise, Idaho, U.S.A.; immunity was evaluated using two definitions: serotypes 1, 2 and 3 positive, or serotypes 1 and 3 positive. This survey evaluated 795 adult refugees between August 2010 and November 2012. Poliovirus immunity in adults >18 years was 55.3% for serotypes 1, 2 and 3 combined, and 60% for serotypes 1 and 3 only. This study demonstrated a WPV immunity rate of poliovirus immunity in all newly arrived adult refugees, either by expanding pre-departure immunization or by screening for immunity at resettlement and vaccinating when indicated. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. Preliminary Observations on the Uptake of Poliovirus by West Coast Shore Crabs

    Science.gov (United States)

    DiGirolamo, Rudolph; Wiczynski, Leokadia; Daley, Michael; Miranda, Florencio

    1972-01-01

    West Coast shore crabs (Pachygrapsus sp. and Hemigrapsus sp.), when in seawater contaminated with poliovirus or allowed to feed on virus-contaminated mussels (Mytilus californianus), were found to accumulate high titers of virus. PMID:4333894

  14. A Cluster of Paralytic Poliomyelitis Cases Due to Transmission of Slightly Diverged Sabin 2 Vaccine Poliovirus.

    Science.gov (United States)

    Korotkova, Ekaterina A; Gmyl, Anatoly P; Yakovenko, Maria L; Ivanova, Olga E; Eremeeva, Tatyana P; Kozlovskaya, Liubov I; Shakaryan, Armen K; Lipskaya, Galina Y; Parshina, Irina L; Loginovskikh, Nataliya V; Morozova, Nadezhda S; Agol, Vadim I

    2016-07-01

    Four cases of acute flaccid paralysis caused by slightly evolved (Sabin-like) vaccine polioviruses of serotype 2 were registered in July to August 2010 in an orphanage of Biysk (Altai Region, Russia). The Biysk cluster of vaccine-associated paralytic poliomyelitis (VAPP) had several uncommon, if not unique, features. (i) Until this outbreak, Sabin-like viruses (in distinction to more markedly evolved vaccine-derived polioviruses [VDPVs]) were reported to cause only sporadic cases of VAPP. Consequently, VAPP cases were not considered to require outbreak-type responses. However, the Biysk outbreak completely blurred the borderline between Sabin-like viruses and VDPVs in epidemiological terms. (ii) The outbreak demonstrated a very high disease/infection ratio, apparently exceeding even that reported for wild polioviruses. The viral genome structures did not provide any substantial hints as to the underlying reason(s) for such pathogenicity. (iii) The replacement of intestinal poliovirus lineages by other Sabin-like lineages during short intervals after the disease onsets was observed in two patients. Again, the sequences of the respective genomes provided no clues to explain these events. (iv) The polioviruses isolated from the patients and their contacts demonstrated a striking heterogeneity as well as rapid and uneven evolution of the whole genomes and their parts, apparently due to extensive interpersonal contacts in a relatively small closed community, multiple bottlenecking, and recombination. Altogether, the results demonstrate several new aspects of pathogenicity, epidemiology, and evolution of vaccine-related polioviruses and underscore several serious gaps in understanding these problems. The oral poliovirus vaccine largely contributed to the nearly complete disappearance of poliovirus-caused poliomyelitis. Being generally safe, it can, in some cases, result in a paralytic disease. Two types of such outcomes are distinguished: those caused by slightly

  15. Human Circulating Antibody-Producing B Cell as a Predictive Measure of Mucosal Immunity to Poliovirus.

    Science.gov (United States)

    Dey, Ayan; Molodecky, Natalie A; Verma, Harish; Sharma, Prashant; Yang, Jae Seung; Saletti, Giulietta; Ahmad, Mohammad; Bahl, Sunil K; Wierzba, Thomas F; Nandy, Ranjan K; Deshpande, Jagadish M; Sutter, Roland W; Czerkinsky, Cecil

    2016-01-01

    The "gold standard" for assessing mucosal immunity after vaccination with poliovirus vaccines consists in measuring virus excretion in stool after challenge with oral poliovirus vaccine (OPV). This testing is time and resource intensive, and development of alternative methods is a priority for accelerating polio eradication. We therefore evaluated circulating antibody-secreting cells (ASCs) as a potential means to evaluate mucosal immunity to poliovirus vaccine. 199 subjects, aged 10 years, and previously immunized repeatedly with OPV, were selected. Subjects were assigned to receive either a booster dose of inactivated poliovirus vaccine (IPV), bivalent OPV (bOPV), or no vaccine. Using a micro-modified whole blood-based ELISPOT assay designed for field setting, circulating poliovirus type-specific IgA- and IgG-ASCs, including gut homing α4β7+ ASCs, were enumerated on days 0 and 7 after booster immunization. In addition, serum samples collected on days 0, 28 and 56 were tested for neutralizing antibody titers against poliovirus types 1, 2, and 3. Stool specimens were collected on day 28 (day of bOPV challenge), and on days 31, 35 and 42 and processed for poliovirus isolation. An IPV dose elicited blood IgA- and IgG-ASC responses in 84.8 to 94.9% of subjects, respectively. In comparison, a bOPV dose evoked corresponding blood ASC responses in 20.0 to 48.6% of subjects. A significant association was found between IgA- and IgG-ASC responses and serum neutralizing antibody titers for poliovirus type 1, 2, 3 (ppoliovirus types 1, 2 and 3 was 62.7%, 89.8% and 45.8%, respectively. A significant association was observed between virus excretion and α4β7+ IgA- and/or IgG-ASC responses to poliovirus type 3 among immunized children; however, only a weak association was found for type 1 poliovirus. Our results suggest that virus-specific blood ASCs, especially for type 3 poliovirus, can serve as surrogate of mucosal immunity after vaccination. Further studies are needed to

  16. Translation of Polioviral mRNA Is Inhibited by Cleavage of Polypyrimidine Tract-Binding Proteins Executed by Polioviral 3Cpro

    Science.gov (United States)

    Back, Sung Hoon; Kim, Yoon Ki; Kim, Woo Jae; Cho, Sungchan; Oh, Hoe Rang; Kim, Jung-Eun; Jang, Sung Key

    2002-01-01

    The translation of polioviral mRNA occurs through an internal ribosomal entry site (IRES). Several RNA-binding proteins, such as polypyrimidine tract-binding protein (PTB) and poly(rC)-binding protein (PCBP), are required for the poliovirus IRES-dependent translation. Here we report that a poliovirus protein, 3Cpro (and/or 3CDpro), cleaves PTB isoforms (PTB1, PTB2, and PTB4). Three 3Cpro target sites (one major target site and two minor target sites) exist in PTBs. PTB fragments generated by poliovirus infection are redistributed to the cytoplasm from the nucleus, where most of the intact PTBs are localized. Moreover, these PTB fragments inhibit polioviral IRES-dependent translation in a cell-based assay system. We speculate that the proteolytic cleavage of PTBs may contribute to the molecular switching from translation to replication of polioviral RNA. PMID:11836431

  17. Genetic relationships and epidemiological links between wild type 1 poliovirus isolates in Pakistan and Afghanistan

    OpenAIRE

    Angez, Mehar; Shaukat, Shahzad; Alam, Muhammad M; Sharif, Salmaan; Khurshid, Adnan; Zahoor Zaidi, Syed Sohail

    2012-01-01

    Abstract Background/Aim Efforts have been made to eliminate wild poliovirus transmission since 1988 when the World Health Organization began its global eradication campaign. Since then, the incidence of polio has decreased significantly. However, serotype 1 and serotype 3 still circulate endemically in Pakistan and Afghanistan. Both countries constitute a single epidemiologic block representing one of the three remaining major global reservoirs of poliovirus transmission. In this study we use...

  18. Virus removal during groundwater recharge: effects of infiltration rate on adsorption of poliovirus to soil.

    OpenAIRE

    Vaughn, J M; Landry, E F; Beckwith, C A; Thomas, M Z

    1981-01-01

    Studies were conducted to determine the influence of infiltration rate on poliovirus removal during groundwater recharge with tertiary-treated wastewater effluents. Experiments were conducted at a uniquely designed, field-situated test recharge basin facility through which some 62,000 m3 of sewage had been previously applied. Recharge at high infiltration rates (75 to 100 cm/h) resulted in the movement of considerable numbers of seeded poliovirus to the groundwater. Moderately reduced infiltr...

  19. Wild and vaccine-derived poliovirus circulation, and implications for polio eradication.

    Science.gov (United States)

    Lopalco, P L

    2017-02-01

    Polio cases due to wild virus are reported by only three countries in the world. Poliovirus type 2 has been globally eradicated and the last detection of poliovirus type 3 dates to November 2012. Poliovirus type 1 remains the only circulating wild strain; between January and September 2016 it caused 26 cases (nine in Afghanistan, 14 in Pakistan, three in Nigeria). The use of oral polio vaccine (OPV) has been the key to success in the eradication effort. However, paradoxically, moving towards global polio eradication, the burden caused by vaccine-derived polioviruses (VDPVs) becomes increasingly important. In this paper circulation of both wild virus and VDPVs is reviewed and implications for the polio eradication endgame are discussed. Between April and May 2016 OPV2 cessation has been implemented globally, in a coordinated switch from trivalent OPV to bivalent OPV. In order to decrease the risk for cVDPV2 re-emergence inactivated polio vaccine (IPV) has been introduced in the routine vaccine schedule of all countries. The likelihood of re-emergence of cVDPVs should markedly decrease with time after OPV cessation, but silent circulation of polioviruses cannot be ruled out even a long time after cessation. For this reason, immunity levels against polioviruses should be kept as high as possible in the population by the use of IPV, and both clinical and environmental surveillance should be maintained at a high level.

  20. Modeling options to manage type 1 wild poliovirus imported into Israel in 2013.

    Science.gov (United States)

    Kalkowska, Dominika A; Duintjer Tebbens, Radboud J; Grotto, Itamar; Shulman, Lester M; Anis, Emilia; Wassilak, Steven G F; Pallansch, Mark A; Thompson, Kimberly M

    2015-06-01

    After 25 years without poliomyelitis cases caused by circulating wild poliovirus (WPV) in Israel, sewage sampling detected WPV type 1 (WPV1) in April 2013, despite high vaccination coverage with only inactivated poliovirus vaccine (IPV) since 2005. We used a differential equation-based model to simulate the dynamics of poliovirus transmission and population immunity in Israel due to past exposure to WPV and use of oral poliovirus vaccine (OPV) in addition to IPV. We explored the influences of various immunization options to stop imported WPV1 circulation in Israel. We successfully modeled the potential for WPVs to circulate without detected cases in Israel. Maintaining a sequential IPV/OPV schedule instead of switching to an IPV-only schedule in 2005 would have kept population immunity high enough in Israel to prevent WPV1 circulation. The Israeli response to WPV1 detection prevented paralytic cases; a more rapid response might have interrupted transmission more quickly. IPV-based protection alone might not provide sufficient population immunity to prevent poliovirus transmission after an importation. As countries transition to IPV in immunization schedules, they may need to actively manage population immunity and consider continued use of OPV, to avoid the potential circulation of imported live polioviruses before globally coordinated cessation of OPV use. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  1. Contribution of Environmental Surveillance Toward Interruption of Poliovirus Transmission in Nigeria, 2012-2015.

    Science.gov (United States)

    Johnson Muluh, Ticha; Hamisu, Abdullahi Walla; Craig, Kehinde; Mkanda, Pascal; Andrew, Etsano; Adeniji, Johnson; Akande, Adefunke; Musa, Audu; Ayodeji, Isiaka; Nicksy, Gumede; Banda, Richard; Tegegne, Sisay G; Nsubuga, Peter; Oyetunji, Ajiboye; Diop, Ousmane; Vaz, Rui G; Muhammad, Ado J G

    2016-05-01

    Cases of paralysis caused by poliovirus have decreased by >99% since the 1988 World Health Assembly's resolution to eradicate polio. The World Health Organization identified environmental surveillance (ES) of poliovirus in the poliomyelitis eradication strategic plan as an activity that can complement acute flaccid paralysis (AFP) surveillance. This article summarizes key public health interventions that followed the isolation of polioviruses from ES between 2012 and 2015. The grap method was used to collect 1.75 L of raw flowing sewage every 2-4 weeks. Once collected, samples were shipped at 4 °C to a polio laboratory for concentration. ES data were then used to guide program implementation. From 2012 to 2015, ES reported 97 circulating vaccine-derived polioviruses (cVDPV2) and 14 wild polioviruses. In 2014 alone, 54 cVDPV type 2 cases and 1 WPV type 1 case were reported. In Sokoto State, 58 cases of AFP were found from a search of 9426 households. A total of 2 252 059 inactivated polio vaccine and 2 460 124 oral polio vaccine doses were administered to children aged poliovirus transmission in Nigeria. © 2016 World Health Organization; licensee Oxford Journals.

  2. Can post-eradication laboratory containment of wild polioviruses be achieved?

    Science.gov (United States)

    Dowdle, Walter R.; Gary, Howard E.; Sanders, Raymond; van Loon, Anton M.

    2002-01-01

    The purpose of containment is to prevent reintroduction of wild polioviruses from laboratories into polio-free communities. In order to achieve global commitment to laboratory containment the rationale should be clear and compelling; the biosafety levels should be justified by the risks; and the objectives should be realistic. Absolute containment can never be assured. Questions of intentional or unintentional non-compliance can never be wholly eliminated. Effective laboratory containment is, however, a realistic goal. Prevention of virus transmission through contaminated laboratory materials is addressed by WHO standards for biosafety. The principal challenge is to prevent transmission through unrecognized infectious laboratory workers. Such transmission is possible only if the following conditions occur: infectious and potentially infectious materials carrying wild poliovirus are present in the laboratory concerned; a laboratory operation exposes a worker to poliovirus; a worker is susceptible to an infection that results in the shedding of poliovirus; and the community is susceptible to poliovirus infections. At present it is difficult to envisage the elimination of any of these conditions. However, the risks of the first three can be greatly reduced so as to create a formidable barrier against poliovirus transmission to the community. Final biosafety recommendations must await post-eradication immunization policies adopted by the international community. PMID:12075368

  3. Poliovirus and other enteroviruses in children infected with intestinal parasites in Nigeria.

    Science.gov (United States)

    Adekolujo, Daniel R; Olayinka, Suraj O; Adeniji, Johnson A; Oyeyemi, Oyetunde T; Odaibo, Alexander B

    2015-10-29

    Poliovirus, an enterovirus, still persists in Nigeria despite the global efforts tailored towards its eradication. This study aimed to assess the impacts of poliovirus and other enteroviruses on the susceptibility of individuals to intestinal parasite infections. A cross-sectional study on the prevalence of intestinal parasites was conducted on two-sample stool specimens of 717 Nigerian children (between 1 and 19 years of age) whose poliovirus/other enteroviruses infection status had been determined. The overall prevalence of Sabin poliovirus and other related enteroviruses infections were 6.6% and 13.8%, respectively. The prevalence of Ascaris lumbricoides was significantly higher than that of other intestinal parasites (p parasitic infection (OR = 11.7, CI = 9.2-15.0). While the prevalence of all species of parasites except S. mansoni showed no significant variations in children with Sabin poliovirus (p > 0.05), the prevalence of hookworms and Taenia spp. was significantly higher in children with other enteroviral infections (p parasites is an indication of possible association of the parasites in a more poliovirus-endemic population. A combined intervention approach for the two infections is advocated.

  4. Sabin Vaccine Reversion in the Field: a Comprehensive Analysis of Sabin-Like Poliovirus Isolates in Nigeria

    OpenAIRE

    Famulare, Michael; Chang, Stewart; Iber, Jane; Zhao, Kun; Adeniji, Johnson A.; Bukbuk, David; Baba, Marycelin; Behrend, Matthew; Burns, Cara C.; Oberste, M. Steven

    2015-01-01

    To assess the dynamics of genetic reversion of live poliovirus vaccine in humans, we studied molecular evolution in Sabin-like poliovirus isolates from Nigerian acute flaccid paralysis cases obtained from routine surveillance. We employed a novel modeling approach to infer substitution and recombination rates from whole-genome sequences and information about poliovirus infection dynamics and the individual vaccination history. We confirmed observations from a recent vaccine trial that VP1 sub...

  5. Identification of a consistent pattern of mutations in neurovirulent variants derived from the sabin vaccine strain of poliovirus type 2.

    OpenAIRE

    Equestre, M; Genovese, D; Cavalieri, F; Fiore, L; Santoro, R; Perez Bercoff, R

    1991-01-01

    Complete nucleotide sequencing of the RNAs of two unrelated neurovirulent isolates of Sabin-related poliovirus type 2 revealed that two nucleotides and one amino acid (amino acid 143 in the major capsid protein VP1) consistently departed from the sequences of the nonneurovirulent poliovirus type 2 712 and Sabin vaccine strains. This pattern of mutation appeared to be a feature common to all neurovirulent variants of poliovirus type 2.

  6. Global Polio Eradication Initiative (GPEI): Future Perspectives and Need for a New Generation of Inactivated Poliovirus Vaccine

    OpenAIRE

    VASHISHTHA, Vipin; NAVEEN, Thacker

    2010-01-01

    More than two decade-old Global Polio Eradication Initiative (GPEI) has finally tasted success and wild poliovirus is now on the verge of eradication. The pre-eradication era was full of challenges and a great learning experience for all those involved with this tedious process. Many new phenomena emerged and new information about poliovirus learned during this campaign. Many new developments such as vaccine-derived polioviruses (VDPVs) were not anticipated and resulted in serious thinking re...

  7. In vivo interactions between the proteins of infectious bursal disease virus: capsid protein VP3 interacts with the RNA dependent polymerase VP1

    NARCIS (Netherlands)

    Tacken, M.G.J.; Rottier, P.J.M.; Gielkens, A.L.J.; Peeters, B.P.H.

    2000-01-01

    Little is known about the intermolecular interactions between the viral proteins of infectious bursal disease virus (IBDV). By using the yeast two-hybrid system, which allows the detection of protein-protein interactions in vivo, all possible interactions were tested by fusing the viral proteins to

  8. Interactions in vivo between the proteins of infectious bursal disease virus: capsid protein VP3 interacts with the RNA-dependent polymerase, VP1

    NARCIS (Netherlands)

    Tacken, M.G.J.; Rottier, P.J.M.; Gielkens, A.L.J.; Peeters, B.P.H.

    2000-01-01

    Little is known about the intermolecular interactions between the viral proteins of infectious bursal disease virus (IBDV). By using the yeast two-hybrid system, which allows the detection of protein-protein interactions in vivo, all possible interactions were tested by fusing the viral proteins to

  9. The Epstein-Barr virus BILF1 gene encodes a G protein-coupled receptor that inhibits phosphorylation of RNA-dependent protein kinase

    NARCIS (Netherlands)

    Beisser, P.S.; Verzijl, D.; Gruijthuijsen, Y.K.; Beuken, E.V.; Smit, M.J.; Leurs, R.; Bruggeman, C.A.; Vink, C.

    2005-01-01

    Epstein-Barr vires (EBV) infection is associated with many lymphoproliferative diseases, such as infectious mononucleosis and Burkitt's lymphoma. Consequently, EBV is one of the most extensively studied herpesvirases. Surprisingly, a putative G protein-coupled receptor (GPCR) gene of EBV, BILF1, has

  10. Poliovirus Studies during the Endgame of the Polio Eradication Program.

    Science.gov (United States)

    Arita, Minetaro

    2017-01-24

    Since the beginning of Global Polio Eradication Initiative in 1988, poliomyelitis cases caused by wild poliovirus (PV) have been drastically reduced, with only 74 cases reported in 2 endemic countries in 2015. The current limited PV transmission suggests that we are in the endgame of the polio eradication program. However, specific challenges have emerged in the endgame, including tight budget, switching of the vaccines, and changes in biorisk management of PV. To overcome these challenges, several PV studies have been implemented in the eradication program. Some of the responses to the emerging challenges in the polio endgame might be valuable in other infectious diseases eradication programs. Here, I will review challenges that confront the polio eradication program and current research to address these challenges.

  11. Poliovirus excretion among persons with primary immune deficiency disorders: summary of a seven-country study series.

    Science.gov (United States)

    Li, Li; Ivanova, Olga; Driss, Nadia; Tiongco-Recto, Marysia; da Silva, Rajiva; Shahmahmoodi, Shohreh; Sazzad, Hossain M S; Mach, Ondrej; Kahn, Anna-Lea; Sutter, Roland W

    2014-11-01

    Persons with primary immune deficiency disorders (PID), especially those disorders affecting the B-cell system, are at substantially increased risk of paralytic poliomyelitis and can excrete poliovirus chronically. However, the risk of prolonged or chronic excretion is not well characterized in developing countries. We present a summary of a country study series on poliovirus excretion among PID cases. Cases with PID from participating institutions were enrolled during the first year and after obtaining informed consent were tested for polioviruses in stool samples. Those cases excreting poliovirus were followed on a monthly basis during the second year until 2 negative stool samples were obtained. A total of 562 cases were enrolled in Bangladesh, China, Iran, Philippines, Russia, Sri Lanka, and Tunisia during 2008-2013. Of these, 17 (3%) shed poliovirus, including 2 cases with immunodeficient vaccine-derived poliovirus. Poliovirus was detected in a single sample from 5/17 (29%) cases. One case excreted for more than 6 months. None of the cases developed paralysis during the study period. Chronic polioviruses excretion remains a rare event even among individuals with PID. Nevertheless, because these individuals were not paralyzed they would have been missed by current surveillance; therefore, surveillance for polioviruses among PID should be established. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  12. Brunenders: a partially attenuated historic poliovirus type I vaccine strain.

    Science.gov (United States)

    Sanders, Barbara P; Liu, Ying; Brandjes, Alies; van Hoek, Vladimir; de Los Rios Oakes, Isabel; Lewis, John; Wimmer, Eckard; Custers, Jerome H H V; Schuitemaker, Hanneke; Cello, Jeronimo; Edo-Matas, Diana

    2015-09-01

    Brunenders, a type I poliovirus (PV) strain, was developed in 1952 by J. F. Enders and colleagues through serial in vitro passaging of the parental Brunhilde strain, and was reported to display partial neuroattenuation in monkeys. This phenotype of attenuation encouraged two vaccine manufacturers to adopt Brunenders as the type I component for their inactivated poliovirus vaccines (IPVs) in the 1950s, although today no licensed IPV vaccine contains Brunenders. Here we confirmed, in a transgenic mouse model, the report of Enders on the reduced neurovirulence of Brunenders. Although dramatically neuroattenuated relative to WT PV strains, Brunenders remains more virulent than the attenuated oral vaccine strain, Sabin 1. Importantly, the neuroattenuation of Brunenders does not affect in vitro growth kinetics and in vitro antigenicity, which were similar to those of Mahoney, the conventional type I IPV vaccine strain. We showed, by full nucleotide sequencing, that Brunhilde and Brunenders differ at 31 nucleotides, eight of which lead to amino acid changes, all located in the capsid. Upon exchanging the Brunenders capsid sequence with that of the Mahoney capsid, WT neurovirulence was regained in vivo, suggesting a role for the capsid mutations in Brunenders attenuation. To date, as polio eradication draws closer, the switch to using attenuated strains for IPV is actively being pursued. Brunenders preceded this novel strategy as a partially attenuated IPV strain, accompanied by decades of successful use in the field. Providing data on the attenuation of Brunenders may be of value in the further construction of attenuated PV strains to support the grand pursuit of the global eradication of poliomyelitis.

  13. Nonhomologous Recombination between Defective Poliovirus and Coxsackievirus Genomes Suggests a New Model of Genetic Plasticity for Picornaviruses

    Science.gov (United States)

    Holmblat, Barbara; Jégouic, Sophie; Muslin, Claire; Blondel, Bruno; Joffret, Marie-Line

    2014-01-01

    ABSTRACT Most of the circulating vaccine-derived polioviruses (cVDPVs) implicated in poliomyelitis outbreaks in Madagascar have been shown to be recombinants between the type 2 poliovirus (PV) strain of the oral polio vaccine (Sabin 2) and another species C human enterovirus (HEV-C), such as type 17 coxsackie A virus (CA17) in particular. We studied intertypic genetic exchanges between PV and non-PV HEV-C by developing a recombination model, making it possible to rescue defective type 2 PV RNA genomes with a short deletion at the 3′ end by the cotransfection of cells with defective or infectious CA17 RNAs. We isolated over 200 different PV/CA17 recombinants, using murine cells expressing the human PV receptor (PVR) and selecting viruses with PV capsids. We found some homologous (H) recombinants and, mostly, nonhomologous (NH) recombinants presenting duplications of parental sequences preferentially located in the regions encoding proteins 2A, 2B, and 3A. Short duplications appeared to be stable, whereas longer duplications were excised during passaging in cultured cells or after multiplication in PVR-transgenic mice, generating H recombinants with diverse sites of recombination. This suggests that NH recombination events may be a transient, intermediate step in the generation and selection of the fittest H recombinants. In addition to the classical copy-choice mechanism of recombination thought to generate mostly H recombinants, there may also be a modular mechanism of recombination, involving NH recombinant precursors, shaping the genomes of recombinant enteroviruses and other picornaviruses. PMID:25096874

  14. Recombination between Poliovirus and Coxsackie A Viruses of Species C: A Model of Viral Genetic Plasticity and Emergence

    Directory of Open Access Journals (Sweden)

    Francis Delpeyroux

    2011-08-01

    Full Text Available Genetic recombination in RNA viruses was discovered many years ago for poliovirus (PV, an enterovirus of the Picornaviridae family, and studied using PV or other picornaviruses as models. Recently, recombination was shown to be a general phenomenon between different types of enteroviruses of the same species. In particular, the interest for this mechanism of genetic plasticity was renewed with the emergence of pathogenic recombinant circulating vaccine-derived polioviruses (cVDPVs, which were implicated in poliomyelitis outbreaks in several regions of the world with insufficient vaccination coverage. Most of these cVDPVs had mosaic genomes constituted of mutated poliovaccine capsid sequences and part or all of the non-structural sequences from other human enteroviruses of species C (HEV-C, in particular coxsackie A viruses. A study in Madagascar showed that recombinant cVDPVs had been co-circulating in a small population of children with many different HEV-C types. This viral ecosystem showed a surprising and extensive biodiversity associated to several types and recombinant genotypes, indicating that intertypic genetic recombination was not only a mechanism of evolution for HEV-C, but an usual mode of genetic plasticity shaping viral diversity. Results suggested that recombination may be, in conjunction with mutations, implicated in the phenotypic diversity of enterovirus strains and in the emergence of new pathogenic strains. Nevertheless, little is known about the rules and mechanisms which govern genetic exchanges between HEV-C types, as well as about the importance of intertypic recombination in generating phenotypic variation. This review summarizes our current knowledge of the mechanisms of evolution of PV, in particular recombination events leading to the emergence of recombinant cVDPVs.

  15. Recombination between poliovirus and coxsackie A viruses of species C: a model of viral genetic plasticity and emergence.

    Science.gov (United States)

    Combelas, Nicolas; Holmblat, Barbara; Joffret, Marie-Line; Colbère-Garapin, Florence; Delpeyroux, Francis

    2011-08-01

    Genetic recombination in RNA viruses was discovered many years ago for poliovirus (PV), an enterovirus of the Picornaviridae family, and studied using PV or other picornaviruses as models. Recently, recombination was shown to be a general phenomenon between different types of enteroviruses of the same species. In particular, the interest for this mechanism of genetic plasticity was renewed with the emergence of pathogenic recombinant circulating vaccine-derived polioviruses (cVDPVs), which were implicated in poliomyelitis outbreaks in several regions of the world with insufficient vaccination coverage. Most of these cVDPVs had mosaic genomes constituted of mutated poliovaccine capsid sequences and part or all of the non-structural sequences from other human enteroviruses of species C (HEV-C), in particular coxsackie A viruses. A study in Madagascar showed that recombinant cVDPVs had been co-circulating in a small population of children with many different HEV-C types. This viral ecosystem showed a surprising and extensive biodiversity associated to several types and recombinant genotypes, indicating that intertypic genetic recombination was not only a mechanism of evolution for HEV-C, but an usual mode of genetic plasticity shaping viral diversity. Results suggested that recombination may be, in conjunction with mutations, implicated in the phenotypic diversity of enterovirus strains and in the emergence of new pathogenic strains. Nevertheless, little is known about the rules and mechanisms which govern genetic exchanges between HEV-C types, as well as about the importance of intertypic recombination in generating phenotypic variation. This review summarizes our current knowledge of the mechanisms of evolution of PV, in particular recombination events leading to the emergence of recombinant cVDPVs.

  16. Seroprevalence of poliovirus antibodies in the Kansas City metropolitan area, 2012–2013

    Science.gov (United States)

    Wallace, Gregory S.; Pahud, Barbara A.; Weldon, William C.; Curns, Aaron T.; Oberste, M. Steven; Harrison, Christopher J.

    2017-01-01

    ABSTRACT No indigenous cases of poliomyelitis have occurred in the US since 1979; however the risk of importation persists until global eradication is achieved. The seropositivity rate for different age cohorts with exposures to different poliovirus vaccine types and wild virus in the US are not presently known. A convenience sample was conducted in the Kansas City metropolitan area during 2012–2103 with approximately 100 participants enrolled for each of 5 age cohorts categorized based on vaccine policy changes over time in the US. Immunization records for poliovirus vaccination were required for participants poliovirus serotypes. Seroprevalence was evaluated by demographics as well as between polio serotypes. The overall seroprevalence to poliovirus was 90.7%, 94.4%, and 83.3%, for types 1, 2, and 3, respectively. Seroprevalence was high (88.6%–96.2%) for all 3 types of poliovirus for the 6–10 y old age group that was likely to have received a complete schedule of IPV-only vaccination. Children 2–3 y of age, who have not yet completed their full IPV series, had lower seroprevalence compared with all older age groups for types 1 and 2 (p-value poliovirus in the population surveyed. Seroprevalence for subjects aged 2–3 y was lower than all other age groups for serotypes 1 and 2 highlighting the importance of completing the recommended poliovirus vaccine series with a booster dose at age 4–6 y. PMID:28059613

  17. World Health Organization Guidelines for Containment of Poliovirus Following Type-Specific Polio Eradication - Worldwide, 2015.

    Science.gov (United States)

    Previsani, Nicoletta; Tangermann, Rudolph H; Tallis, Graham; Jafari, Hamid S

    2015-08-28

    In 1988, the World Health Assembly of the World Health Organization (WHO) resolved to eradicate polio worldwide. Among the three wild poliovirus (WPV) types (type 1, type 2, and type 3), WPV type 2 (WPV2) has been eliminated in the wild since 1999, and WPV type 3 (WPV3) has not been reported since 2012. In 2015, only Afghanistan and Pakistan have reported WPV transmission. On May 25, 2015, all WHO Member States endorsed World Health Assembly resolution 68.3 on full implementation of the Polio Eradication and Endgame Strategic Plan 2013-2018 (the Endgame Plan), and with it, the third Global Action Plan to minimize poliovirus facility-associated risk (GAPIII). All WHO Member States have committed to implementing appropriate containment of WPV2 in essential laboratory and vaccine production facilities* by the end of 2015 and of type 2 oral poliovirus vaccine (OPV2) within 3 months of global withdrawal of OPV2, which is planned for April 2016. This report summarizes critical steps for essential laboratory and vaccine production facilities that intend to retain materials confirmed to contain or potentially containing type-specific WPV, vaccine-derived poliovirus (VDPV), or OPV/Sabin viruses, and steps for nonessential facilities† that process specimens that contain or might contain polioviruses. National authorities will need to certify that the essential facilities they host meet the containment requirements described in GAPIII. After certification of WPV eradication, the use of all OPV will cease; final containment of all polioviruses after polio eradication and OPV cessation will minimize the risk for reintroduction of poliovirus into a polio-free world.

  18. Identification of special fragments containing the 5' end of polivirus RNA after ribonuclease III digestion

    Energy Technology Data Exchange (ETDEWEB)

    Harris, T.J.R.; Dunn, J.J.; Wimmer, E.

    1978-11-01

    The small protein (VPg) covalently linked to the 5' end of the poliovirus Type 1 (PV-1) RNA has been labeled in vitro with /sup 125/I usingthe Bolton and Hunter reagent. The RNA is not degraded under the conditions used and nearly all the label enters VPg and not the polynucleotide chain. When this /sup 125/I-labeled RNA is cleaved with RNase III at low monovalent salt concentrations, one major /sup 125/I-labeled fragment, approximately 100 nucleotides long, is produced. The corresponding fragment from similar digests of /sup 32/P-labeled RNA has also been identified. The /sup 32/P-labeled fragment changes electrophoretic mobility after protease treatment indicating that it contains VPg. Furthermore, the RNase T1 oligonucleotide known to be at the 5' terminus of poliovirus RNA is found in T1 digests of the purified fragment. These results confirm that the fragment is derived from the 5' end of the RNA. This fragment will be useful in studies concerning the initiation of protein synthesis during poliovirus infection.

  19. Synthetic virus seeds for improved vaccine safety: Genetic reconstruction of poliovirus seeds for a PER.C6 cell based inactivated poliovirus vaccine.

    Science.gov (United States)

    Sanders, Barbara P; Edo-Matas, Diana; Papic, Natasa; Schuitemaker, Hanneke; Custers, Jerome H H V

    2015-10-13

    Safety of vaccines can be compromised by contamination with adventitious agents. One potential source of adventitious agents is a vaccine seed, typically derived from historic clinical isolates with poorly defined origins. Here we generated synthetic poliovirus seeds derived from chemically synthesized DNA plasmids encoding the sequence of wild-type poliovirus strains used in marketed inactivated poliovirus vaccines. The synthetic strains were phenotypically identical to wild-type polioviruses as shown by equivalent infectious titers in culture supernatant and antigenic content, even when infection cultures are scaled up to 10-25L bioreactors. Moreover, the synthetic seeds were genetically stable upon extended passaging on the PER.C6 cell culture platform. Use of synthetic seeds produced on the serum-free PER.C6 cell platform ensures a perfectly documented seed history and maximum control over starting materials. It provides an opportunity to maximize vaccine safety which increases the prospect of a vaccine end product that is free from adventitious agents. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. Genetic relationships and epidemiological links between wild type 1 poliovirus isolates in Pakistan and Afghanistan

    Directory of Open Access Journals (Sweden)

    Angez Mehar

    2012-02-01

    Full Text Available Abstract Background/Aim Efforts have been made to eliminate wild poliovirus transmission since 1988 when the World Health Organization began its global eradication campaign. Since then, the incidence of polio has decreased significantly. However, serotype 1 and serotype 3 still circulate endemically in Pakistan and Afghanistan. Both countries constitute a single epidemiologic block representing one of the three remaining major global reservoirs of poliovirus transmission. In this study we used genetic sequence data to investigate transmission links among viruses from diverse locations during 2005-2007. Methods In order to find the origins and routes of wild type 1 poliovirus circulation, polioviruses were isolated from faecal samples of Acute Flaccid Paralysis (AFP patients. We used viral cultures, two intratypic differentiation methods PCR, ELISA to characterize as vaccine or wild type 1 and nucleic acid sequencing of entire VP1 region of poliovirus genome to determine the genetic relatedness. Results One hundred eleven wild type 1 poliovirus isolates were subjected to nucleotide sequencing for genetic variation study. Considering the 15% divergence of the sequences from Sabin 1, Phylogenetic analysis by MEGA software revealed that active inter and intra country transmission of many genetically distinct strains of wild poliovirus type 1 belonged to genotype SOAS which is indigenous in this region. By grouping wild type 1 polioviruses according to nucleotide sequence homology, three distinct clusters A, B and C were obtained with multiple chains of transmission together with some silent circulations represented by orphan lineages. Conclusion Our results emphasize that there was a persistent transmission of wild type1 polioviruses in Pakistan and Afghanistan during 2005-2007. The epidemiologic information provided by the sequence data can contribute to the formulation of better strategies for poliomyelitis control to those critical areas

  1. Pathogenic Events in a Nonhuman Primate Model of Oral Poliovirus Infection Leading to Paralytic Poliomyelitis.

    Science.gov (United States)

    Shen, Ling; Chen, Crystal Y; Huang, Dan; Wang, Richard; Zhang, Meihong; Qian, Lixia; Zhu, Yanfen; Zhang, Alvin Zhuoran; Yang, Enzhuo; Qaqish, Arwa; Chumakov, Konstantin; Kouiavskaia, Diana; Vignuzzi, Marco; Nathanson, Neal; Macadam, Andrew J; Andino, Raul; Kew, Olen; Xu, Junfa; Chen, Zheng W

    2017-07-15

    Despite a great deal of prior research, the early pathogenic events in natural oral poliovirus infection remain poorly defined. To establish a model for study, we infected 39 macaques by feeding them single high doses of the virulent Mahoney strain of wild type 1 poliovirus. Doses ranging from 10 7 to 10 9 50% tissue culture infective doses (TCID 50 ) consistently infected all the animals, and many monkeys receiving 10 8 or 10 9 TCID 50 developed paralysis. There was no apparent difference in the susceptibilities of the three macaque species (rhesus, cynomolgus, and bonnet) used. Virus excretion in stool and nasopharynges was consistently observed, with occasional viremia, and virus was isolated from tonsils, gut mucosa, and draining lymph nodes. Viral replication proteins were detected in both epithelial and lymphoid cell populations expressing CD155 in the tonsil and intestine, as well as in spinal cord neurons. Necrosis was observed in these three cell types, and viral replication in the tonsil/gut was associated with histopathologic destruction and inflammation. The sustained response of neutralizing antibody correlated temporally with resolution of viremia and termination of virus shedding in oropharynges and feces. For the first time, this model demonstrates that early in the infectious process, poliovirus replication occurs in both epithelial cells (explaining virus shedding in the gastrointestinal tract) and lymphoid/monocytic cells in tonsils and Peyer's patches (explaining viremia), extending previous studies of poliovirus pathogenesis in humans. Because the model recapitulates human poliovirus infection and poliomyelitis, it can be used to study polio pathogenesis and to assess the efficacy of candidate antiviral drugs and new vaccines. IMPORTANCE Early pathogenic events of poliovirus infection remain largely undefined, and there is a lack of animal models mimicking natural oral human infection leading to paralytic poliomyelitis. All 39 macaques fed with

  2. Bacterial RNA induces myocyte cellular dysfunction through the activation of PKR

    OpenAIRE

    Bleiblo, Farag; Michael, Paul; Brabant, Danielle; Ramana, Chilakamarti V.; Tai, TC; Saleh, Mazen; Parrillo, Joseph E.; Kumar, Anand; Kumar, Aseem

    2012-01-01

    Severe sepsis and the ensuing septic shock are serious life threatening conditions. These diseases are triggered by the host's over exuberant systemic response to the infecting pathogen. Several surveillance mechanisms have evolved to discriminate self from foreign RNA and accordingly trigger effective cellular responses to target the pathogenic threats. The RNA-dependent protein kinase (PKR) is a key component of the cytoplasmic RNA sensors involved in the recognition of viral double-strande...

  3. Radiation sensitivity of poliovirus, a model for norovirus, inoculated in oyster (Crassostrea gigas) and culture broth under different conditions

    Energy Technology Data Exchange (ETDEWEB)

    Jung, Pil-Mun [Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup 580-185 (Korea, Republic of); Park, Jae Seok [Korea Food and Drug Administration, Seoul 122-704 (Korea, Republic of); Park, Jin-Gyu; Park, Jae-Nam; Han, In-Jun; Song, Beom-Seok; Choi, Jong-il; Kim, Jae-Hun; Byun, Myung-Woo [Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup 580-185 (Korea, Republic of); Baek, Min [Atomic Energy Policy Division, Ministry of Education, Science and Technology, Gwacheon 427-715 (Korea, Republic of); Chung, Young-Jin [Department of Food and Nutrition, Chungnam National University, Daejeon 305-764 (Korea, Republic of); Lee, Ju-Woon [Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup 580-185 (Korea, Republic of)], E-mail: sjwlee@kaeri.re.kr

    2009-07-15

    Poliovirus is a recognized surrogate for norovirus, pathogen in water and food, due to the structural and genetic similarity. Although radiation sensitivity of poliovirus in water or media had been reported, there has been no research in food model such as shellfish. In this study, oyster (Crassostrea gigas) was incubated in artificial seawater contaminated with poliovirus, and thus radiation sensitivity of poliovirus was determined in inoculated oyster. The effects of ionizing radiation on the sensitivity of poliovirus were also evaluated under different conditions such as pH (4-7) and salt concentration (1-15%) in culture broth, and temperature during irradiation. The D{sub 10} value of poliovirus in PBS buffer, virus culture broth and oyster was determined to 0.46, 2.84 and 2.94 kGy, respectively. The initial plaque forming unit (PFU) of poliovirus in culture broth was slightly decreased as the decrease of pH and the increase of salt concentration, but radiation sensitivity was not affected by pH and salt contents. However, radiation resistance of poliovirus was increased at frozen state. These results provide the basic information for the inactivation of pathogenic virus in foods by using irradiation.

  4. Radiation sensitivity of poliovirus, a model for norovirus, inoculated in oyster (Crassostrea gigas) and culture broth under different conditions

    International Nuclear Information System (INIS)

    Jung, Pil-Mun; Park, Jae Seok; Park, Jin-Gyu; Park, Jae-Nam; Han, In-Jun; Song, Beom-Seok; Choi, Jong-il; Kim, Jae-Hun; Byun, Myung-Woo; Baek, Min; Chung, Young-Jin; Lee, Ju-Woon

    2009-01-01

    Poliovirus is a recognized surrogate for norovirus, pathogen in water and food, due to the structural and genetic similarity. Although radiation sensitivity of poliovirus in water or media had been reported, there has been no research in food model such as shellfish. In this study, oyster (Crassostrea gigas) was incubated in artificial seawater contaminated with poliovirus, and thus radiation sensitivity of poliovirus was determined in inoculated oyster. The effects of ionizing radiation on the sensitivity of poliovirus were also evaluated under different conditions such as pH (4-7) and salt concentration (1-15%) in culture broth, and temperature during irradiation. The D 10 value of poliovirus in PBS buffer, virus culture broth and oyster was determined to 0.46, 2.84 and 2.94 kGy, respectively. The initial plaque forming unit (PFU) of poliovirus in culture broth was slightly decreased as the decrease of pH and the increase of salt concentration, but radiation sensitivity was not affected by pH and salt contents. However, radiation resistance of poliovirus was increased at frozen state. These results provide the basic information for the inactivation of pathogenic virus in foods by using irradiation.

  5. Simple quantitative protein A micro-immunoprecipitation method; assay of antibodies to the N and H antigens of poliovirus

    Energy Technology Data Exchange (ETDEWEB)

    Vrijsen, R.; Rombaut, B.; Boeye, A. (Brussels Univ. (Belgium))

    1983-04-29

    Staphylococcus aureus (Cowan strain I) was used to absorb immune complexes from antiserum to poliovirus to which labeled N or H poliovirus antigens had been added, and the radioactivity in the pelleted organisms and in the supernatant was measured. Excellent agreement was obtained between values calculated separately from the pellet and supernatant readings, validating the use of supernatant measurements from a microtitration plate method.

  6. Switch from oral to inactivated poliovirus vaccine in Yogyakarta Province, Indonesia: summary of coverage, immunity, and environmental surveillance.

    Science.gov (United States)

    Wahjuhono, Gendro; Revolusiana; Widhiastuti, Dyah; Sundoro, Julitasari; Mardani, Tri; Ratih, Woro Umi; Sutomo, Retno; Safitri, Ida; Sampurno, Ondri Dwi; Rana, Bardan; Roivainen, Merja; Kahn, Anna-Lea; Mach, Ondrej; Pallansch, Mark A; Sutter, Roland W

    2014-11-01

    Inactivated poliovirus vaccine (IPV) is rarely used in tropical developing countries. To generate additional scientific information, especially on the possible emergence of vaccine-derived polioviruses (VDPVs) in an IPV-only environment, we initiated an IPV introduction project in Yogyakarta, an Indonesian province. In this report, we present the coverage, immunity, and VDPV surveillance results. In Yogyakarta, we established environmental surveillance starting in 2004; and conducted routine immunization coverage and seroprevalence surveys before and after a September 2007 switch from oral poliovirus vaccine (OPV) to IPV, using standard coverage and serosurvey methods. Rates and types of polioviruses found in sewage samples were analyzed, and all poliovirus isolates after the switch were sequenced. Vaccination coverage (>95%) and immunity (approximately 100%) did not change substantially before and after the IPV switch. No VDPVs were detected. Before the switch, 58% of environmental samples contained Sabin poliovirus; starting 6 weeks after the switch, Sabin polioviruses were rarely isolated, and if they were, genetic sequencing suggested recent introductions. This project demonstrated that under almost ideal conditions (good hygiene, maintenance of universally high IPV coverage, and corresponding high immunity against polioviruses), no emergence and circulation of VDPV could be detected in a tropical developing country setting. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  7. Radiation sensitivity of poliovirus, a model for norovirus, inoculated in oyster ( Crassostrea gigas) and culture broth under different conditions

    Science.gov (United States)

    Jung, Pil-Mun; Park, Jae Seok; Park, Jin-Gyu; Park, Jae-Nam; Han, In-Jun; Song, Beom-Seok; Choi, Jong-il; Kim, Jae-Hun; Byun, Myung-Woo; Baek, Min; Chung, Young-Jin; Lee, Ju-Woon

    2009-07-01

    Poliovirus is a recognized surrogate for norovirus, pathogen in water and food, due to the structural and genetic similarity. Although radiation sensitivity of poliovirus in water or media had been reported, there has been no research in food model such as shellfish. In this study, oyster ( Crassostrea gigas) was incubated in artificial seawater contaminated with poliovirus, and thus radiation sensitivity of poliovirus was determined in inoculated oyster. The effects of ionizing radiation on the sensitivity of poliovirus were also evaluated under different conditions such as pH (4-7) and salt concentration (1-15%) in culture broth, and temperature during irradiation. The D10 value of poliovirus in PBS buffer, virus culture broth and oyster was determined to 0.46, 2.84 and 2.94 kGy, respectively. The initial plaque forming unit (PFU) of poliovirus in culture broth was slightly decreased as the decrease of pH and the increase of salt concentration, but radiation sensitivity was not affected by pH and salt contents. However, radiation resistance of poliovirus was increased at frozen state. These results provide the basic information for the inactivation of pathogenic virus in foods by using irradiation.

  8. Molecular Properties of Poliovirus Isolates: Nucleotide Sequence Analysis, Typing by PCR and Real-Time RT-PCR.

    Science.gov (United States)

    Burns, Cara C; Kilpatrick, David R; Iber, Jane C; Chen, Qi; Kew, Olen M

    2016-01-01

    Virologic surveillance is essential to the success of the World Health Organization initiative to eradicate poliomyelitis. Molecular methods have been used to detect polioviruses in tissue culture isolates derived from stool samples obtained through surveillance for acute flaccid paralysis. This chapter describes the use of realtime PCR assays to identify and serotype polioviruses. In particular, a degenerate, inosine-containing, panpoliovirus (panPV) PCR primer set is used to distinguish polioviruses from NPEVs. The high degree of nucleotide sequence diversity among polioviruses presents a challenge to the systematic design of nucleic acid-based reagents. To accommodate the wide variability and rapid evolution of poliovirus genomes, degenerate codon positions on the template were matched to mixed-base or deoxyinosine residues on both the primers and the TaqMan™ probes. Additional assays distinguish between Sabin vaccine strains and non-Sabin strains. This chapter also describes the use of generic poliovirus specific primers, along with degenerate and inosine-containing primers, for routine VP1 sequencing of poliovirus isolates. These primers, along with nondegenerate serotype-specific Sabin primers, can also be used to sequence individual polioviruses in mixtures.

  9. Rioolwateronderzoek op locaties met een verhoogde kans op import van wild poliovirus ; een pilot in drie asielzoekerscentra

    NARCIS (Netherlands)

    van der Avoort HGAM; Bijen M; Ras A; Koopmans MPG; van Loon AM; LIO; LIS

    1997-01-01

    Watermonsters, verkregen uit het riool op of nabij een drietal asielzoekerscentra werden onderzocht op de aanwezigheid van wild poliovirus, als experiment voor de surveillance van import van wild poliovirus in Nederland. In geen van de 73 monsters, die waren verkregen in een periode van vier

  10. Development and introduction of inactivated poliovirus vaccines derived from Sabin strains in Japan.

    Science.gov (United States)

    Shimizu, Hiroyuki

    2016-04-07

    During the endgame of global polio eradication, the universal introduction of inactivated poliovirus vaccines is urgently required to reduce the risk of vaccine-associated paralytic poliomyelitis and polio outbreaks due to wild and vaccine-derived polioviruses. In particular, the development of inactivated poliovirus vaccines (IPVs) derived from the attenuated Sabin strains is considered to be a highly favorable option for the production of novel IPV that reduce the risk of facility-acquired transmission of poliovirus to the communities. In Japan, Sabin-derived IPVs (sIPVs) have been developed and introduced for routine immunization in November 2012. They are the first licensed sIPVs in the world. Consequently, trivalent oral poliovirus vaccine was used for polio control in Japan for more than half a century but has now been removed from the list of vaccines licensed for routine immunization. This paper reviews the development, introduction, characterization, and global status of IPV derived from attenuated Sabin strains. Copyright © 2014 Elsevier Ltd. All rights reserved.

  11. Estimated Effect of Inactivated Poliovirus Vaccine Campaigns, Nigeria and Pakistan, January 2014-April 2016.

    Science.gov (United States)

    Shirreff, George; Wadood, Mufti Zubair; Vaz, Rui Gama; Sutter, Roland W; Grassly, Nicholas C

    2017-02-01

    In 2014, inactivated poliovirus vaccine (IPV) campaigns were implemented in Nigeria and Pakistan after clinical trials showed that IPV boosts intestinal immunity in children previously given oral poliovirus vaccine (OPV). We estimated the effect of these campaigns by using surveillance data collected during January 2014-April 2016. In Nigeria, campaigns with IPV and trivalent OPV (tOPV) substantially reduced the incidence of poliomyelitis caused by circulating serotype-2 vaccine-derived poliovirus (incidence rate ratio [IRR] 0.17 for 90 days after vs. 90 days before campaigns, 95% CI 0.04-0.78) and the prevalence of virus in environmental samples (prevalence ratio [PR] 0.16, 95% CI 0.02-1.33). Campaigns with tOPV alone resulted in similar reductions (IRR 0.59, 95% CI 0.18-1.97; PR 0.45, 95% CI 0.21-0.95). In Pakistan, the effect of IPV+tOPV campaigns on wild-type poliovirus was not significant. Results suggest that administration of IPV alongside OPV can decrease poliovirus transmission if high vaccine coverage is achieved.

  12. Public health response to the silent reintroduction of wild poliovirus to Israel, 2013-2014.

    Science.gov (United States)

    Moran-Gilad, J; Kaliner, E; Gdalevich, M; Grotto, I

    2016-12-01

    During 2013/14, Israel witnessed the silent reintroduction and sustained transmission of wild poliovirus type 1 (WPV1) detected through routine environmental surveillance performed on sewage samples. The public health response to silent poliovirus transmission in a population with high inactivated polio vaccine (IPV) coverage poses an emerging challenge towards the 'End Game' of global poliovirus eradication. This paper reviews the risk assessment, risk management and risk communication aspects of this poliovirus incident. Special emphasis is placed on the use of scientific data generated in the risk assessment phase to inform the public health response. Reintroducing a live vaccine in supplemental immunization activities in response to transmission of WPV or vaccine-derived poliovirus should be considered close to the 'End Game' of polio eradication, especially if targeting the population at risk is feasible. Such circumstances require a comprehensive contingency plan that will support the generation of important public health evidence at the risk assessment stage, thereby allowing to tailor the risk management approaches and underpin appropriate risk communication. Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

  13. Estimated Effect of Inactivated Poliovirus Vaccine Campaigns, Nigeria and Pakistan, January 2014–April 2016

    Science.gov (United States)

    Shirreff, George; Wadood, Mufti Zubair; Vaz, Rui Gama; Sutter, Roland W.

    2017-01-01

    In 2014, inactivated poliovirus vaccine (IPV) campaigns were implemented in Nigeria and Pakistan after clinical trials showed that IPV boosts intestinal immunity in children previously given oral poliovirus vaccine (OPV). We estimated the effect of these campaigns by using surveillance data collected during January 2014–April 2016. In Nigeria, campaigns with IPV and trivalent OPV (tOPV) substantially reduced the incidence of poliomyelitis caused by circulating serotype-2 vaccine–derived poliovirus (incidence rate ratio [IRR] 0.17 for 90 days after vs. 90 days before campaigns, 95% CI 0.04–0.78) and the prevalence of virus in environmental samples (prevalence ratio [PR] 0.16, 95% CI 0.02–1.33). Campaigns with tOPV alone resulted in similar reductions (IRR 0.59, 95% CI 0.18–1.97; PR 0.45, 95% CI 0.21–0.95). In Pakistan, the effect of IPV+tOPV campaigns on wild-type poliovirus was not significant. Results suggest that administration of IPV alongside OPV can decrease poliovirus transmission if high vaccine coverage is achieved. PMID:27861118

  14. EFFECT OF TRIIODOTHYRONINE ON CELLS AND ON THEIR RESPONSE TO INFECTION BY POLIOVIRUSES1

    Science.gov (United States)

    Murphy, William H.; Bullis, Cora

    1962-01-01

    Murphy, W. H. (The University of Michigan, Ann Arbor) and Cora Bullis. Effect of triiodothyronine on cells and on their response to infection by polioviruses. J. Bacteriol. 83:641–648. 1962.—An analysis was made of the effect of triiodothyronine (T3) at physiological (1 μg/ml) and maximal subliminal toxic levels (35 μg/ml) on HeLa-S3, HeLa-Gey, Chang-liver, and Maben cells, and on their response to infection by cytopathic and submoderate (noncytopathic) mutants of type 2 poliovirus. Assays of cell response to T3 alone, or in combination with the mutants of poliovirus, were made by conventional monolayer cell culture techniques, by study of the effect of T3 on plating efficiency of cells, and by study of its influence on colonies of cell variants. Cellular response to liminal doses of T3 was characterized by agglutination of cells and thickening of the cell membrane. Compact colonies of Chang-liver and Maben cells were the most sensitive to maximal subliminal amounts of T3. T3 in combination with cytopathic or submoderate (noncytopathic) mutants of poliovirus slightly increased the rate of destruction of cells susceptible to virus, but did not influence yield of virus from cell cultures. T3 at physiological or subliminal concentrations did not induce cytopathic response of cell cultures latently infected by submoderate poliovirus. Images PMID:14477441

  15. Preventing Vaccine-Derived Poliovirus Emergence during the Polio Endgame.

    Directory of Open Access Journals (Sweden)

    Margarita Pons-Salort

    2016-07-01

    Full Text Available Reversion and spread of vaccine-derived poliovirus (VDPV to cause outbreaks of poliomyelitis is a rare outcome resulting from immunisation with the live-attenuated oral poliovirus vaccines (OPVs. Global withdrawal of all three OPV serotypes is therefore a key objective of the polio endgame strategic plan, starting with serotype 2 (OPV2 in April 2016. Supplementary immunisation activities (SIAs with trivalent OPV (tOPV in advance of this date could mitigate the risks of OPV2 withdrawal by increasing serotype-2 immunity, but may also create new serotype-2 VDPV (VDPV2. Here, we examine the risk factors for VDPV2 emergence and implications for the strategy of tOPV SIAs prior to OPV2 withdrawal. We first developed mathematical models of VDPV2 emergence and spread. We found that in settings with low routine immunisation coverage, the implementation of a single SIA increases the risk of VDPV2 emergence. If routine coverage is 20%, at least 3 SIAs are needed to bring that risk close to zero, and if SIA coverage is low or there are persistently "missed" groups, the risk remains high despite the implementation of multiple SIAs. We then analysed data from Nigeria on the 29 VDPV2 emergences that occurred during 2004-2014. Districts reporting the first case of poliomyelitis associated with a VDPV2 emergence were compared to districts with no VDPV2 emergence in the same 6-month period using conditional logistic regression. In agreement with the model results, the odds of VDPV2 emergence decreased with higher routine immunisation coverage (odds ratio 0.67 for a 10% absolute increase in coverage [95% confidence interval 0.55-0.82]. We also found that the probability of a VDPV2 emergence resulting in poliomyelitis in >1 child was significantly higher in districts with low serotype-2 population immunity. Our results support a strategy of focused tOPV SIAs before OPV2 withdrawal in areas at risk of VDPV2 emergence and in sufficient number to raise population

  16. A Specific Hepatic Transfer RNA for Phosphoserine*

    Science.gov (United States)

    Mäenpää, Pekka H.; Bernfield, Merton R.

    1970-01-01

    Radioactive O-phosphoryl-L-serine was detected after alkaline deacylation of rat and rooster liver [3H]seryl-tRNA acylated in vitro with homologous synthetases. Ribonuclease treatment of this tRNA yielded a compound with the properties of phosphoseryl-adenosine. Benzoylated DEAE-cellulose chromatography of seryl-tRNA yielded four distinct peaks, only one of which contained phosphoserine. A unique fraction for phosphoserine was also found on chromatography of nonacylated tRNA. In ribosome binding studies, this fraction responded very slightly with poly(U,C), but not with any of the known serine trinucleotide codons. Substantial incorporation of [3H]-serine into protein from this tRNA species was observed in an aminoacyl-tRNA dependent polysomal system derived from chick oviducts. No phosphoserine was found in Escherichia coli or yeast seryl-tRNA acylated with homologous enzymes, nor in E. coli seryl-tRNA acylated with liver synthetase. In the absence of tRNA, free phosphoserine was not formed in reaction mixtures, which suggests that phosphoseryl-tRNA arises by phosphorylation of the unique seryl-tRNA species. These results demonstrate a discrete tRNASer species in rat and rooster liver containing phosphoserine and suggest that this tRNA is involved in ribosomal polypeptide synthesis. PMID:4943179

  17. Poliovirus surveillance by examining sewage specimens. Quantitative recovery of virus after introduction into sewerage at remote upstream location.

    Science.gov (United States)

    Hovi, T; Stenvik, M; Partanen, H; Kangas, A

    2001-08-01

    In order to assess the feasibility of environmental poliovirus surveillance, known amounts of poliovirus type 1, strain Sabin, were flushed into the sewage network of Helsinki. Grab specimens collected at a remote downstream location and concentrated about a 100-fold revealed infectious poliovirus on four successive days in all three separate experiments. As for concentration, a simple two-phase separation method was found to be at least as useful as a several-fold more resource-demanding polyethylene glycol (PEG) precipitation method. Recovery of the introduced virus was remarkably high (more than 10%). Using the current system, it might be possible to detect poliovirus circulation in a population of 700,000 people by examining a single 400 ml sewage specimen, if 1 out of 10,000 inhabitants were excreting the virus. It is concluded that environmental surveillance is a sensitive approach to monitor silent poliovirus circulation in populations served by a sewage network.

  18. Glutathione is a highly efficient thermostabilizer of poliovirus Sabin strains.

    Science.gov (United States)

    Abdelnabi, Rana; Delang, Leen; Neyts, Johan

    2017-03-07

    Glutathione (GSH) is the most abundant thiol peptide in animal cells and has a critical role in antioxidation. GSH was reported to be essential for stabilization of some enteroviruses, including poliovirus (PV), during viral morphogenesis. Here, we explored the potential use of GSH as a thermostabilizer of oral poliomyelitis vaccine (OPV) formulations. GSH significantly protected the three types of PV from heat-inactivation in a concentration-dependent manner. At a GSH concentration of 20mM, nearly complete protection was observed against heating temperatures up to 53°C for 2min.GSH also markedly protected PV1 from heat-inactivation and this up to 6 h at temperatures of 44°C and 46°C and 3 h at 48°C. The fact that GSH is naturally present at high concentration in the human body makes it an efficient candidate stabilizer for OPV formulations. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. [The role of Sabin inactivated poliovirus vaccine in the final phase of global polio eradication].

    Science.gov (United States)

    Dong, S Z; Zhu, W B

    2016-12-06

    Global polio eradication has entered its final phase, but still faces enormous challenges. The Polio Eradication and Endgame Strategic Plan (2013-2018) set the target for making the world polio-free by 2018. Meanwhile, the World Heath Organization Global Action Plan (GAP Ⅲ) recommended that polioviruses be stored under strict conditions after eradication of the wild poliovirus. At least one dose of inactivated poliovirus vaccine (IPV) would be required for each newborn baby in the world to ensure successful completion of the final strategy and GAP Ⅲ. The Sabin IPV has a high production safety and low production cost, compared with the wild-virus IPV and, therefore, can play an important role in the final stage of global polio eradication.

  20. Transformation and Tumorigenicity Testing of Simian Cell Lines and Evaluation of Poliovirus Replication.

    Directory of Open Access Journals (Sweden)

    Silvia Dotti

    Full Text Available The key role of cell cultures in different scientific fields is worldwide recognized, both as in vitro research models alternative to laboratory animals and substrates for biological production. However, many safety concerns rise from the use of animal/human cell lines that may be tumorigenic, leading to potential adverse contaminations in cell-derived biologicals. In order to evaluate the suitability of 13 different cell lines for Poliovirus vaccine production, safety and quality, in vitro/in vivo tumorigenicity and Poliovirus propagation properties were evaluated. Our results revealed that non-human primate cell lines CYNOM-K1, FRhK-4, 4MBr-5 and 4647 are free of tumorigenic features and represent highly susceptible substrates for attenuated Sabin Poliovirus strains. In particular, FRhK-4 and 4647 cell lines are characterized by a higher in vitro replication, resulting indicated for the use in large-scale production field.

  1. Isolation of sabin-like polioviruses from wastewater in a country using inactivated polio vaccine.

    Science.gov (United States)

    Zurbriggen, Sebastian; Tobler, Kurt; Abril, Carlos; Diedrich, Sabine; Ackermann, Mathias; Pallansch, Mark A; Metzler, Alfred

    2008-09-01

    From 2001 to 2004, Switzerland switched from routine vaccination with oral polio vaccine (OPV) to inactivated polio vaccine (IPV), using both vaccines in the intervening period. Since IPV is less effective at inducing mucosal immunity than OPV, this change might allow imported poliovirus to circulate undetected more easily in an increasingly IPV-immunized population. Environmental monitoring is a recognized tool for identifying polioviruses in a community. To look for evidence of poliovirus circulation following cessation of OPV use, two sewage treatment plants located in the Zurich area were sampled from 2004 to 2006. Following virus isolation using either RD or L20B cells, enteroviruses and polioviruses were identified by reverse transcription-PCR. A total of 20 out of 174 wastewater samples were positive for 62 Sabin-like isolates. One isolate from each poliovirus-positive sample was analyzed in more detail. Sequencing the complete viral protein 1 (VP1) capsid coding region, as well as intratypic differentiation (ITD), identified 3 Sabin type 1, 13 Sabin type 2, and 4 Sabin type 3 strains. One serotype 1 strain showed a discordant result in the ITD. Three-quarters of the strains showed mutations within the 5' untranslated region and VP1, known to be associated with reversion to virulence. Moreover, three strains showed heterotypic recombination (S2/S1 and S3/S2/S3). The low number of synonymous mutations and the partial temperature sensitivity are not consistent with extended circulation of these Sabin virus strains. Nevertheless, the continuous introduction of polioviruses into the community emphasizes the necessity for uninterrupted child vaccination to maintain high herd immunity.

  2. Modulation of gene expression in a human cell line caused by poliovirus, vaccinia virus and interferon

    Directory of Open Access Journals (Sweden)

    Hoddevik Gunnar

    2007-03-01

    Full Text Available Abstract Background The project was initiated to describe the response of a human embryonic fibroblast cell line to the replication of two different viruses, and, more specifically, to look for candidate genes involved in viral defense. For this purpose, the cells were synchronously infected with poliovirus in the absence or presence of interferon-alpha, or with vaccinia virus, a virus that is not inhibited by interferon. By comparing the changes in transcriptosome due to these different challenges, it should be possible to suggest genes that might be involved in defense. Results The viral titers were sufficient to yield productive infection in a majority of the cells. The cells were harvested in triplicate at various time-points, and the transcriptosome compared with mock infected cells using oligo-based, global 35 k microarrays. While there was very limited similarities in the response to the different viruses, a large proportion of the genes up-regulated by interferon-alpha were also up-regulated by poliovirus. Interferon-alpha inhibited poliovirus replication, but there were no signs of any interferons being induced by poliovirus. The observations suggest that the cells do launch an antiviral response to poliovirus in the absence of interferon. Analyses of the data led to a list of candidate antiviral genes. Functional information was limited, or absent, for most of the candidate genes. Conclusion The data are relevant for our understanding of how the cells respond to poliovirus and vaccinia virus infection. More annotations, and more microarray studies with related viruses, are required in order to narrow the list of putative defence-related genes.

  3. A C-terminal, cysteine-rich site in poliovirus 2C(ATPase) is required for morphogenesis.

    Science.gov (United States)

    Wang, Chunling; Ma, Hsin-Chieh; Wimmer, Eckard; Jiang, Ping; Paul, Aniko V

    2014-06-01

    The morphogenesis of viruses belonging to the genus Enterovirus in the family Picornaviridae is still poorly understood despite decades-long investigations. However, we recently provided evidence that 2C(ATPase) gives specificity to poliovirus encapsidation through an interaction with capsid protein VP3. The polypeptide 2C(ATPase) is a highly conserved non-structural protein of enteroviruses with important roles in RNA replication, encapsidation and uncoating. We have identified a site (K279/R280) near the C terminus of the polypeptide that is required for morphogenesis. The aim of the current project was to search for additional functional sites near the C terminus of the 2C(ATPase) polypeptide, with particular interest in those that are required for encapsidation. We selected for analysis a cysteine-rich site of the polypeptide and constructed four mutants in which cysteines or a histidine was changed to an alanine. The RNA transcripts were transfected into HeLa cells yielding two lethal, one temperature-sensitive and one quasi-infectious mutants. All four mutants exhibited normal protein translation in vitro and three of them possessed severe RNA replication defects. The quasi-infectious mutant (C286A) yielded variants with a pseudo-reversion at the original site (A286D), but some also contained one additional mutation: A138V or M293V. The temperature-sensitive mutant (C272A/H273A) exhibited an encapsidation and possibly also an uncoating defect at 37 °C. Variants of this mutant revealed suppressor mutations at three different sites in the 2C(ATPase) polypeptide: A138V, M293V and K295R. We concluded that the cysteine-rich site near the C terminus of 2C(ATPase) is involved in encapsidation, possibly through an interaction with an upstream segment located between boxes A and B of the nucleotide-binding domain. © 2014 The Authors.

  4. Characterization of human monoclonal antibodies that neutralize multiple poliovirus serotypes.

    Science.gov (United States)

    Puligedda, Rama Devudu; Kouiavskaia, Diana; Al-Saleem, Fetweh H; Kattala, Chandana Devi; Nabi, Usman; Yaqoob, Hamid; Bhagavathula, V Sandeep; Sharma, Rashmi; Chumakov, Konstantin; Dessain, Scott K

    2017-10-04

    Following the eradication of wild poliovirus (PV), achieving and maintaining a polio-free status will require eliminating potentially pathogenic PV strains derived from the oral attenuated vaccine. For this purpose, a combination of non-cross-resistant drugs, such as small molecules and neutralizing monoclonal antibodies (mAbs), may be ideal. We previously isolated chimpanzee and human mAbs capable of neutralizing multiple PV types (cross-neutralization). Here, we describe three additional human mAbs that neutralize types 1 and 2 PV and one mAb that neutralizes all three types. Most bind conformational epitopes and have unusually long heavy chain complementarity determining 3 domains (HC CDR3). We assessed the ability of the mAbs to neutralize A12 escape mutant PV strains, and found that the neutralizing activities of the mAbs were disrupted by different amino acid substitutions. Competitive binding studies further suggested that the specific mAb:PV interactions that enable cross-neutralization differ among mAbs and serotypes. All of the cloned mAbs bind PV in the vicinity of the "canyon", a circular depression around the 5-fold axis of symmetry through which PV recognizes its cellular receptor. We were unable to generate escape mutants to two of the mAbs, suggesting that their epitopes are important for the PV life cycle. These data indicate that PV cross-neutralization involves binding to highly conserved structures within the canyon that binds to the cellular receptor. These may be facilitated by the long HC CDR3 domains, which may adopt alternative binding configurations. We propose that the human and chimpanzee mAbs described here could have potential as anti-PV therapeutics. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. [Polyadenylated RNA and mRNA export factors in extrachromosomal nuclear domains of vitellogenic oocytes of the insect Tenebrio molitor].

    Science.gov (United States)

    Bogoliubov, D S; Kiselev, A M; Shabel'nikov, S V; Parfenov, V N

    2012-01-01

    The nucleus ofvitellogenic oocytes of the yellow mealworm, Tenebrio molitor, contains a karyosphere that consists of the condensed chromatin embedded in an extrachromosomal fibrogranular material. Numerous nuclear bodies located freely in the nucleoplasm are also observed. Amongst these bodies, counterparts of nuclear speckles (= interchromatin granule clusters, IGCs) can be identified by the presence of the marker protein SC35. Microinjections of fluorescently tagged methyloligoribonucleotide probes 2'-O-Me(U)22, complementary to poly(A) tails of RNAs, revealed poly(A)+ RNA in the vast majority of IGCs. We found that all T. molitor oocyte IGCs contain heterogeneous ribonucleoprotein (hnRNP) core protein Al that localizes to IGCs in an RNA-dependent manner. The extrachromosomal material of the karyosphere and a part of nucleoplasmic IGCs also contain the adapter protein Aly that is known to provide a link between pre-mRNA splicing and mRNA export. The essential mRNA export factor/receptor NXF1 was observed to colocalize with Aly. In nucleoplasmic IGCs, NXF1 was found to localize in an RNA-dependent manner whereas it is RNA-independently located in the extrachromosomal material of the karyosphere. We believe our data suggest on a role of the nucleoplasmic IGCs in mRNA biogenesis and retention in a road to nuclear export.

  6. Human Enterovirus Nonstructural Protein 2CATPase Functions as Both an RNA Helicase and ATP-Independent RNA Chaperone

    Science.gov (United States)

    Xia, Hongjie; Wang, Peipei; Wang, Guang-Chuan; Yang, Jie; Sun, Xianlin; Wu, Wenzhe; Qiu, Yang; Shu, Ting; Zhao, Xiaolu; Yin, Lei; Qin, Cheng-Feng; Hu, Yuanyang; Zhou, Xi

    2015-01-01

    RNA helicases and chaperones are the two major classes of RNA remodeling proteins, which function to remodel RNA structures and/or RNA-protein interactions, and are required for all aspects of RNA metabolism. Although some virus-encoded RNA helicases/chaperones have been predicted or identified, their RNA remodeling activities in vitro and functions in the viral life cycle remain largely elusive. Enteroviruses are a large group of positive-stranded RNA viruses in the Picornaviridae family, which includes numerous important human pathogens. Herein, we report that the nonstructural protein 2CATPase of enterovirus 71 (EV71), which is the major causative pathogen of hand-foot-and-mouth disease and has been regarded as the most important neurotropic enterovirus after poliovirus eradication, functions not only as an RNA helicase that 3′-to-5′ unwinds RNA helices in an adenosine triphosphate (ATP)-dependent manner, but also as an RNA chaperone that destabilizes helices bidirectionally and facilitates strand annealing and complex RNA structure formation independently of ATP. We also determined that the helicase activity is based on the EV71 2CATPase middle domain, whereas the C-terminus is indispensable for its RNA chaperoning activity. By promoting RNA template recycling, 2CATPase facilitated EV71 RNA synthesis in vitro; when 2CATPase helicase activity was impaired, EV71 RNA replication and virion production were mostly abolished in cells, indicating that 2CATPase-mediated RNA remodeling plays a critical role in the enteroviral life cycle. Furthermore, the RNA helicase and chaperoning activities of 2CATPase are also conserved in coxsackie A virus 16 (CAV16), another important enterovirus. Altogether, our findings are the first to demonstrate the RNA helicase and chaperoning activities associated with enterovirus 2CATPase, and our study provides both in vitro and cellular evidence for their potential roles during viral RNA replication. These findings increase our

  7. Intratypic differentiation of polioviruses isolated from suspected cases of poliomyelitis in Brazil during the period of 1990 to 1993

    Directory of Open Access Journals (Sweden)

    A. M. B. de Filippis

    1994-12-01

    Full Text Available This study analyzed 3129 fecal samples derived from 1626 patients with sudden onset acute flaccid paralysis clinically compatible with poliomyelitis. The samples were collected in the period ranging from January 1990 to September 1993 in all regions of Brazil. Among the 1626 cases studied, 196 had isolation of poliovirus. Nevertheless, it was observed that some factors influenced the isolation rate and the intratypic characterization of these polioviruses. No cases of acute flaccid paralysis has been found to be etiologically related with wild polioviruses.

  8. Flock House virus subgenomic RNA3 is replicated and its replication correlates with transactivation of RNA2

    International Nuclear Information System (INIS)

    Eckerle, Lance D.; Albarino, Cesar G.; Ball, L. Andrew.

    2003-01-01

    The nodavirus Flock House virus has a bipartite genome composed of RNAs 1 and 2, which encode the catalytic component of the RNA-dependent RNA polymerase (RdRp) and the capsid protein precursor, respectively. In addition to catalyzing replication of the viral genome, the RdRp also transcribes from RNA1 a subgenomic RNA3, which is both required for and suppressed by RNA2 replication. Here, we show that in the absence of RNA1 replication, FHV RdRp replicated positive-sense RNA3 transcripts fully and copied negative-sense RNA3 transcripts into positive strands. The two nonstructural proteins encoded by RNA3 were dispensable for replication, but sequences in the 3'-terminal 58 nucleotides were required. RNA3 variants that failed to replicate also failed to transactivate RNA2. These results imply that RNA3 is naturally produced both by transcription from RNA1 and by subsequent RNA1-independent replication and that RNA3 replication may be necessary for transactivation of RNA2

  9. [Eradication of poliomyelitis and emergence of pathogenic vaccine-derived polioviruses: from Madagascar to Cameroon].

    Science.gov (United States)

    Delpeyroux, Francis; Colbère-Garapin, Florence; Razafindratsimandresy, Richter; Sadeuh-Mba, Serge; Joffret, Marie-Line; Rousset, Dominique; Blondel, Bruno

    2013-11-01

    The oral poliovaccine, a live vaccine made of attenuated poliovirus strains, is the main tool of the vaccination campaigns organised for eradicating poliomyelitis. these campaigns had led to the decline and, thereafter, to the disappearance of wild poliovirus strains of the three serotypes (1-3) in most parts of the world. However, when the poliovaccine coverage becomes too low, vaccine polioviruses can circulate in insufficiently immunized populations and become then pathogenic by mutations and genetic recombination with other enteroviruses of the same species, in particular some coxsackievirus A. These mutated and recombinant vaccine strains have been implicated in several epidemics of paralytic poliomyelitis. Two polio outbreaks associated with these pathogenic circulating vaccine-derived poliovirus (cVDPV) occurred in 2001-2002 and 2005 in the South of Madagascar where vaccine coverage was low. These cVDPV, of serotype 2 or 3, were isolated from paralyzed children and some of their healthy contacts. Other cVDPV were isolated in the same region from healthy children in 2011, indicating that these viruses were circulating again. Vaccination campaigns could stop the outbreaks in 2002 and 2005, and most probably prevent another one in 2011. Therefore, the genetic plasticity of poliovaccine strains that threatens the benefit of vaccination campaigns is the target of an accurate surveillance and an important theme of studies in the virology laboratories of the Institut Pasteur international network. © 2013 médecine/sciences – Inserm.

  10. Innovative IPV from attenuated Sabin poliovirus or newly designed alternative seed strains.

    Science.gov (United States)

    Hamidi, Ahd; Bakker, Wilfried A M

    2012-11-01

    This article gives an overview of the patent literature related to innovative inactivated polio vaccine (i-IPV) based on using Sabin poliovirus strains and newly developed alternative recombinant poliovirus strains. This innovative approach for IPV manufacturing is considered to attribute to the requirement for affordable IPV in the post-polio-eradication era, which is on the horizon. Although IPV is a well-established vaccine, the number of patent applications in this field was seen to have significantly increased in the past decade. Currently, regular IPV appears to be too expensive for universal use. Future affordability may be achieved by using alternative cell lines, alternative virus seed strains, improved and optimized processes, dose sparing, or the use of adjuvants. A relatively short-term option to achieve cost-price reduction is to work on regular IPV, using wild-type poliovirus strains, or on Sabin-IPV, based on using attenuated poliovirus strains. This price reduction can be achieved by introducing efficiency in processing. There are also multiple opportunities to work on dose sparing, for example, by using adjuvants or fractional doses. Renewed interest in this field was clearly reflected in the number and diversity of patent applications. In a later stage, several innovative approaches may become even more attractive, for example the use of recombinant virus strains or even a totally synthetic vaccine. Currently, such work is mainly carried out by research institutes and universities and therefore clinical data are not available.

  11. Differential depuration of poliovirus, Escherichia coli, and a coliphage by the common mussel, Mytilus edulis

    International Nuclear Information System (INIS)

    Power, U.F.; Collins, J.K.

    1989-01-01

    The elimination of sewage effluent-associated poliovirus, Escherichia coli, and a 22-nm icosahedral coliphage by the common mussel, Mytilus edulis, was studied. Both laboratory-and commercial-scale recirculating, UV depuration systems were used in this study. In the laboratory system, the logarithms of the poliovirus, E. coli, and coliphage levels were reduced by 1.86, 2.9, and 2.16, respectively, within 52 h of depuration. The relative patterns and rates of elimination of the three organisms suggest that they are eliminated from mussels by different mechanisms during depuration under suitable conditions. Poliovirus was not included in experiments undertaken in the commercial-scale depuration system. The differences in the relative rates and patterns of elimination were maintained for E. coli and coliphage in this system, with the logarithm of the E. coli levels being reduced by 3.18 and the logarithm of the coliphage levels being reduced by 0.87. The results from both depuration systems suggest that E. coli is an inappropriate indicator of the efficiency of virus elimination during depuration. The coliphage used appears to be a more representative indicator. Depuration under stressful conditions appeared to have a negligible affect on poliovirus and coliphage elimination rates from mussels. However, the rate and pattern of E. coli elimination were dramatically affected by these conditions. Therefore, monitoring E. coli counts might prove useful in ensuring that mussels are functioning well during depuration

  12. Update on Vaccine-Derived Polioviruses - Worldwide, January 2015-May 2016.

    Science.gov (United States)

    Jorba, Jaume; Diop, Ousmane M; Iber, Jane; Sutter, Roland W; Wassilak, Steven G; Burns, Cara C

    2016-08-05

    In 1988, the World Health Assembly resolved to eradicate poliomyelitis worldwide (1). One of the main tools used in polio eradication efforts has been the live, attenuated, oral poliovirus vaccine (OPV) (2), an inexpensive vaccine easily administered by trained volunteers. OPV might require several doses to induce immunity, but provides long-term protection against paralytic disease. Through effective use of OPV, the Global Polio Eradication Initiative (GPEI) has brought wild polioviruses to the threshold of eradication (1). However, OPV use, particularly in areas with low routine vaccination coverage, is associated with the emergence of genetically divergent vaccine-derived polioviruses (VDPVs) whose genetic drift from the parental OPV strains indicates prolonged replication or circulation (3). VDPVs can emerge among immunologically normal vaccine recipients and their contacts as well as among persons with primary immunodeficiencies (PIDs). Immunodeficiency-associated VDPVs (iVDPVs) can replicate for years in some persons with PIDs. In addition, circulating vaccine-derived polioviruses (cVDPVs) (3) can emerge in areas with low OPV coverage and can cause outbreaks of paralytic polio. This report updates previous summaries regarding VDPVs (4).

  13. Laboratory analysis of environmental samples taken following the reported release of live poliovirus

    NARCIS (Netherlands)

    Duizer E; REV; I&V

    2015-01-01

    Analyse poliomonsters na lozingsincident

    Op 2 september 2014 heeft het bedrijf GlaxoSmithKline (GSK), dat vaccins produceert, in België door een menselijke fout 45 liter geconcentreerd poliovirus op het riool geloosd. Via de nabijgelegen rioolwaterzuivering is dit water onder andere in de

  14. Hematopoietic Cancer Cell Lines Can Support Replication of Sabin Poliovirus Type 1

    Science.gov (United States)

    van Eikenhorst, Gerco; de Gruijl, Tanja D.; van der Pol, Leo A.; Bakker, Wilfried A. M.

    2015-01-01

    Viral vaccines can be produced in adherent or in suspension cells. The objective of this work was to screen human suspension cell lines for the capacity to support viral replication. As the first step, it was investigated whether poliovirus can replicate in such cell lines. Sabin poliovirus type 1 was serially passaged on five human cell lines, HL60, K562, KG1, THP-1, and U937. Sabin type 1 was capable of efficiently replicating in three cell lines (K562, KG1, and U937), yielding high viral titers after replication. Expression of CD155, the poliovirus receptor, did not explain susceptibility to replication, since all cell lines expressed CD155. Furthermore, we showed that passaged virus replicated more efficiently than parental virus in KG1 cells, yielding higher virus titers in the supernatant early after infection. Infection of cell lines at an MOI of 0.01 resulted in high viral titers in the supernatant at day 4. Infection of K562 with passaged Sabin type 1 in a bioreactor system yielded high viral titers in the supernatant. Altogether, these data suggest that K562, KG1, and U937 cell lines are useful for propagation of poliovirus. PMID:25815312

  15. Anti-idiotypic antibodies to poliovirus antibodies in commercial immunoglubulin preparations, human serum and milk.

    NARCIS (Netherlands)

    M. Hahn-Zoric; B. Carlsson; S. Jeansson; H.P. Ekre; A.D.M.E. Osterhaus (Albert); D. Roberton; L.A. Hanson

    1993-01-01

    textabstractOur previous studies have suggested that fetal antibody production can be induced by maternal antiidiotypic antibodies transferred to the fetus via the placenta. We tested commercial Ig, sera, and milk for the presence of anti-idiotypic antibodies to poliovirus type 1, using affinity

  16. Different virucidal activities of hyperbranched quaternary ammonium coatings on poliovirus and influenza virus

    NARCIS (Netherlands)

    Tuladhar, E.; Koning, de M.C.; Fundeanu, I.; Beumer, R.R.; Duizer, E.

    2012-01-01

    Virucidal activity of immobilized quaternary ammonium compounds (IQACs) coated onto glass and plastic surfaces was tested against enveloped influenza A (H1N1) virus and nonenveloped poliovirus Sabin1. The IQACs tested were virucidal against the influenza virus within 2 min, but no virucidal effect

  17. Update on vaccine-derived polioviruses - worldwide, July 2012-December 2013.

    Science.gov (United States)

    Diop, Ousmane M; Burns, Cara C; Wassilak, Steven G; Kew, Olen M

    2014-03-21

    In 1988, the World Health Assembly resolved to eradicate poliomyelitis worldwide. One of the main tools used in polio eradication efforts has been live, attenuated oral poliovirus vaccine (OPV), an inexpensive vaccine easily administered by trained volunteers. OPV might require several doses to induce immunity, but then it provides long-term protection against paralytic disease through durable humoral immunity. Rare cases of vaccine-associated paralytic poliomyelitis can occur among immunologically normal OPV recipients, their contacts, and persons who are immunodeficient. In addition, vaccine-derived polioviruses (VDPVs) can emerge in areas with low OPV coverage to cause polio outbreaks and can replicate for years in persons who have primary, B-cell immunodeficiencies. This report updates previous surveillance summaries and describes VDPVs detected worldwide during July 2012-December 2013. Those include a new circulating VDPV (cVDPV) outbreak identified in Pakistan in 2012, with spread to Afghanistan; an outbreak in Afghanistan previously identified in 2009 that continued into 2013; a new outbreak in Chad that spread to Cameroon, Niger, and northeastern Nigeria; and an outbreak that began in Somalia in 2008 that continued and spread to Kenya in 2013. A large outbreak in Nigeria that was identified in 2005 was nearly stopped by the end of 2013. Additionally, 10 newly identified persons in eight countries were found to excrete immunodeficiency-associated VDPVs (iVDPVs), and VDPVs were found among immunocompetent persons and environmental samples in 13 countries. Because the majority of VDPV isolates are type 2, the World Health Organization has developed a plan for coordinated worldwide replacement of trivalent OPV (tOPV) with bivalent OPV (bOPV; types 1 and 3) by 2016, preceded by introduction of at least 1 dose of inactivated poliovirus vaccine (IPV) containing all three poliovirus serotypes into routine immunization schedules worldwide to ensure high population

  18. Five of Five VHHs Neutralizing Poliovirus Bind the Receptor-Binding Site.

    Science.gov (United States)

    Strauss, Mike; Schotte, Lise; Thys, Bert; Filman, David J; Hogle, James M

    2016-01-13

    Nanobodies, or VHHs, that recognize poliovirus type 1 have previously been selected and characterized as candidates for antiviral agents or reagents for standardization of vaccine quality control. In this study, we present high-resolution cryo-electron microscopy reconstructions of poliovirus with five neutralizing VHHs. All VHHs bind the capsid in the canyon at sites that extensively overlap the poliovirus receptor-binding site. In contrast, the interaction involves a unique (and surprisingly extensive) surface for each of the five VHHs. Five regions of the capsid were found to participate in binding with all five VHHs. Four of these five regions are known to alter during the expansion of the capsid associated with viral entry. Interestingly, binding of one of the VHHs, PVSS21E, resulted in significant changes of the capsid structure and thus seems to trap the virus in an early stage of expansion. We describe the cryo-electron microscopy structures of complexes of five neutralizing VHHs with the Mahoney strain of type 1 poliovirus at resolutions ranging from 3.8 to 6.3Å. All five VHHs bind deep in the virus canyon at similar sites that overlap extensively with the binding site for the receptor (CD155). The binding surfaces on the VHHs are surprisingly extensive, but despite the use of similar binding surfaces on the virus, the binding surface on the VHHs is unique for each VHH. In four of the five complexes, the virus remains essentially unchanged, but for the fifth there are significant changes reminiscent of but smaller in magnitude than the changes associated with cell entry, suggesting that this VHH traps the virus in a previously undescribed early intermediate state. The neutralizing mechanisms of the VHHs and their potential use as quality control agents for the end game of poliovirus eradication are discussed. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  19. Cytotoxicity and antiviral activity of electrochemical - synthesized silver nanoparticles against poliovirus.

    Science.gov (United States)

    Huy, Tran Quang; Hien Thanh, Nguyen Thi; Thuy, Nguyen Thanh; Chung, Pham Van; Hung, Pham Ngoc; Le, Anh-Tuan; Hong Hanh, Nguyen Thi

    2017-03-01

    Silver nanoparticles (AgNPs) have been proven to have noticeable cytotoxicity in vitro and antiviral activity against some types of enveloped viruses. This paper presents the cytotoxicity and antiviral activity of pure AgNPs synthesized by the electrochemical method, towards cell culture and poliovirus (a non-enveloped virus). Prepared AgNPs were characterized by ultraviolet-visible spectroscopy, energy-dispersive X-ray spectroscopy and transmission electron microscopy. Before incubation with poliovirus, different concentrations of AgNPs were added to human rhabdomyosarcoma (RD) cell monolayers seeded in 96 well plates for testing their cytotoxicity. The in vitro cytotoxicity and anti-poliovirus activity of AgNPs were daily assessed for cytopathic effect (CPE) through inverted light microscopy. CPE in the tested wells was determined in comparison with those in wells of negative and positive control. Structure analysis showed that AgNPs were formed with a quasi-spherical shape with mean size about 7.1nm and high purity. No CPE of RD cells was seen in wells at the time point of 48h post-incubation with AgNPs at concentration up to 100ppm. The anti-poliovirus activity of AgNPs was determined at 3.13ppm corresponding to the viral concentration of 1TCID 50 (Tissue Culture Infective Dose) after 30min, and 10TCID 50 after 60min, the cell viability was found up to 98% at 48h post-infection, with no CPE found. Whereas, a strong CPE of RD cells was found at 48h post-infection with the mixture of AgNPs and poliovirus at concentration of 100TCID 50 , and in wells of positive controls. With mentioned advantages, electrochemical-synthesized AgNPs are promising candidate for advanced biomedical and disinfection applications. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. An acute flaccid paralysis surveillance-based serosurvey of poliovirus antibodies in Western Uttar Pradesh, India.

    Science.gov (United States)

    Bahl, Sunil; Gary, Howard E; Jafari, Hamid; Sarkar, Bidyut K; Pathyarch, Surendra K; Sethi, Raman; Deshpande, Jagadish

    2014-11-01

    Despite intensified use of monovalent oral poliovirus type 1 vaccine and improved coverage of immunization campaigns, wild poliovirus type 1 persisted in Indian states of Uttar Pradesh and Bihar during 2006 to 2009. A serosurvey was conducted among cases of acute flaccid paralysis in the 25 high-polio-incidence districts of western Uttar Pradesh. Children were recruited by age group (6-11 months, 12-24 months, and 25-69 months) from among cases reported through the acute flaccid paralysis surveillance system between November 2008 and August 2009. Seroprevalence for type 1 wild poliovirus was >96.4% for each age group. The seroprevalence of wild poliovirus types 2 and 3 increased with age, from 36.7% to 73.4% for type 2 and from 39.0% to 74.1% for type 3. In addition to the number of type-specific vaccine doses, father's level of education, being from a Muslim family, height for age, and female sex were the socioeconomic risk factors associated with seronegativity to poliovirus. The seroprevalence and risk factors identified in this study were consistent with the epidemiology of polio, and the findings were instrumental in optimizing vaccination strategy in western Uttar Pradesh with respect to the choice of OPV types, the frequency of supplementary immunization campaigns, and the urgency to improve routine immunization services. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  1. A simple quantitative protein A micro-immunoprecipitation method; assay of antibodies to the N and H antigens of poliovirus

    International Nuclear Information System (INIS)

    Vrijsen, R.; Rombaut, B.; Boeye, A.

    1983-01-01

    Staphylococcus aureus (Cowan strain I) was used to absorb immune complexes from antiserum to poliovirus to which labeled N or H poliovirus antigens had been added, and the radioactivity in the pelleted organisms and in the supernatant was measured. Excellent agreement was obtained between values calculated separately from the pellet and supernatant readings, validating the use of supernatant measurements from a microtitration plate method. (Auth.)

  2. Real-time reverse transcription-polymerase chain reaction assays for identification of wild poliovirus 1 & 3.

    Science.gov (United States)

    Sharma, Deepa K; Nalavade, Uma P; Deshpande, Jagadish M

    2015-10-01

    The poliovirus serotype identification and intratypic differentiation by real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay is suitable for serotype mixtures but not for intratypic mixtures of wild and vaccine poliovirus strains. This study was undertaken to develop wild poliovirus 1 and 3 (WPV1 and WPV3) specific rRT-PCR assays for use. Specific primers and probes for rRT-PCR were designed based on VP1 sequences of WPV1 and WPV3 isolated in India since 2000. The specificity of the rRT-PCR assays was evaluated using WPV1 and WPV3 of different genetic lineages, non-polio enteroviruses (NPEVs) and mixtures of wild/wild and wild/Sabin vaccine strains. The sensitivity of the assays was determined by testing serial 10-fold dilutions of wild poliovirus 1 and 3 stock suspensions of known titre. No cross-reactivity with Sabin strains, intertypic wild poliovirus isolates or 27 types of NPEVs across all the four Enterovirus species was found for both the wild poliovirus 1 and 3 rRT-PCR assays. All WPV1 and WPV3 strains isolated since 2000 were successfully amplified. The rRT-PCR assays detected 10 4.40 CCID 50 /ml of WPV1 and 10 4.00 CCID 50 /ml of WPV3, respectively either as single isolate or mixture with Sabin vaccine strains or intertypic wild poliovirus. rRT-PCR assays for WPV1 and WPV3 have been validated to detect all the genetic variations of the WPV1 and WPV3 isolated in India for the last decade. When used in combination with the current rRT-PCR assay testing was complete for confirmation of the presence of wild poliovirus in intratypic mixtures.

  3. Poliovirus-associated protein kinase: Destabilization of the virus capsid and stimulation of the phosphorylation reaction by Zn2+

    International Nuclear Information System (INIS)

    Ratka, M.; Lackmann, M.; Ueckermann, C.; Karlins, U.; Koch, G.

    1989-01-01

    The previously described poliovirus-associated protein kinase activity phosphorylates viral proteins VP0 and VP2 as well as exogenous proteins in the presence of Mg 2+ . In this paper, the effect of Zn 2+ on the phosphorylation reaction and the stability of the poliovirus capsid has been studied in detail and compared to that of Mg 2+ . In the presence of Zn 2+ , phosphorylation of capsid proteins VP2 and VP4 is significantly higher while phosphorylation of VP0 and exogenous phosphate acceptor proteins is not detected. The results indicate the activation of more than one virus-associated protein kinase by Zn 2+ . The ion-dependent behavior of the enzyme activities is observed independently of whether the virus was obtained from HeLa or green monkey kidney cells. The poliovirus capsid is destabilized by Zn 2+ . This alteration of the poliovirus capsid structure is a prerequisite for effective phosphorylation of viral capsid proteins. The increased level of phosphorylation of viral capsid proteins results in further destabilization of the viral capsid. As a result of the conformational changes, poliovirus-associated protein kinase activities dissociate from the virus particle. The authors suggest that the destabilizing effect of phosphorylation on the viral capsid plays a role in uncoating of poliovirus

  4. Prolonged Excretion of Poliovirus among Individuals with Primary Immunodeficiency Disorder: An Analysis of the World Health Organization Registry

    Directory of Open Access Journals (Sweden)

    Grace Macklin

    2017-09-01

    Full Text Available Individuals with primary immunodeficiency disorder may excrete poliovirus for extended periods and will constitute the only remaining reservoir of virus after eradication and withdrawal of oral poliovirus vaccine. Here, we analyzed the epidemiology of prolonged and chronic immunodeficiency-related vaccine-derived poliovirus cases in a registry maintained by the World Health Organization, to identify risk factors and determine the length of excretion. Between 1962 and 2016, there were 101 cases, with 94/101 (93% prolonged excretors and 7/101 (7% chronic excretors. We documented an increase in incidence in recent decades, with a shift toward middle-income countries, and a predominance of poliovirus type 2 in 73/101 (72% cases. The median length of excretion was 1.3 years (95% confidence interval: 1.0, 1.4 and 90% of individuals stopped excreting after 3.7 years. Common variable immunodeficiency syndrome and residence in high-income countries were risk factors for long-term excretion. The changing epidemiology of cases, manifested by the greater incidence in recent decades and a shift to from high- to middle-income countries, highlights the expanding risk of poliovirus transmission after oral poliovirus vaccine cessation. To better quantify and reduce this risk, more sensitive surveillance and effective antiviral therapies are needed.

  5. Effect of buffer on the immune response to trivalent oral poliovirus vaccine in Bangladesh: a community based randomized controlled trial.

    Science.gov (United States)

    Chandir, Subhash; Ahamed, Kabir U; Baqui, Abdullah H; Sutter, Roland W; Okayasu, Hiromasa; Pallansch, Mark A; Oberste, Mark S; Moulton, Lawrence H; Halsey, Neal A

    2014-11-01

    Polio eradication efforts have been hampered by low responses to trivalent oral poliovirus vaccine (tOPV) in some developing countries. Since stomach acidity may neutralize vaccine viruses, we assessed whether administration of a buffer solution could improve the immunogenicity of tOPV. Healthy infants 4-6 weeks old in Sylhet, Bangladesh, were randomized to receive tOPV with or without a sodium bicarbonate and sodium citrate buffer at age 6, 10, and 14 weeks. Levels of serum neutralizing antibodies for poliovirus types 1, 2, and 3 were measured before and after vaccination, at 6 and 18 weeks of age, respectively. Serologic response rates following 3 doses of tOPV for buffer recipients and control infants were 95% and 88% (P=.065), respectively, for type 1 poliovirus; 95% and 97% (P=.543), respectively, for type 2 poliovirus; and 90% and 89% (P=.79), respectively, for type 3 poliovirus. Administration of a buffer solution prior to vaccination was not associated with statistically significant increases in the immune response to tOPV; however, a marginal 7% increase (P=.065) in serologic response to poliovirus type 1 was observed. NCT01579825. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  6. Development of inactivated poliovirus vaccine from Sabin strains: A progress report.

    Science.gov (United States)

    Okayasu, Hiromasa; Sein, Carolyn; Hamidi, Ahd; Bakker, Wilfried A M; Sutter, Roland W

    2016-11-01

    The Global Polio Eradication Initiative (GPEI) has seen significant progress since it began in 1988, largely due to the worldwide use of oral poliovirus vaccine (OPV). In order to achieve polio eradication the global cessation of OPV is necessary because OPV contains live attenuated poliovirus, which in rare circumstances could re-gain wild poliovirus (WPV) characteristics with potential to establish transmission. The GPEI endgame strategy for the period 2013-2018 recommends the globally synchronised sequential cessation of the Sabin strains contained in the OPV, starting with type 2 Sabin. The withdrawal of Sabin type 2 took place in April 2016, with the introduction of at least one dose of inactivated poliovirus vaccine (IPV) as a risk mitigation strategy. The introduction of IPV into 126 countries since 2013 has required a rapid scale-up of IPV production by the two manufacturers supplying the global public sector market. This scale-up has been fraught with challenges, resulting in reductions of 40-50% of initial supply commitments. Consequently, 22 countries will not be supplied until 2018, and another 23 countries will experience serious stock-outs. In the last decade repeated calls-for-action were made to the global community to invigorate their vision and investment in developing "new poliovirus vaccines" including the development of IPV from less-virulent strains, such as Sabin-IPV (S-IPV). The conventional Salk-IPV production is limited to high-income industrialized-country manufacturers due to the containment requirements (i.e., high sanitation, low force-of-poliovirus-infection, and high population immunity). The use of Sabin strains in the production of S-IPV carries a lower biosafety risk, and was determined to be suitable for production in developing countries, expanding the manufacturing base and making IPV more affordable and accessible in the long term. Significant progress in the S-IPV has been made since 2006. S-IPV is now licensed as S-IPV in

  7. Survey of poliovirus antibodies in Borno and Yobe States, North-Eastern Nigeria.

    Science.gov (United States)

    Gofama, Mustapha Modu; Verma, Harish; Abdullahi, Hamisu; Molodecky, Natalie A; Craig, Kehinde T; Urua, Utibe-Abasi; Garba, Mohammed Ashir; Alhaji, Mohammed Arab; Weldon, William C; Oberste, M Steven; Braka, Fiona; Muhammad, Ado J G; Sutter, Roland W

    2017-01-01

    Nigeria remains one of only three polio-endemic countries in the world. In 2016, after an absence of 2 years, wild poliovirus serotype 1 was again detected in North-Eastern Nigeria. To better guide programmatic action, we assessed the immunity status of infants and children in Borno and Yobe states, and evaluated the impact of recently introduced inactivated poliovirus vaccine (IPV) on antibody seroprevalence. We conducted a facility-based study of seroprevalence to poliovirus serotypes 1, 2 and 3 among health-seeking patients in two sites each of Borno and Yobe States. Enrolment was conducted amongst children 6-9 and 36-47 months of age attending the paediatrics outpatient department of the selected hospitals in the two states between 11 January and 5 February 2016. Detailed demographic and immunization history of the child was taken and an assessment of the child's health and nutritional state was conducted via physical examination. Blood was collected to test for levels of neutralizing antibody titres against the three poliovirus serotypes. The seroprevalence in the two age groups, potential determinants of seropositivity and the impact of one dose of IPV on humoral immunity were assessed. A total of 583 subjects were enrolled and provided sufficient quantities of serum for testing. Among 6-9-month-old infants, the seroprevalence was 81% (74-87%), 86% (79-91%), and 72% (65-79%) in Borno State, and 75% (67-81%), 74% (66-81%) and 69% (61-76%) in Yobe States, for serotypes-1, 2 and 3, respectively. Among children aged 36-47 months, the seroprevalence was >90% in both states for all three serotypes, with the exception of type 3 seroprevalence in Borno [87% (80-91%)]. Median reciprocal anti-polio neutralizing antibody titers were consistently >900 for serotypes 1 and 2 across age groups and states; with lower estimates for serotype 3, particularly in Borno. IPV received in routine immunization was found to be a significant determinant of seropositivity and anti

  8. Survey of poliovirus antibodies in Borno and Yobe States, North-Eastern Nigeria.

    Directory of Open Access Journals (Sweden)

    Mustapha Modu Gofama

    Full Text Available Nigeria remains one of only three polio-endemic countries in the world. In 2016, after an absence of 2 years, wild poliovirus serotype 1 was again detected in North-Eastern Nigeria. To better guide programmatic action, we assessed the immunity status of infants and children in Borno and Yobe states, and evaluated the impact of recently introduced inactivated poliovirus vaccine (IPV on antibody seroprevalence.We conducted a facility-based study of seroprevalence to poliovirus serotypes 1, 2 and 3 among health-seeking patients in two sites each of Borno and Yobe States. Enrolment was conducted amongst children 6-9 and 36-47 months of age attending the paediatrics outpatient department of the selected hospitals in the two states between 11 January and 5 February 2016. Detailed demographic and immunization history of the child was taken and an assessment of the child's health and nutritional state was conducted via physical examination. Blood was collected to test for levels of neutralizing antibody titres against the three poliovirus serotypes. The seroprevalence in the two age groups, potential determinants of seropositivity and the impact of one dose of IPV on humoral immunity were assessed. A total of 583 subjects were enrolled and provided sufficient quantities of serum for testing. Among 6-9-month-old infants, the seroprevalence was 81% (74-87%, 86% (79-91%, and 72% (65-79% in Borno State, and 75% (67-81%, 74% (66-81% and 69% (61-76% in Yobe States, for serotypes-1, 2 and 3, respectively. Among children aged 36-47 months, the seroprevalence was >90% in both states for all three serotypes, with the exception of type 3 seroprevalence in Borno [87% (80-91%]. Median reciprocal anti-polio neutralizing antibody titers were consistently >900 for serotypes 1 and 2 across age groups and states; with lower estimates for serotype 3, particularly in Borno. IPV received in routine immunization was found to be a significant determinant of seropositivity and

  9. Evolution of the Sabin vaccine into pathogenic derivatives without appreciable changes in antigenic properties: need for improvement of current poliovirus surveillance.

    Science.gov (United States)

    Yakovenko, Maria L; Korotkova, Ekaterina A; Ivanova, Olga E; Eremeeva, Tatyana P; Samoilovich, Elena; Uhova, Iryna; Gavrilin, Gene V; Agol, Vadim I

    2009-04-01

    The Sabin oral polio vaccine (OPV) may evolve into pathogenic viruses, causing sporadic cases and outbreaks of poliomyelitis. Such vaccine-derived polioviruses (VDPV) generally exhibit altered antigenicity. The current paradigm to distinguish VDPV from OPV and wild polioviruses is to characterize primarily those poliovirus isolates that demonstrate deviations from OPV in antigenic and genetic intratypic differentiation (ITD) tests. Here we report on two independent cases of poliomyelitis caused by VDPVs with "Sabin-like" properties in several ITD assays. The results suggest the existence of diverse pathways of OPV evolution and necessitate improvement of poliovirus surveillance, which currently potentially misses this class of VDPV.

  10. Epithelial Distribution and Replication of Foot-and-Mouth Disease Virus RNA in Infected Pigs

    DEFF Research Database (Denmark)

    Durand, S.; Murphy, C.; Zhang, Z.

    2008-01-01

    experimentally with FMDV serotype O UKG 34/2001 and tissue samples were collected from I to 4 clays post-infection. Samples were stored at -70 degrees C and frozen sections were prepared for in-situ hybridization (ISH). A digoxigenin-labelled RNA probe complementary to a coding part of the RNA-dependent RNA...... negative strand RNA was observed in basal cells above the basement membrane and along the dermal papillae. The basal cells therefore demonstrate the highest signal for detection of the FMDV positive and negative strand RNAs in both tongue and foot epithelium. These novel results Suggest that the epithelial...

  11. Natural minus-strand RNAs of alfalfa mosaic virus as in vitro templates for viral RNA polymerase. 3'-Terminal non-coded guanosine and coat protein are insufficient factors for full-size plus-strand synthesis

    NARCIS (Netherlands)

    Houwing, C.J.; Huis in 't Veld, M.; Zuidema, D.; Graaff, de M.; Jaspars, E.M.J.

    2001-01-01

    Replication complexes of alfalfa mosaic virus produce in vivo large quantities of plus-strand RNAs, but this production is fully dependent on the presence of coat protein. In order to study this process of RNA-dependent and coat protein-regulated RNA synthesis we have isolated the three natural

  12. The cis-acting replication signal at the 3' end of Flock House virus RNA2 is RNA3-dependent

    International Nuclear Information System (INIS)

    Albarino, Cesar G.; Eckerle, Lance D.; Ball, L. Andrew

    2003-01-01

    The nodavirus Flock House virus has a bipartite positive-sense RNA genome consisting of RNAs 1 and 2, which encode the viral RNA-dependent RNA polymerase (RdRp) and capsid protein precursor, respectively. The RdRp catalyzes replication of both genome segments and produces from RNA1 a subgenomic RNA (RNA3) that transactivates RNA2 replication. Here, we replaced internal sequences of RNAs 1 and 2 with a common heterologous core and were thereby able to test the RNA termini for compatibility in supporting the replication of chimeric RNAs. The results showed that the 3' 50 nt of RNA2 contained an RNA3-dependent cis-acting replication signal. Since covalent RNA dimers can direct the synthesis of monomeric replication products, the RdRp can evidently respond to cis-acting replication signals located internally. Accordingly, RNA templates containing the 3' termini of both RNAs 1 and 2 in tandem generated different replication products depending on the presence or absence of RNA3

  13. Clinical development of a novel inactivated poliomyelitis vaccine based on attenuated Sabin poliovirus strains.

    Science.gov (United States)

    Verdijk, Pauline; Rots, Nynke Y; Bakker, Wilfried A M

    2011-05-01

    Following achievement of polio eradication, the routine use of all live-attenuated oral poliovirus vaccines should be discontinued. However, the costs per vaccine dose for the alternative inactivated poliovirus vaccine (IPV) are significantly higher and the current production capacity is not sufficient for worldwide distribution of the vaccine. In order to achieve cost-prize reduction and improve affordability, IPV production processes and dose-sparing strategies should be developed to facilitate local manufacture at a relatively lower cost. The use of attenuated Sabin instead of wild-type polio strains will provide additional safety during vaccine production and permits production in low-cost settings. Sabin-IPV is under development by several manufacturers. This article gives an overview of results from clinical trials with Sabin-IPV and discusses the requirements and challenges in the clinical development of this novel IPV.

  14. Pseudogenes regulate parental gene expression via ceRNA network.

    Science.gov (United States)

    An, Yang; Furber, Kendra L; Ji, Shaoping

    2017-01-01

    The concept of competitive endogenous RNA (ceRNA) was first proposed by Salmena and colleagues. Evidence suggests that pseudogene RNAs can act as a 'sponge' through competitive binding of common miRNA, releasing or attenuating repression through sequestering miRNAs away from parental mRNA. In theory, ceRNAs refer to all transcripts such as mRNA, tRNA, rRNA, long non-coding RNA, pseudogene RNA and circular RNA, because all of them may become the targets of miRNA depending on spatiotemporal situation. As binding of miRNA to the target RNA is not 100% complementary, it is possible that one miRNA can bind to multiple target RNAs and vice versa. All RNAs crosstalk through competitively binding to miRNAvia miRNA response elements (MREs) contained within the RNA sequences, thus forming a complex regulatory network. The ratio of a subset of miRNAs to the corresponding number of MREs determines repression strength on a given mRNA translation or stability. An increase in pseudogene RNA level can sequester miRNA and release repression on the parental gene, leading to an increase in parental gene expression. A massive number of transcripts constitute a complicated network that regulates each other through this proposed mechanism, though some regulatory significance may be mild or even undetectable. It is possible that the regulation of gene and pseudogene expression occurring in this manor involves all RNAs bearing common MREs. In this review, we will primarily discuss how pseudogene transcripts regulate expression of parental genes via ceRNA network and biological significance of regulation. © 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  15. MicroRNA Related Polymorphisms and Breast Cancer Risk

    DEFF Research Database (Denmark)

    Khan, Sofia; Greco, Dario; Michailidou, Kyriaki

    2014-01-01

    Genetic variations, such as single nucleotide polymorphisms (SNPs) in microRNAs (miRNA) or in the miRNA binding sites may affect the miRNA dependent gene expression regulation, which has been implicated in various cancers, including breast cancer, and may alter individual susceptibility to cancer....... We investigated associations between miRNA related SNPs and breast cancer risk. First we evaluated 2,196 SNPs in a case-control study combining nine genome wide association studies (GWAS). Second, we further investigated 42 SNPs with suggestive evidence for association using 41,785 cases and 41......,880 controls from 41 studies included in the Breast Cancer Association Consortium (BCAC). Combining the GWAS and BCAC data within a meta-analysis, we estimated main effects on breast cancer risk as well as risks for estrogen receptor (ER) and age defined subgroups. Five miRNA binding site SNPs associated...

  16. Vaccine-Derived Poliovirus Outbreaks and Events - Three Provinces, Democratic Republic of the Congo, 2017.

    Science.gov (United States)

    Alleman, Mary M; Chitale, Rohit; Burns, Cara C; Iber, Jane; Dybdahl-Sissoko, Naomi; Chen, Qi; Van Koko, Djo-Roy; Ewetola, Raimi; Riziki, Yogolelo; Kavunga-Membo, Hugo; Dah, Cheikh; Andriamihantanirina, Rija

    2018-03-16

    The last confirmed wild poliovirus (WPV) case in Democratic Republic of the Congo (DRC) had paralysis onset in December 2011 (1). DRC has had cases of vaccine-derived polioviruses (VDPVs) documented since 2004 (Table 1) (1-6). After an outbreak of 30 circulating VDPV type 2 (cVDPV2) cases during 2011-2012, only five VDPV2 cases were reported during 2013-2016 (Table 1) (1-6). VDPVs can emerge from oral poliovirus vaccine (OPV types 1, 2, or 3; Sabin) polioviruses that have genetically mutated resulting in reversion to neurovirulence. This process occurs during extensive person-to-person transmission in populations with low immunity or after extended replication in the intestines of immune-deficient persons following vaccination (1-6). During 2017 (as of March 8, 2018), 25 VDPV cases were reported in three provinces in DRC: in Tanganyika province, an emergence with one VDPV2 case (pending final classification) in Kabalo health zone and an emergence with one ambiguous VDPV type 1 (aVDPV1) case in Ankoro health zone; in Maniema province, an emergence with two cVDPV2 cases; and in Haut Lomami province, an emergence with 20 cVDPV2 cases that originated in Haut Lomami province and later spread to Tanganyika province (hereafter referred to as the Haut Lomami outbreak area) and an emergence with one aVDPV type 2 (aVDPV2) case in Lwamba health zone (Table 1) (Figure) (6). Outbreak response supplementary immunization activities (SIAs) were conducted during June-December 2017 (Table 2) (6). Because of limitations in surveillance and suboptimal SIA quality and geographic scope, cVDPV2 circulation is likely continuing in 2018, requiring additional SIAs. DRC health officials and Global Polio Eradication Initiative (GPEI) partners are increasing human and financial resources to improve all aspects of outbreak response.

  17. [Investigation of a Patient with Pre-vaccine-derived Poliovirus in Shandong Province, China].

    Science.gov (United States)

    Lin, Xiaojuan; Liu, Yao; Wang, Suting; Zhang Xiao; Song, Lizhi; Tao, Zexin; Ji, Feng; Xiong, Ping; Xu, Aiqiang

    2015-09-01

    To analyze the genetic characteristics of a polio-I highly variant vaccine recombinant virus in Shandong Province (China) in 2011 and to identify isolates from healthy contacts, two stool specimens from one patient with acute flaccid paralysis (AFP) and 40 stool specimens from his contacts were collected for virus isolation. The complete genome of poliovirus and VP1 coding region of the non-polio enterovirus were sequenced. Homologous comparison and phylogenetic analyses based on VP1 sequences were undertaken among coxsackievirus (CV) B1, CV-B3 isolates, and those in GenBank. One poliovirus (P1/11186), CV-A4 and CV-A8 were isolated from the AFP patient; one CV-A2, Echovirus 3 (E-3), E-12 and E-14, ten CV-B1, and five CV-B3 strains were isolated from his contacts. These results led us to believe that there may be a human enterovirus epidemic in this area, and that surveillance must be enhanced. P1/11186 was a type-1 vaccine-related poliovirus; it combined with type-2 and type-3 polioviruses in 2A and 3A regions, respectively. There were 25 nucleotide mutations with 9 amino-acid alterations in the entire genome. There were 8 nucleotide mutations with 5 amino-acid alterations in the VP1 region compared with the corresponding Sabin strains. Homology analyses suggested that the ten CV-B1 isolates had 97.0%-100% nucleotide and 98.9%-100% amino-acid identities with each other, as well as 92.6%-100% nucleotide and 99.2%-100% amino-acid identities among the five CV-B3 isolates. Phylogenetic analyses on the complete sequences of VP1 among CV-B1 and CV-B3 isolates showed that Shandong strains, together with strains from other provinces in China, had a close relationship and belonged to the same group.

  18. Fractional-Dose Inactivated Poliovirus Vaccine Campaign - Sindh Province, Pakistan, 2016.

    Science.gov (United States)

    Pervaiz, Aslam; Mbaeyi, Chukwuma; Baig, Mirza Amir; Burman, Ashley; Ahmed, Jamal A; Akter, Sharifa; Jatoi, Fayaz A; Mahamud, Abdirahman; Asghar, Rana Jawad; Azam, Naila; Shah, Muhammad Nadeem; Laghari, Mumtaz Ali; Soomro, Kamaluddin; Wadood, Mufti Zubair; Ehrhardt, Derek; Safdar, Rana M; Farag, Noha

    2017-12-01

    Following the declaration of eradication of wild poliovirus (WPV) type 2 in September 2015, trivalent oral poliovirus vaccine (tOPV) was withdrawn globally to reduce the risk for type 2 vaccine-derived poliovirus (VDPV2) transmission; all countries implemented a synchronized switch to bivalent OPV (type 1 and 3) in April 2016 (1,2). Any isolation of VDPV2 after the switch is to be treated as a potential public health emergency and might indicate the need for supplementary immunization activities (3,4). On August 9, 2016, VDPV2 was isolated from a sewage sample taken from an environmental surveillance site in Hyderabad, Sindh province, Pakistan. Possible vaccination activities in response to VDPV2 isolation include the use of injectable inactivated polio vaccine (IPV), which poses no risk for vaccine-derived poliovirus transmission. Fractional-dose, intradermal IPV (fIPV), one fifth of the standard intramuscular dose, has been developed to more efficiently manage limited IPV supplies. fIPV has been shown in some studies to be noninferior to full-dose IPV (5,6) and was used successfully in response to a similar detection of a single VDPV2 isolate from sewage in India (7). Injectable fIPV was used for response activities in Hyderabad and three neighboring districts. This report describes the findings of an assessment of preparatory activities and subsequent implementation of the fIPV campaign. Despite achieving high coverage (>80%), several operational challenges were noted. The lessons learned from this campaign could help to guide the planning and implementation of future fIPV vaccination activities.

  19. Molecular Evolution of a Type 1 Wild-Vaccine Poliovirus Recombinant during Widespread Circulation in China

    Science.gov (United States)

    Liu, Hong-Mei; Zheng, Du-Ping; Zhang, Li-Bi; Oberste, M. Steven; Pallansch, Mark A.; Kew, Olen M.

    2000-01-01

    Type 1 wild-vaccine recombinant polioviruses were isolated from poliomyelitis patients in China from 1991 to 1993. We compared the sequences of 34 recombinant isolates over the 1,353-nucleotide (nt) genomic interval (nt 2480 to 3832) encoding the major capsid protein, VP1, and the protease, 2A. All recombinants had a 367-nt block of sequence (nt 3271 to 3637) derived from the Sabin 1 oral poliovirus vaccine strain spanning the 3′-terminal sequences of VP1 (115 nt) and the 5′ half of 2A (252 nt). The remaining VP1 sequences were closely (up to 99.5%) related to those of a major genotype of wild type 1 poliovirus endemic to China up to 1994. In contrast, the non-vaccine-derived sequences at the 3′ half of 2A were more distantly related (polioviruses from China. The vaccine-derived sequences of the earliest (April 1991) isolates completely matched those of Sabin 1. Later isolates diverged from the early isolates primarily by accumulation of synonymous base substitutions (at a rate of ∼3.7 × 10−2 substitutions per synonymous site per year) over the entire VP1-2A interval. Distinct evolutionary lineages were found in different Chinese provinces. From the combined epidemiologic and evolutionary analyses, we propose that the recombinant virus arose during mixed infection of a single individual in northern China in early 1991 and that its progeny spread by multiple independent chains of transmission into some of the most populous areas of China within a year of the initiating infection. PMID:11070012

  20. Regulatory Aspects of Sabin Type 2 Withdrawal From Trivalent Oral Poliovirus Vaccine: Process and Lessons Learned.

    Science.gov (United States)

    Decina, Daniela; Fournier-Caruana, Jacqueline; Takane, Marina; Ostad Ali Dehaghi, Razieh; Sutter, Roland

    2017-07-01

    Withdrawal of type 2 oral poliovirus vaccine (OPV) in OPV-using countries required regulatory approval for use of inactivated poliovirus vaccine and bivalent OPV in routine immunization. Worldwide, a variety of mechanisms were used by member states, with some differences in approach observed between inactivated poliovirus vaccine and bivalent OPV. These included acceptance for use of World Health Organization (WHO) prequalified vaccines, registration and licensure pathways, participation in WHO-convened joint reviews of licensing dossiers, as well as pragmatic application of alternatively available mechanisms, when appropriate. Simple but effective tools were used to monitor progress and to record, authenticate, and share information. Essential to achievement of regulatory targets was ongoing communication with key stakeholders, including switch-country national regulatory authorities, vaccine manufacturers, partner organizations, and relevant units within WHO. Understanding of the regulatory environment gained through the OPV switch can be helpful in supporting further stages of the polio end game and other time-sensitive vaccine introduction programs. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America.

  1. Comparative inactivation of enteric adenoviruses, poliovirus and coliphages by ultraviolet irradiation

    International Nuclear Information System (INIS)

    Meng, Q.S.; Gerba, C.P.

    1996-01-01

    The inactivation of enteric adenoviruses 40 and 41 by ultraviolet (UV) radiation was investigated and compared with poliovirus type 1 (strain LSc-2ab) and coliphages MS-2 and PRD-1. Purified stocks of the viruses were exposed to collimated ultraviolet radiation in a stirred reactor for a total dose of up to 140 mW s/cm 2 . The doses of UV to achieve a 90% inactivation of adenovirus 40, adenovirus 41, coliphages MS-2 and PRD-1 and poliovirus type 1 were 30, 23.6, 14, 8.7 and 4.1 mW s/cm 2 , respectively. Adenovirus 40 was significantly more resistant than coliphage MS-2 to UV irradiation (P < 0.01). Adenovirus 41 appeared slightly more sensitive than adenovirus 40, but the difference was not significant (P>0.05). The resistance of PRD-1 was less than MS-2 (P < 0.01), but greater than poliovirus type 1 (P < 0.01). Adenoviruses 40 and 41 were more resistant than Bacillus subtilis spores, often suggested as an indicator of UV light performance. The double-stranded DNA adenoviruses appear to be the most resistant of all potentially water-borne enteric viruses to UV light disinfection. (author)

  2. Simian virus 40, poliovirus vaccines, and human cancer: research progress versus media and public interests

    Science.gov (United States)

    Butel, J. S.

    2000-01-01

    From 1955 through early 1963, millions of people were inadvertently exposed to simian virus 40 (SV40) as a contaminant of poliovirus vaccines; the virus had been present in the monkey kidney cultures used to prepare the vaccines and had escaped detection. SV40 was discovered in 1960 and subsequently eliminated from poliovirus vaccines. This article reviews current knowledge about SV40 and considers public responses to reports in the media. SV40 is a potent tumour virus with broad tissue tropism that induces tumours in rodents and transforms cultured cells from many species. It is also an important laboratory model for basic studies of molecular processes in eukaryotic cells and mechanisms of neoplastic transformation. SV40 neutralizing antibodies have been detected in individuals not exposed to contaminated poliovirus vaccines. There have been many reports of detection of SV40 DNA in human tumours, especially mesotheliomas, brain tumours and osteosarcomas; and DNA sequence analyses have ruled out the possibility that the viral DNA in tumours was due to laboratory contamination or that the virus had been misidentified. However, additional studies are necessary to prove that SV40 is the cause of certain human cancers. A recently published review article evaluated the status of the field and received much media attention. The public response emphasized that there is great interest in the possibility of health risks today from vaccinations received in the past.

  3. Cross-sectional serologic assessment of immunity to poliovirus infection in high-risk areas of northern India.

    Science.gov (United States)

    Bahl, Sunil; Estívariz, Concepción F; Sutter, Roland W; Sarkar, Bidyut K; Verma, Harish; Jain, Vibhor; Agrawal, Ashutosh; Rathee, Mandeep; Shukla, Hemant; Pathyarch, Surendra K; Sethi, Raman; Wannemuehler, Kathleen A; Jafari, Hamid; Deshpande, Jagadish M

    2014-11-01

    The objectives of this survey were to assess the seroprevalence of antibodies to poliovirus types 1 and 3 and the impact of bivalent (types 1 and 3) oral poliovirus vaccine (bOPV) use in immunization campaigns in northern India. In August 2010, a 2-stage stratified cluster sampling method identified infants aged 6-7 months in high-risk blocks for wild poliovirus infection. Vaccination history, weight and length, and serum were collected to test for neutralizing antibodies to poliovirus types 1, 2, and 3. Seroprevalences of antibodies to poliovirus types 1, 2, and 3 were 98% (95% confidence interval [CI], 97%-99%), 66% (95% CI, 62%-69%), and 77% (95% CI, 75%-79%), respectively, among 664 infants from Bihar and 616 infants from Uttar Pradesh. Infants had received a median of 3 bOPV doses and 2 monovalent type 1 OPV (mOPV1) doses through campaigns and 3 trivalent OPV (tOPV) doses through routine immunization. Among subjects with 0 tOPV doses, the seroprevalences of antibodies to type 3 were 50%, 77%, and 82% after 2, 3, and 4 bOPV doses, respectively. In multivariable analysis, malnutrition was associated with a lower seroprevalence of type 3 antibodies. This study confirmed that replacing mOPV1 with bOPV in campaigns was successful in maintaining very high population immunity to type 1 poliovirus and substantially decreasing the immunity gap to type 3 poliovirus. Published by Oxford University Press on behalf of the Infectious Diseases Society of America 2014. This work is written by (a) US Government employee(s) and is in the public domain in the US.

  4. Localization of foot-and-mouth disease - RNA synthesis on newly formed cellular smooth membranous vacuoles

    International Nuclear Information System (INIS)

    Polatnick, J.; Wool, S.H.

    1982-01-01

    Viral RNA synthesis in foot-and-mouth disease infected bovine kidney cell cultures was associated throughout the infectious period with newly formed smooth membranous vacuoles. Membrane formation was measured by choline uptake. The site of RNA synthesis was determined by electron microscopic examination of autoradiograms of incorporated [ 3 H] uridine. Both membrane formation and RNA synthesis became signifcant at 2.5 hours postinfection, but membrane formation increased steadily to 4.5 hours while RNA synthesis peaked at 3.5 hours. Percent density distributions of developed silver grains on autoradiograms showed that almost all RNA synthesis was concentrated on the smooth vacuoles of infected cells. Histogram analysis of grain density distributions established that the site of RNA synthesis was the vacuolar membrane. The newly formed smooth membrane-bound vacuoles were not seen to coalesce into the large vacuolated areas typical of poliovirus cytopathogenicity. (Author)

  5. Localization of foot-and-mouth disease - RNA synthesis on newly formed cellular smooth membranous vacuoles

    Energy Technology Data Exchange (ETDEWEB)

    Polatnick, J.; Wool, S.H. (United States Department of Agriculture, Science and Education, Greenport, New York (USA). Agricultural Research, Plum Island Animal Disease Center)

    1982-01-01

    Viral RNA synthesis in foot-and-mouth disease infected bovine kidney cell cultures was associated throughout the infectious period with newly formed smooth membranous vacuoles. Membrane formation was measured by choline uptake. The site of RNA synthesis was determined by electron microscopic examination of autoradiograms of incorporated (/sup 3/H) uridine. Both membrane formation and RNA synthesis became signifcant at 2.5 hours postinfection, but membrane formation increased steadily to 4.5 hours while RNA synthesis peaked at 3.5 hours. Percent density distributions of developed silver grains on autoradiograms showed that almost all RNA synthesis was concentrated on the smooth vacuoles of infected cells. Histogram analysis of grain density distributions established that the site of RNA synthesis was the vacuolar membrane. The newly formed smooth membrane-bound vacuoles were not seen to coalesce into the large vacuolated areas typical of poliovirus cytopathogenicity.

  6. Ausencia de circulación de poliovirus en departamentos colombianos con coberturas vacunales inferiores a 80% Absence of poliovirus circulation in Colombian departments with vaccination coverage below 80%

    Directory of Open Access Journals (Sweden)

    María Mercedes González

    2012-08-01

    Full Text Available El presente estudio se propuso explorar la posible circulación silente de poliovirus salvajes y derivados de la vacuna (VDPV, por sus siglas en inglés, en departamentos de Colombia con cobertura de vacunación para polio (OPV, por sus siglas en inglés menor de 80%. Se colectaron 52 muestras de aguas residuales que se concentraron mediante precipitación con polietilenglicol y cloruro de sodio. La detección viral se realizó mediante aislamiento y la identificación por neutralización del efecto citopático, así como mediante reacción en cadena de la polimerasa convencional y en tiempo real, posterior a la transcripción reversa (TR-RCP y rTR-RCP. Los poliovirus aislados se caracterizaron por secuenciación del gen VP1. En dos de las 52 muestras hubo presencia de poliovirus Sabin 2 con más de 99% de similitud de secuencia con la cepa OPV Sabin 2. Se detectó circulación de enterovirus no polio en 17,3% de las muestras. Los serotipos identificados correspondieron a coxsackievirus B1, echovirus 30 y echovirus 11. No se detectaron evidencias de circulación de VDPV ni poliovirus salvaje en los departamentos de Colombia con coberturas de OPV inferiores a 80%.This study aims to explore a possible silent circulation of wild and vaccine-derived polioviruses in departments of Colombia with polio vaccination coverage of below 80%. The study collected 52 samples of wastewater concentrated as a result of precipitation with polyethylene glycol and sodium chloride. The viral detection was carried out through isolation and the identification through neutralization of the cytopathic effect, as well as through a conventional polymerase chain reaction following reverse transcription. The isolated polioviruses were characterized by the VP1 gene sequence. In two of the 52 samples, there was a presence of the Sabin type 2 poliovirus with more than 99% sequence similarity with the Sabin type 2 strain polio. Circulation of the nonpolio enterovirus was

  7. [Genetic recombination in vaccine poliovirus: comparative study in strains excreted in course of vaccination by oral poliovirus vaccine and circulating strains].

    Science.gov (United States)

    Haddad-Boubaker, S; Ould-Mohamed-Abdallah, M V; Ben-Yahia, A; Triki, H

    2010-12-01

    Recombination is one of the major mechanisms of evolution in poliovirus. In this work, recombination was assessed in children during vaccination with OPV and among circulating vaccine strains isolated in Tunisia during the last 15 years in order to identify a possible role of recombination in the response to the vaccine or the acquisition of an increased transmissibility. This study included 250 poliovirus isolates: 137 vaccine isolates, excreted by children during primary vaccination with OPV and 113 isolates obtained from acute flaccid paralytic (AFP) cases and healthy contacts. Recombination was first assessed using a double PCR-RFLP, and sequencing. Nineteen per cent of recombinant strains were identified: 20% of strains excreted by vaccinees among 18% of circulating strains. The proportion of recombinant in isolates of serotype1 was very low in the two groups while the proportions of recombinants in serotypes 2 and 3 were different. In vaccinees, the frequency of recombinants in serotype3 decreased during the course of vaccination: 54% after the first dose, 32% after the second and 14% after the third dose. These results suggest that recombination enhances the ability of serotype3 vaccine strains to induce an immune response. Apart from recent vaccination, it may contribute to a more effective transmissibility of vaccine strains among human population. Copyright © 2009 Elsevier Masson SAS. All rights reserved.

  8. Nuclear entry of poliovirus protease-polymerase precursor 3CD: implications for host cell transcription shut-off

    International Nuclear Information System (INIS)

    Sharma, Rakhi; Raychaudhuri, Santanu; Dasgupta, Asim

    2004-01-01

    Host cell transcription mediated by all three RNA polymerases is rapidly inhibited after infection of mammalian cells with poliovirus (PV). Both genetic and biochemical studies have shown that the virus-encoded protease 3C cleaves the TATA-binding protein and other transcription factors at glutamine-glycine sites and is directly responsible for host cell transcription shut-off. PV replicates in the cytoplasm of infected cells. To shut-off host cell transcription, 3C or a precursor of 3C must enter the nucleus of infected cells. Although the 3C protease itself lacks a nuclear localization signal (NLS), amino acid sequence examination of 3D identified a potential single basic type NLS, KKKRD, spanning amino acids 125-129 within this polypeptide. Thus, a plausible scenario is that 3C enters the nucleus in the form of its precursor, 3CD, which then generates 3C by auto-proteolysis ultimately leading to cleavage of transcription factors in the nucleus. Using transient transfection of enhanced green fluorescent protein (EGFP) fusion polypeptides, we demonstrate here that both 3CD and 3D are capable of entering the nucleus in PV-infected cells. However, both polypeptides remain in the cytoplasm in uninfected HeLa cells. Mutagenesis of the NLS sequence in 3D prevents nuclear entry of 3D and 3CD in PV-infected cells. We also demonstrate that 3CD can be detected in the nuclear fraction from PV-infected HeLa cells as early as 2 h postinfection. Significant amount of 3CD is found associated with the nuclear fraction by 3-4 h of infection. Taken together, these results suggest that both the 3D NLS and PV infection are required for the entry of 3CD into the nucleus and that this may constitute a means by which viral protease 3C is delivered into the nucleus leading to host cell transcription shut-off

  9. A ribonuclease coordinates siRNA amplification and mRNA cleavage during RNAi.

    Science.gov (United States)

    Tsai, Hsin-Yue; Chen, Chun-Chieh G; Conte, Darryl; Moresco, James J; Chaves, Daniel A; Mitani, Shohei; Yates, John R; Tsai, Ming-Daw; Mello, Craig C

    2015-01-29

    Effective silencing by RNA-interference (RNAi) depends on mechanisms that amplify and propagate the silencing signal. In some organisms, small-interfering RNAs (siRNAs) are amplified from target mRNAs by RNA-dependent RNA polymerase (RdRP). Both RdRP recruitment and mRNA silencing require Argonaute proteins, which are generally thought to degrade RNAi targets by directly cleaving them. However, in C. elegans, the enzymatic activity of the primary Argonaute, RDE-1, is not required for silencing activity. We show that RDE-1 can instead recruit an endoribonuclease, RDE-8, to target RNA. RDE-8 can cleave RNA in vitro and is needed for the production of 3' uridylated fragments of target mRNA in vivo. We also find that RDE-8 promotes RdRP activity, thereby ensuring amplification of siRNAs. Together, our findings suggest a model in which RDE-8 cleaves target mRNAs to mediate silencing, while generating 3' uridylated mRNA fragments to serve as templates for the RdRP-directed amplification of the silencing signal. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. RNA Crystallization

    Science.gov (United States)

    Golden, Barbara L.; Kundrot, Craig E.

    2003-01-01

    RNA molecules may be crystallized using variations of the methods developed for protein crystallography. As the technology has become available to syntheisize and purify RNA molecules in the quantities and with the quality that is required for crystallography, the field of RNA structure has exploded. The first consideration when crystallizing an RNA is the sequence, which may be varied in a rational way to enhance crystallizability or prevent formation of alternate structures. Once a sequence has been designed, the RNA may be synthesized chemically by solid-state synthesis, or it may be produced enzymatically using RNA polymerase and an appropriate DNA template. Purification of milligram quantities of RNA can be accomplished by HPLC or gel electrophoresis. As with proteins, crystallization of RNA is usually accomplished by vapor diffusion techniques. There are several considerations that are either unique to RNA crystallization or more important for RNA crystallization. Techniques for design, synthesis, purification, and crystallization of RNAs will be reviewed here.

  11. Bacterial RNA induces myocyte cellular dysfunction through the activation of PKR

    Science.gov (United States)

    Bleiblo, Farag; Michael, Paul; Brabant, Danielle; Ramana, Chilakamarti V.; Tai, TC; Saleh, Mazen; Parrillo, Joseph E.; Kumar, Anand

    2012-01-01

    Severe sepsis and the ensuing septic shock are serious life threatening conditions. These diseases are triggered by the host's over exuberant systemic response to the infecting pathogen. Several surveillance mechanisms have evolved to discriminate self from foreign RNA and accordingly trigger effective cellular responses to target the pathogenic threats. The RNA-dependent protein kinase (PKR) is a key component of the cytoplasmic RNA sensors involved in the recognition of viral double-stranded RNA (dsRNA). Here, we identify bacterial RNA as a distinct pathogenic pattern recognized by PKR. Our results indicate that natural RNA derived from bacteria directly binds to and activates PKR. We further show that bacterial RNA induces human cardiac myocyte apoptosis and identify the requirement for PKR in mediating this response. In addition to bacterial immunity, the results presented here may also have implications in cardiac pathophysiology. PMID:22833816

  12. Natural Type 3/Type 2 Intertypic Vaccine-Related Poliovirus Recombinants with the First Crossover Sites within the VP1 Capsid Coding Region

    DEFF Research Database (Denmark)

    Zhang, Yong; Zhu, Shuangli; Yan, Dongmei

    2010-01-01

    Ten uncommon natural type 3/type 2 intertypic poliovirus recombinants were isolated from stool specimens from nine acute flaccid paralysis case patients and one healthy vaccinee in China from 2001 to 2008.......Ten uncommon natural type 3/type 2 intertypic poliovirus recombinants were isolated from stool specimens from nine acute flaccid paralysis case patients and one healthy vaccinee in China from 2001 to 2008....

  13. Reactogenicity and immunogenicity of inactivated poliovirus vaccine produced from Sabin strains: a phase I Trial in healthy adults in Cuba.

    Science.gov (United States)

    Resik, Sonia; Tejeda, Alina; Fonseca, Magilé; Alemañi, Nilda; Diaz, Manuel; Martinez, Yenisleidys; Garcia, Gloria; Okayasu, Hiromasa; Burton, Anthony; Bakker, Wilfried A M; Verdijk, Pauline; Sutter, Roland W

    2014-09-22

    To ensure that developing countries have the option to produce inactivated poliovirus vaccine (IPV), the Global Polio Eradication Initiative has promoted the development of an IPV using Sabin poliovirus strains (Sabin IPV). This trial assessed the reactogenicity and immunogenicity of Sabin IPV and adjuvanted Sabin IPV in healthy adults in Cuba. This is a randomized, controlled phase I trial, enrolling 60 healthy (previously vaccinated) male human volunteers, aged 19-23 years to receive one dose of either Sabin IPV (20:32:64 DU/dose), adjuvanted Sabin IPV (10:16:32 DU/dose), or conventional Salk IPV (40:8:32 DU/dose). The primary endpoint for reactogenicity relied on monitoring of adverse events. The secondary endpoint measured boosting immune responses (i.e. seroconversion or 4-fold rise) of poliovirus antibody, assessed by neutralization assays. Sixty subjects fulfilled the study requirements. No serious adverse events reported were attributed to trial interventions during the 6-month follow-up period. Twenty-eight days after vaccination, boosting immune responses against poliovirus types 1-3 were between 90% and 100% in all vaccination groups. There was a more than 6-fold increase in median antibody titers between pre- and post-vaccination titers in all vaccination groups. Both Sabin IPV and adjuvanted Sabin IPV were well tolerated and immunogenic against all poliovirus serotypes. This result suggests that the aluminum adjuvant may allow a 50% (or higher) dose reduction. Copyright © 2014. Published by Elsevier Ltd.

  14. A poliomyelitis model through mucosal infection in transgenic mice bearing human poliovirus receptor, TgPVR21

    International Nuclear Information System (INIS)

    Nagata, Noriyo; Iwasaki, Takuya; Ami, Yasushi; Sato, Yuko; Hatano, Ikuyoshi; Harashima, Ayako; Suzaki, Yuriko; Yoshii, Takao; Hashikawa, Tsutomu; Sata, Tetsutaro; Horiuchi, Yoshinobu; Koike, Satoshi; Kurata, Takeshi; Nomoto, Akio

    2004-01-01

    Transgenic mice bearing the human poliovirus receptor (TgPVR) are less susceptible to oral inoculation, although they are susceptible to parenteral inoculation. We investigated the susceptibility of TgPVR 21 line [Arch. Virol. 130 (1994) 351] to poliovirus through various mucosal routes. Intranasal inoculation of a neurovirulent Mahoney strain (OM1) caused flaccid paralysis with viral replication in the central nervous system at a dose of 10 6 cell culture infectious dose (CCID 50 ), in contrast, no paralysis following oral or intragastric inoculation of the same dose. Intranasal inoculation of a vaccine strain, Sabin 1, at 10 6 CCID 50 , resulted in no paralysis. Initial replication of poliovirus in the nasal cavity was confirmed by virus isolation and detection of negative-stranded replicative intermediates by RT-PCR and viral antigens using a high-sensitive immunohistochemistry and genome/transcripts by in situ hybridization. Poliovirus-specific IgG antibodies were elevated in the sera of surviving TgPVR21. This model can be used as a mucosal infection model and for differentiation of neurovirulent and attenuated poliovirus strains

  15. Insights from a Systematic Search for Information on Designs, Costs, and Effectiveness of Poliovirus Environmental Surveillance Systems.

    Science.gov (United States)

    Duintjer Tebbens, Radboud J; Zimmermann, Marita; Pallansch, Mark A; Thompson, Kimberly M

    2017-12-01

    Poliovirus surveillance plays a critical role in achieving and certifying eradication and will play a key role in the polio endgame. Environmental surveillance can provide an opportunity to detect circulating polioviruses prior to the observation of any acute flaccid paralysis cases. We completed a systematic review of peer-reviewed publications on environmental surveillance for polio including the search terms "environmental surveillance" or "sewage," and "polio," "poliovirus," or "poliomyelitis," and compared characteristics of the resulting studies. The review included 146 studies representing 101 environmental surveillance activities from 48 countries published between 1975 and 2016. Studies reported taking samples from sewage treatment facilities, surface waters, and various other environmental sources, although they generally did not present sufficient details to thoroughly evaluate the sewage systems and catchment areas. When reported, catchment areas varied from 50 to over 7.3 million people (median of 500,000 for the 25% of activities that reported catchment areas, notably with 60% of the studies not reporting this information and 16% reporting insufficient information to estimate the catchment area population size). While numerous studies reported the ability of environmental surveillance to detect polioviruses in the absence of clinical cases, the review revealed very limited information about the costs and limited information to support quantitative population effectiveness of conducting environmental surveillance. This review motivates future studies to better characterize poliovirus environmental surveillance systems and the potential value of information that they may provide in the polio endgame.

  16. RNA Origami

    DEFF Research Database (Denmark)

    Sparvath, Steffen Lynge

    introducerede vores gruppe den enkeltstrengede RNA-origami metode, der giver mulighed for cotranscriptional foldning af veldefinerede nanostrukturer, og er en central del af arbejdet præsenteret heri. Denne ph.d.-afhandling udforsker potentielle anvendelser af RNA-origami nanostrukturer, som nanomedicin eller...... biosensorer. Afhandlingen består af en introduktion til RNA-nanoteknologi feltet, en introduktion af enkeltstrenget RNA-origami design, og fire studier, der beskriver design, produktion og karakterisering af både strukturelle og funktionelle RNA-origamier. Flere RNA-origami designs er blevet undersøgt, og...... projekterne, der indgår i denne afhandling, inkluderer de nyeste fremskridt indenfor strukturel RNA-nanoteknologi og udvikling af funktionelle RNA-baserede enheder. Det første studie beskriver konstruktion og karakterisering af en enkeltstrenget 6-helix RNA-origami stuktur, som er den første demonstration af...

  17. Sporadic isolation of sabin-like polioviruses and high-level detection of non-polio enteroviruses during sewage surveillance in seven Italian cities, after several years of inactivated poliovirus vaccination.

    Science.gov (United States)

    Battistone, A; Buttinelli, G; Fiore, S; Amato, C; Bonomo, P; Patti, A M; Vulcano, A; Barbi, M; Binda, S; Pellegrinelli, L; Tanzi, M L; Affanni, P; Castiglia, P; Germinario, C; Mercurio, P; Cicala, A; Triassi, M; Pennino, F; Fiore, L

    2014-08-01

    Sewage surveillance in seven Italian cities between 2005 and 2008, after the introduction of inactivated poliovirus vaccination (IPV) in 2002, showed rare polioviruses, none that were wild-type or circulating vaccine-derived poliovirus (cVDPV), and many other enteroviruses among 1,392 samples analyzed. Two of five polioviruses (PV) detected were Sabin-like PV2 and three PV3, based on enzyme-linked immunosorbent assay (ELISA) and PCR results. Neurovirulence-related mutations were found in the 5'noncoding region (5'NCR) of all strains and, for a PV2, also in VP1 region 143 (Ile>Thr). Intertypic recombination in the 3D region was detected in a second PV2 (Sabin 2/Sabin 1) and a PV3 (Sabin 3/Sabin 2). The low mutation rate in VP1 for all PVs suggests limited interhuman virus passages, consistent with efficient polio immunization in Italy. Nonetheless, these findings highlight the risk of wild or Sabin poliovirus reintroduction from abroad. Non-polio enteroviruses (NPEVs) were detected, 448 of which were coxsackievirus B (CVB) and 294 of which were echoviruses (Echo). Fifty-six NPEVs failing serological typing were characterized by sequencing the VP1 region (nucleotides [nt] 2628 to 2976). A total of 448 CVB and 294 Echo strains were identified; among those strains, CVB2, CVB5, and Echo 11 predominated. Environmental CVB5 and CVB2 strains from this study showed high sequence identity with GenBank global strains. The high similarity between environmental NPEVs and clinical strains from the same areas of Italy and the same periods indicates that environmental strains reflect the viruses circulating in the population and highlights the potential risk of inefficient wastewater treatments. This study confirmed that sewage surveillance can be more sensitive than acute flaccid paralysis (AFP) surveillance in monitoring silent poliovirus circulation in the population as well as the suitability of molecular approaches to enterovirus typing.

  18. CpG oligodeoxynucleotides are a potent adjuvant for an inactivated polio vaccine produced from Sabin strains of poliovirus.

    Science.gov (United States)

    Yang, Chunting; Shi, Huiying; Zhou, Jun; Liang, Yanwen; Xu, Honglin

    2009-11-05

    Poliovirus transmission is controlled globally through world-wide use of a live attenuated oral polio vaccine (OPV). However, the imminence of global poliovirus eradication calls for a switch to the inactivated polio vaccine (IPV). Given the limited manufacturing capacity and high cost of IPV, this switch is unlikely in most developing and undeveloped countries. Adjuvantation is an effective strategy for antigen sparing. In this study, we evaluated the adjuvanticity of CpG oligodeoxynucleotides (CpG-ODN) for an experimental IPV produced from Sabin strains of poliovirus. Our results showed that CpG-ODN, alone or in combination with alum, can significantly enhance both the humoral and cellular immune responses to IPV in mice, and, consequently, the antigen dose could be reduced substantially. Therefore, our study suggests that the global use of IPV could be facilitated by using CpG-ODN or other feasible adjuvants.

  19. Acute flaccid paralysis surveillance in bosnia and herzegovina: Recent isolation of two sabin like type 2 poliovirus.

    Science.gov (United States)

    Fontana, Stefano; Buttinelli, Gabriele; Fiore, Stefano; Mulaomerovic, Mirsada; Aćimović, Jela; Amato, Concetta; Delogu, Roberto; Rezza, Giovanni; Stefanelli, Paola

    2017-09-01

    The WHO Regional Commission for the Certification of Poliomyelitis Eradication has recently indicated Bosnia and Herzegovina (B&H) as a high risk country for transmission, following importation, of wild poliovirus (WPV) or circulating vaccine-derived poliovirus (cVDPV). We analyzed data on Acute Flaccid Paralysis (AFP) surveillance between 2007 to 2016, and the trend of polio immunization coverage in B&H. The majority of AFP cases was recorded in 2016 suggesting an enhancement of the AFP surveillance activities. However, the decline in the immunization coverage, around 74%, and the isolation of two Sabin-like poliovirus type 2 strains, one of them close to a VDPV, require a particular attention in the area. Although B&H has successfully maintained its polio-free status since 2002 several challenges need to be addressed. © 2017 Wiley Periodicals, Inc.

  20. Cross-sectional Serologic Assessment of Immunity to Poliovirus in Differential Risk Areas of India: India Seroprevalence Survey - 2014.

    Science.gov (United States)

    Ahmad, Mohammad; Bahl, Sunil; Kunwar, Abhishek

    2016-08-07

    To assess the seroprevalence against all three poliovirus serotypes in traditional high risk areas in Bihar, lowest routine immunization coverage areas in Madhya Pradesh and migrant population living in Mumbai urban slums. Cross-sectional Survey. Subjects selected by house to house visit (community based) and transported to government health facilities for further study procedures. 1137 randomly selected healthy infants 6-11 months of age residing in the selected high-risk areas. Serum samples from the study site were shipped to Enterovirus Research Centre (ERC), Mumbai to determine the neutralizing antibodies against all three poliovirus serotypes. Children with a reciprocal antibody titer ≥1:8 were considered seropositive to the specific poliovirus. Overall, seroprevalence in all the three study areas was 98%, 98% and 91% against poliovirus type-1, type-2 and type-3, respectively. Bihar had a seroprevalence of 99%, 99% and 92% against type-1, type-2 and type-3 respectively. Corresponding figures for Madhya Pradesh and Mumbai were 98%, 99% and 88% and 98%, 97% and 94%, respectively. The study found high seroprevalence against all three poliovirus types not only in the traditional high-risk areas for polio in India, but even in the areas known to have low routine immunization coverage and among the migratory clusters living in Mumbai urban slums. Type-2 seroprevalence was found to be high. These findings are reassuring against the threat of emergence of circulating vaccine derived polioviruses (cVDPVs) in the country subsequent to switch from trivalent oral polio vaccine to bivalent oral polio vaccine in the routine immunization schedule from April 2016.

  1. Twenty-Eight Years of Poliovirus Replication in an Immunodeficient Individual: Impact on the Global Polio Eradication Initiative.

    Science.gov (United States)

    Dunn, Glynis; Klapsa, Dimitra; Wilton, Thomas; Stone, Lindsay; Minor, Philip D; Martin, Javier

    2015-08-01

    There are currently huge efforts by the World Health Organization and partners to complete global polio eradication. With the significant decline in poliomyelitis cases due to wild poliovirus in recent years, rare cases related to the use of live-attenuated oral polio vaccine assume greater importance. Poliovirus strains in the oral vaccine are known to quickly revert to neurovirulent phenotype following replication in humans after immunisation. These strains can transmit from person to person leading to poliomyelitis outbreaks and can replicate for long periods of time in immunodeficient individuals leading to paralysis or chronic infection, with currently no effective treatment to stop excretion from these patients. Here, we describe an individual who has been excreting type 2 vaccine-derived poliovirus for twenty eight years as estimated by the molecular clock established with VP1 capsid gene nucleotide sequences of serial isolates. This represents by far the longest period of excretion described from such a patient who is the only identified individual known to be excreting highly evolved vaccine-derived poliovirus at present. Using a range of in vivo and in vitro assays we show that the viruses are very virulent, antigenically drifted and excreted at high titre suggesting that such chronic excreters pose an obvious risk to the eradication programme. Our results in virus neutralization assays with human sera and immunisation-challenge experiments using transgenic mice expressing the human poliovirus receptor indicate that while maintaining high immunisation coverage will likely confer protection against paralytic disease caused by these viruses, significant changes in immunisation strategies might be required to effectively stop their occurrence and potential widespread transmission. Eventually, new stable live-attenuated polio vaccines with no risk of reversion might be required to respond to any poliovirus isolation in the post-eradication era.

  2. The role of supplementary environmental surveillance to complement acute flaccid paralysis surveillance for wild poliovirus in Pakistan - 2011-2013.

    Directory of Open Access Journals (Sweden)

    Tori L Cowger

    Full Text Available More than 99% of poliovirus infections are non-paralytic and therefore, not detected by acute flaccid paralysis (AFP surveillance. Environmental surveillance (ES can detect circulating polioviruses from sewage without relying on clinical presentation. With extensive ES and continued circulation of polioviruses, Pakistan presents a unique opportunity to quantify the impact of ES as a supplement to AFP surveillance on overall completeness and timeliness of poliovirus detection.Genetic, geographic and temporal data were obtained for all wild poliovirus (WPV isolates detected in Pakistan from January 2011 through December 2013. We used viral genetics to assess gaps in AFP surveillance and ES as measured by detection of 'orphan viruses' (≥1.5% different in VP1 capsid nucleotide sequence. We compared preceding detection of closely related circulating isolates (≥99% identity detected by AFP surveillance or ES to determine which surveillance system first detected circulation before the presentation of each polio case.A total of 1,127 WPV isolates were detected by AFP surveillance and ES in Pakistan from 2011-2013. AFP surveillance and ES combined exhibited fewer gaps (i.e., % orphan viruses in detection than AFP surveillance alone (3.3% vs. 7.7%, respectively. ES detected circulation before AFP surveillance in nearly 60% of polio cases (200 of 346. For polio cases reported from provinces conducting ES, ES detected circulation nearly four months sooner on average (117.6 days than did AFP surveillance.Our findings suggest ES in Pakistan is providing earlier, more sensitive detection of wild polioviruses than AFP surveillance alone. Overall, targeted ES through strategic selection of sites has important implications in the eradication endgame strategy.

  3. Synthesis of double-stranded RNA in a virus-enriched fraction from Agaricus bisporus

    International Nuclear Information System (INIS)

    Sriskantha, A.; Wach, P.; Schlagnhaufer, B.; Romaine, C.P.

    1986-01-01

    Partially purified virus preparations from sporophores of Agaricus bisporus affected with LaFrance disease had up to a 15-fold-higher RNA-dependent RNA polymerase activity than did comparable preparations from health sporophores. Enzyme activity was dependent upon the presence of Mg 2+ and the four nucleoside triphosphates and was insensitive to actinomycin D, α-amanitin, and rifampin. The 3 H-labeled enzyme reaction products were double-stranded RNA (dsRNA) as indicated by CF-11 cellulose column chromatography and by their ionic-strength-dependent sensitivity to hydrolysis by RNase A. The principal dsRNA products had estimated molecular weights of 4.3 /times/ 10 6 and 1.4 /times/ 10 6 . Cs 2 SO 4 equilibrium centrifugation of the virus preparation resolved a single peak of RNA polymerase activity that banded with a 35-nm spherical virus particle containing dsRNAs with molecular weights of 4.3 /times/ 10 6 and 1.4 /times/ 10 6 . The data suggest that the RNA-dependent RNA polymerase associated with the 35-nm spherical virus is a replicase which catalyzes the synthesis of the genomic dsRNAs

  4. A rapid microassay for detecting antibodies against poliovirus based on [14C]thymidine uptake of treated cell cultures

    International Nuclear Information System (INIS)

    Hilfenhaus, J.; Damm, H.; Ziegelmaier, R.; Gruschkau, H.

    1977-01-01

    DNA synthesis of mammalian cells propagated in microplates can easily be measured if cell cultures incubated with [ 14 C]thymidine are harvested on to glass fibre filters by a semiautomatic harvesting technique. Soon after infection with poliovirus, [ 14 C]thymidine uptake of U cells (established, human amniotic cell line) is inhibited. This inhibition can be prevented by previous virus neutralization with antibody. Based on this effect a rapid, precise assay method was set up to determine neutralizing antibody titres against poliovirus. There was a good correlation between titres obtained by this assay and those obtained by 50% endpoint titrations in cytopathogenic effect inhibition assays

  5. Stool screening of Syrian refugees and asylum seekers in Germany, 2013/2014: Identification of Sabin like polioviruses.

    Science.gov (United States)

    Böttcher, Sindy; Neubauer, Katrin; Baillot, Armin; Rieder, Gabriele; Adam, Maja; Diedrich, Sabine

    2015-10-01

    Germany is a partner of the Global Polio Eradication Initiative. Assurance of polio free status is based on enterovirus surveillance, which focuses on patients with signs of acute flaccid paralysis or aseptic meningitis/encephalitis, representing the key symptoms of poliovirus infection. In response to the wild poliovirus outbreak in Syria 2013 and high number of refugees coming from Syria to Germany, stool samples from 629 Syrian refugees/asylum seekers aged Syrian refugees and asylum seekers at that time. Copyright © 2015 Elsevier GmbH. All rights reserved.

  6. Immunogenicity of type 2 monovalent oral and inactivated poliovirus vaccines for type 2 poliovirus outbreak response: an open-label, randomised controlled trial.

    Science.gov (United States)

    Zaman, Khalequ; Estívariz, Concepción F; Morales, Michelle; Yunus, Mohammad; Snider, Cynthia J; Gary, Howard E; Weldon, William C; Oberste, M Steven; Wassilak, Steven G; Pallansch, Mark A; Anand, Abhijeet

    2018-03-20

    Monovalent type 2 oral poliovirus vaccine (mOPV2) and inactivated poliovirus vaccine (IPV) are used to respond to type 2 poliovirus outbreaks. We aimed to assess the effect of two mOPV2 doses on the type 2 immune response by varying the time interval between mOPV2 doses and IPV co-administration with mOPV2. We did a randomised, controlled, parallel, open-label, non-inferiority, inequality trial at two study clinics in Dhaka, Bangladesh. Healthy infants aged 6 weeks (42-48 days) at enrolment were randomly assigned (1:1:1:1) to receive two mOPV2 doses (each dose consisting of two drops [0·1 mL in total] of about 10 5 50% cell culture infectious dose of type 2 Sabin strain) at intervals of 1 week, 2 weeks, 4 weeks (standard or control group), or 4 weeks with IPV (0·5 mL of type 1 [Mahoney, 40 D-antigen units], type 2 [MEF-1, 8 D-antigen units], and type 3 [Saukett, 32 D-antigen units]) administered intramuscularly with the first mOPV2 dose. We used block randomisation, randomly selecting blocks of sizes four, eight, 12, or 16 stratified by study sites. We concealed randomisation assignment from staff managing participants in opaque, sequentially numbered, sealed envelopes. Parents and clinic staff were unmasked to assignment after the randomisation envelope was opened. Laboratory staff analysing sera were masked to assignment, but investigators analysing data and assessing outcomes were not. The primary outcome was type 2 immune response measured 4 weeks after mOPV2 administration. The primary modified intention-to-treat analysis included participants with testable serum samples before and after vaccination. A non-inferiority margin of 10% and p=0·05 (one-tailed) was used. This trial is registered at ClinicalTrials.gov, number NCT02643368, and is closed to accrual. Between Dec 7, 2015, and Jan 5, 2016, we randomly assigned 760 infants to receive two mOPV2 doses at intervals of 1 week (n=191), 2 weeks (n=191), 4 weeks (n=188), or 4 weeks plus IPV (n=190). Immune

  7. The origin and effect of small RNA signaling in plants

    Directory of Open Access Journals (Sweden)

    Jean-Sébastien eParent

    2012-08-01

    Full Text Available Given their sessile condition, land plants need to integrate environmental cues rapidly and send signal throughout the organism to modify their metabolism accordingly. Small RNA (sRNA molecules are among the messengers that plant cells use to carry such signals. These molecules originate from fold-back stem-loops transcribed from endogenous loci or from perfect double-stranded RNA produced through the action of RNA-dependent RNA polymerases. Once produced, sRNAs associate with Argonaute and other proteins to form the RNA-induced silencing complex (RISC that executes silencing of complementary RNA molecules. Depending on the nature of the RNA target and the Argonaute protein involved, RISC triggers either DNA methylation and chromatin modification (leading to transcriptional gene silencing, TGS or RNA cleavage or translational inhibition (leading to post-transcriptional gene silencing, PTGS. In some cases, sRNAs move to neighboring cells and/or to the vascular tissues for long-distance trafficking. Many genes are involved in the biogenesis of sRNAs and recent studies have shown that both their origin and their protein partners have great influence on their activity and range. Here we summarize the work done to uncover the mode of action of the different classes of small RNA with special emphasis on their movement and how plants can take advantage of their mobility. We also review the various genetic requirements needed for production, movement and perception of the silencing signal.

  8. Assessing the risks for poliovirus outbreaks in polio-free countries--Africa, 2012-2013.

    Science.gov (United States)

    2013-09-20

    In 2012, the World Health Assembly of the World Health Organization (WHO) declared the completion of polio eradication a programmatic emergency. Indigenous wild poliovirus (WPV) transmission remains uninterrupted in Nigeria (in the WHO African Region [AFR]) and in Afghanistan and Pakistan (in the WHO Eastern Mediterranean Region [EMR]). In the WHO AFR, multiple WPV outbreaks have occurred since 2003 after importation of indigenous West African WPV into 21 previously polio-free countries in a "WPV importation belt"* that extends across the continent. The Global Polio Eradication Initiative (GPEI) and WHO regional offices have used indicators of population immunity, surveillance quality, and other factors (e.g., high-risk subpopulations and proximity to WPV-affected countries) to assess the risk for outbreaks in polio-free countries and guide the implementation of risk mitigation measures to limit poliovirus transmission after WPV importation and prevent the emergence of circulating vaccine-derived poliovirus (cVDPV). Despite risk mitigation efforts, a polio outbreak, first confirmed in May 2013, is ongoing; as of September 10, a total of 178 WPV type 1 (WPV1) cases have been reported in Somalia† (163 cases), Kenya (14 cases) and Ethiopia (1 case), after importation of WPV1 of West African origin. This report summarizes steps taken by the GPEI to assess and mitigate the risks for outbreaks after WPV importation or the emergence of cVDPV in polio-free countries within the WHO AFR's "WPV importation belt." All countries will continue to have some level of risk for WPV outbreaks as long as endemic circulation continues in Afghanistan, Nigeria, and Pakistan.

  9. Molecular Mechanisms of Attenuation of the Sabin Strain of Poliovirus Type 3

    OpenAIRE

    Guest, Stephen; Pilipenko, Evgeny; Sharma, Kamal; Chumakov, Konstantin; Roos, Raymond P.

    2004-01-01

    Mutations critical for the central nervous system (CNS) attenuation of the Sabin vaccine strains of poliovirus (PV) are located within the viral internal ribosome entry site (IRES). We examined the interaction of the IRESs of PV type 3 (PV3) and Sabin type 3 (Sabin3) with polypyrimidine tract-binding protein (PTB) and a neural cell-specific homologue, nPTB. PTB and nPTB were found to bind to a site directly adjacent to the attenuating mutation, and binding at this site was less efficient on t...

  10. Update on Vaccine-Derived Polioviruses - Worldwide, January 2016-June 2017.

    Science.gov (United States)

    Jorba, Jaume; Diop, Ousmane M; Iber, Jane; Henderson, Elizabeth; Sutter, Roland W; Wassilak, Steven G F; Burns, Cara C

    2017-11-03

    In 1988, the World Health Assembly launched the Global Polio Eradication Initiative (GPEI) (1). Among the three wild poliovirus (WPV) serotypes, only type 1 (WPV1) has been detected since 2012. Since 2014, detection of WPV1 has been limited to three countries, with 37 cases in 2016 and 11 cases in 2017 as of September 27. The >99.99% decline worldwide in polio cases since the launch of the GPEI is attributable to the extensive use of the live, attenuated oral poliovirus vaccine (OPV) in mass vaccination campaigns and comprehensive national routine immunization programs. Despite its well-established safety record, OPV use can be associated with rare emergence of genetically divergent vaccine-derived polioviruses (VDPVs) whose genetic drift from the parental OPV strains indicates prolonged replication or circulation (2). VDPVs can also emerge among persons with primary immunodeficiencies (PIDs). Immunodeficiency-associated VDPVs (iVDPVs) can replicate for years in some persons with PIDs. In addition, circulating vaccine-derived polioviruses (cVDPVs) can emerge very rarely among immunologically normal vaccine recipients and their contacts in areas with inadequate OPV coverage and can cause outbreaks of paralytic polio. This report updates previous summaries regarding VDPVs (3). During January 2016-June 2017, new cVDPV outbreaks were identified, including two in the Democratic Republic of the Congo (DRC) (eight cases), and another in Syria (35 cases), whereas the circulation of cVDPV type 2 (cVDPV2) in Nigeria resulted in cVDPV2 detection linked to a previous emergence. The last confirmed case from the 2015-2016 cVDPV type 1 (cVDPV1) outbreak in Laos occurred in January 2016. Fourteen newly identified persons in 10 countries were found to excrete iVDPVs, and three previously reported patients in the United Kingdom and Iran (3) were still excreting type 2 iVDPV (iVDPV2) during the reporting period. Ambiguous VDPVs (aVDPVs), isolates that cannot be classified

  11. Update on Vaccine-Derived Polioviruses - Worldwide, January 2014-March 2015.

    Science.gov (United States)

    Diop, Ousmane M; Burns, Cara C; Sutter, Roland W; Wassilak, Steven G; Kew, Olen M

    2015-06-19

    Since the World Health Assembly's 1988 resolution to eradicate poliomyelitis, one of the main tools of the World Health Organization (WHO) Global Polio Eradication Initiative (GPEI) has been the live, attenuated oral poliovirus vaccine (OPV). OPV might require several doses to induce immunity but provides long-term protection against paralytic disease. Through effective use of OPV, GPEI has brought polio to the threshold of eradication. Wild poliovirus type 2 (WPV2) was eliminated in 1999, WPV3 has not been detected since November 2012, and WPV1 circulation appears to be restricted to parts of Pakistan and Afghanistan. However, continued use of OPV carries two key risks. The first, vaccine-associated paralytic poliomyelitis (VAPP) has been recognized since the early 1960s. VAPP is a very rare event that occurs sporadically when an administered dose of OPV reverts to neurovirulence and causes paralysis in the vaccine recipient or a nonimmune contact. VAPP can occur among immunologically normal vaccine recipients and their contacts as well as among persons who have primary immunodeficiencies (PIDs) manifested by defects in antibody production; it is not associated with outbreaks. The second, the emergence of genetically divergent, neurovirulent vaccine-derived polioviruses (VDPVs) was recognized more recently. Circulating VDPVs (cVDPVs) resemble WPVs and, in areas with low OPV coverage, can cause polio outbreaks. Immunodeficiency-associated VDPVs (iVDPVs) can replicate and be excreted for years in some persons with PIDs; GPEI maintains a registry of iVDPV cases. Ambiguous VDPVs (aVDPVs) are isolates that cannot be classified definitively. This report updates previous surveillance summaries and describes VDPVs detected worldwide during January 2014-March 2015. Those include new cVDPV outbreaks in Madagascar and South Sudan, and sharply reduced type 2 cVDPV (cVDPV2) circulation in Nigeria and Pakistan during the latter half of 2014. Eight newly identified persons in

  12. Effect of ionic environment on the inactivation of poliovirus in water by chlorine.

    OpenAIRE

    Sharp, D G; Young, D C; Floyd, R; Johnson, J D

    1980-01-01

    The rate of inactivation of poliovirus in water by chlorine is strongly influenced by the pH, which in turn influences the relative amounts of HOCl and OCl- that are present and acting on the virus in the region of pH 6 to 10. The distribution of HOCl and OCl- is influenced to a lesser extent by the addition of NaCl. The major part of the sharp increase in disinfection rate seen with this salt is thought to be due to its effect on the virus itself resulting in an increased chlorine sensitivit...

  13. Sabin Vaccine Reversion in the Field: a Comprehensive Analysis of Sabin-Like Poliovirus Isolates in Nigeria.

    Science.gov (United States)

    Famulare, Michael; Chang, Stewart; Iber, Jane; Zhao, Kun; Adeniji, Johnson A; Bukbuk, David; Baba, Marycelin; Behrend, Matthew; Burns, Cara C; Oberste, M Steven

    2016-01-01

    To assess the dynamics of genetic reversion of live poliovirus vaccine in humans, we studied molecular evolution in Sabin-like poliovirus isolates from Nigerian acute flaccid paralysis cases obtained from routine surveillance. We employed a novel modeling approach to infer substitution and recombination rates from whole-genome sequences and information about poliovirus infection dynamics and the individual vaccination history. We confirmed observations from a recent vaccine trial that VP1 substitution rates are increased for Sabin-like isolates relative to the rate for the wild type due to increased nonsynonymous substitution rates. We also inferred substitution rates for attenuating nucleotides and confirmed that reversion can occur in days to weeks after vaccination. We combine our observations for Sabin-like virus evolution with the molecular clock for VP1 of circulating wild-type strains to infer that the mean time from the initiating vaccine dose to the earliest detection of circulating vaccine-derived poliovirus (cVDPV) is 300 days for Sabin-like virus type 1, 210 days for Sabin-like virus type 2, and 390 days for Sabin-like virus type 3. Phylogenetic relationships indicated transient local transmission of Sabin-like virus type 3 and, possibly, Sabin-like virus type 1 during periods of low wild polio incidence. Comparison of Sabin-like virus recombinants with known Nigerian vaccine-derived poliovirus recombinants shows that while recombination with non-Sabin enteroviruses is associated with cVDPV, the recombination rates are similar for Sabin isolate-Sabin isolate and Sabin isolate-non-Sabin enterovirus recombination after accounting for the time from dosing to the time of detection. Our study provides a comprehensive picture of the evolutionary dynamics of the oral polio vaccine in the field. The global polio eradication effort has completed its 26th year. Despite success in eliminating wild poliovirus from most of the world, polio persists in populations

  14. HIV-1 transcripts use IRES-initiation under conditions where Cap-dependent translation is restricted by poliovirus 2A protease.

    Directory of Open Access Journals (Sweden)

    Raquel Amorim

    Full Text Available The 30 different species of mRNAs synthesized during the HIV-1 replication cycle are all capped and polyadenilated. Internal ribosome entry sites have been recognized in the 5' untranslated region of some mRNA species of HIV-1, which would contribute to an alternative mechanism of initiation of mRNA translation. However, the Cap-dependent translation is assumed to be the main mechanism driving the initiation of HIV-1 protein synthesis. In this work, we describe a cell system in which lower to higher levels of transient expression of the poliovirus 2A protease strongly inhibited cellular Cap-dependent translation with no toxic effect to the cells during a 72-hour time frame. In this system, the synthesis of HIV-1 proteins was inhibited in a temporal dose-dependent way. Higher levels of 2A protease expression severely inhibited HIV-1 protein synthesis during the first 24 hours of infection consequently inhibiting viral production and infectivity. Intermediate to lower levels of 2A Protease expression caused the inhibition of viral protein synthesis only during the first 48 hours of viral replication. After this period both protein synthesis and viral release were recovered to the control levels. However, the infectivity of viral progeny was still partially inhibited. These results indicate that two mechanisms of mRNA translation initiation contribute to the synthesis of HIV-1 proteins; during the first 24-48 hours of viral replication HIV-1 protein synthesis is strongly dependent on Cap-initiation, while at later time points IRES-driven translation initiation is sufficient to produce high amounts of viral particles.

  15. Immunoassay of poliovirus antigens by single-radial-diffusion: development and characteristics of a sensitive autoradiographic zone size enhancement (ZE) technique

    International Nuclear Information System (INIS)

    Schild, G.C.; Wood, J.M.; Minor, P.D.; Dandawate, C.N.; Magrath, D.I.

    1980-01-01

    The reactions of polioviruses in single-radial-immunodiffusion (SRD) tests were investigated with a view to developing accurate and sensitive antigen assay systems. In direct SRD tests, the reactions were type-specific for polioviruses of types 1, 2 and 3 but the tests were of low sensitivity, being applicable only to the assay of virus concentrates. A novel autoradiographic zone size enhancement (ZE) test was developed which increased the sensitivity of the SRD assay 40-to 100-fold. The ZE test was dependent upon the ability of unlabelled poliovirus to co-migrate with the radioactive marker virus and so enhance the zone size detected autoradiographically. The areas of the autoradiographic zones were directly proportional to the concentration of unlabelled antigen. The ZE test was capable of detecting poliovirus D antigens in amounts corresponding to 10sup(3.3) to 10sup(4.3) TCID 50 of infectious virus. Studies with poliovirus type 3 strains indicated that the ZE test was narrowly strain-specific for the D-antigen of poliovirus type 3 strains when homologous type 3 D-antigen was used as radioactive marker, but broadly cross-reactive for the D-antigen of type 3 viruses when heterologous poliovirus type 3 D-antigen was used as marker. (author)

  16. Screening for long-term poliovirus excretion among children with primary immunodeficiency disorders: preparation for the polio posteradication era in Bangladesh.

    Science.gov (United States)

    Sazzad, Hossain M S; Rainey, Jeanette J; Kahn, Anna-Lea; Mach, Ondrej; Liyanage, Jayantha B L; Alam, Ahmed Nawsher; Kawser, Choudhury A; Hossain, Asgar; Sutter, Roland; Luby, Stephen P

    2014-11-01

    Persons with primary immune deficiency disorders (PIDD) who receive oral poliovirus vaccine (OPV) may transmit immunodeficiency-associated vaccine-derived polioviruses (iVDPVs) and cause paralytic polio. The objective of this study was to identify children with PIDD in Bangladesh, and estimate the proportion with chronic poliovirus excretion. Patients admitted at 5 teaching hospitals were screened for PIDD according to standardized clinical case definitions. PIDD was confirmed by age-specific quantitative immunoglobulin levels. Stool specimens were collected from patients with confirmed PIDD. From February 2011 through January 2013, approximately 96 000 children were screened, and 53 patients were identified who met the clinical case definition for PIDD. Thirteen patients (24%) had age-specific quantitative immunoglobulins results that confirmed PIDD. Of these, 9 (69%) received OPV 3-106 months before stool specimen collection. Among 11 patients, stool specimens from 1 patient tested positive for polioviruses 34 months after OPV ingestion. However, the poliovirus isolate was not available for genetic sequencing, and a subsequent stool specimen 45 days later was negative. The risk of chronic poliovirus excretion among children with PIDD in Bangladesh seems to be low. The national polio eradication program should incorporate strategies for screening for poliovirus excretion among patients with PIDD. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  17. Enterovirus Control of Translation and RNA Granule Stress Responses.

    Science.gov (United States)

    Lloyd, Richard E

    2016-03-30

    Enteroviruses such as poliovirus (PV) and coxsackievirus B3 (CVB3) have evolved several parallel strategies to regulate cellular gene expression and stress responses to ensure efficient expression of the viral genome. Enteroviruses utilize their encoded proteinases to take over the cellular translation apparatus and direct ribosomes to viral mRNAs. In addition, viral proteinases are used to control and repress the two main types of cytoplasmic RNA granules, stress granules (SGs) and processing bodies (P-bodies, PBs), which are stress-responsive dynamic structures involved in repression of gene expression. This review discusses these processes and the current understanding of the underlying mechanisms with respect to enterovirus infections. In addition, the review discusses accumulating data suggesting linkage exists between RNA granule formation and innate immune sensing and activation.

  18. PER.C6(®) cells as a serum-free suspension cell platform for the production of high titer poliovirus: a potential low cost of goods option for world supply of inactivated poliovirus vaccine

    NARCIS (Netherlands)

    Sanders, Barbara P.; Edo-Matas, Diana; Custers, Jerome H. H. V.; Koldijk, Martin H.; Klaren, Vincent; Turk, Marije; Luitjens, Alfred; Bakker, Wilfried A. M.; Uytdehaag, Fons; Goudsmit, Jaap; Lewis, John A.; Schuitemaker, Hanneke

    2013-01-01

    There are two highly efficacious poliovirus vaccines: Sabin's live-attenuated oral polio vaccine (OPV) and Salk's inactivated polio vaccine (IPV). OPV can be made at low costs per dose and is easily administrated. However, the major drawback is the frequent reversion of the OPV vaccine strains to

  19. New insights into siRNA amplification and RNAi.

    Science.gov (United States)

    Zhang, Chi; Ruvkun, Gary

    2012-08-01

    In the nematode Caenorhabditis elegans (C. elegans), gene inactivation by RNA interference can achieve remarkable potency due to the amplification of initial silencing triggers by RNA-dependent RNA polymerases (RdRPs). RdRPs catalyze the biogenesis of an abundant species of secondary small interfering RNAs (siRNAs) using the target mRNA as template. The interaction between primary siRNAs derived from the exogenous double-stranded RNA (dsRNA) trigger and the target mRNA is required for the recruitment of RdRPs. Other genetic requirements for RdRP activities have not been characterized. Recent studies have identified the RDE-10/RDE-11 complex which interacts with the primary siRNA bound target mRNA and acts upstream of the RdRPs. rde-10 and rde-11 mutants show an RNAi defective phenotype because the biogenesis of secondary siRNAs is completely abolished. In addition, the RDE-10/RDE-11 complex plays a similar role in the endogenous RNAi pathway for the biogenesis of a subset of siRNAs targeting recently acquired, duplicated genes.

  20. Comparison of culture, single and multiplex real-time PCR for detection of Sabin poliovirus shedding in recently vaccinated Indian children.

    Science.gov (United States)

    Giri, Sidhartha; Rajan, Anand K; Kumar, Nirmal; Dhanapal, Pavithra; Venkatesan, Jayalakshmi; Iturriza-Gomara, Miren; Taniuchi, Mami; John, Jacob; Abraham, Asha Mary; Kang, Gagandeep

    2017-08-01

    Although, culture is considered the gold standard for poliovirus detection from stool samples, real-time PCR has emerged as a faster and more sensitive alternative. Detection of poliovirus from the stool of recently vaccinated children by culture, single and multiplex real-time PCR was compared. Of the 80 samples tested, 55 (68.75%) were positive by culture compared to 61 (76.25%) and 60 (75%) samples by the single and one step multiplex real-time PCR assays respectively. Real-time PCR (singleplex and multiplex) is more sensitive than culture for poliovirus detection in stool, although the difference was not statistically significant. © 2017 Wiley Periodicals, Inc.

  1. [Role of the National Poliovirus Laboratory for the Program of eradication and poliomyelitis surveillance].

    Science.gov (United States)

    Trallero, Gloria; Cabrerizo, María; Avellón, Ana

    2013-01-01

    The Spanish acute flaccid paralysis surveillance network is coordinated by the National Poliovirus Laboratory (NPL), which, since 1998, carries out polioviruses (PV) and other enteroviruses detected characterization by cell culture and molecular techniques. A total of 110,725 (70046+40679) samples were studied between 1998-2012 and enteroviruses were detected in 8% of these. Among these enteroviruses 241 PV were characterized as PV Sabin-like, except samples belong to an imported poliomyelitis case, all of which were characterised as vaccine derived PV type 2. The NPL has carried out the serotyping and the intratypic differentiation of all the isolated PV in Spain of any syndrome. It is shown that wild PV has not circulated in our country during the 15 years studied and that has led to the signing of the Act of the "eradication of poliomyelitis in Spain" by WHO in 2001, and the /"certification of the eradication of wild PV free for European countries" on 21 June 2002. Currently only 3 countries have endemic transmission of wild PV (Pakistan, Afghanistan and Nigeria). Until a complete worldwide eradication, was achieved, Spain will actively continue to participate in the maintenance of the poliomyelitis eradication infrastructure by monitoring and vaccination as well as the wild PV containment plan to avoid the spread of wild PV.

  2. Antigenic characterization of a formalin-inactivated poliovirus vaccine derived from live-attenuated Sabin strains.

    Science.gov (United States)

    Tano, Yoshio; Shimizu, Hiroyuki; Martin, Javier; Nishimura, Yorihiro; Simizu, Bunsiti; Miyamura, Tatsuo

    2007-10-10

    A candidate inactivated poliovirus vaccine derived from live-attenuated Sabin strains (sIPV), which are used in the oral poliovirus vaccine (OPV), was prepared in a large-production scale. The modification of viral antigenic epitopes during the formalin inactivation process was investigated by capture ELISA assays using type-specific and antigenic site-specific monoclonal antibodies (MoAbs). The major antigenic site 1 was modified during the formalin inactivation of Sabin 1. Antigenic sites 1-3 were slightly modified during the formalin inactivation of Sabin 2 strain. Sites 1 and 3 were altered on inactivated Sabin 3 virus. These alterations were different to those shown by wild-type Saukett strain, used in conventional IPV (cIPV). It has been previously reported that type 1 sIPV showed higher immunogenicity to type 1 cIPV whereas types 2 and 3 sIPV induced lower level of immunogenicity than their cIPV counterparts. Our results suggest that the differences in epitope structure after formalin inactivation may account, at least in part, for the observed differences in immunogenicity between Sabin and wild-type inactivated poliovaccines.

  3. Poliovirus mutants excreted by a chronically infected hypogammaglobulinemic patient establish persistent infections in human intestinal cells

    International Nuclear Information System (INIS)

    Labadie, Karine; Pelletier, Isabelle; Saulnier, Aure; Martin, Javier; Colbere-Garapin, Florence

    2004-01-01

    Immunodeficient patients whose gut is chronically infected by vaccine-derived poliovirus (VDPV) may excrete large amounts of virus for years. To investigate how poliovirus (PV) establishes chronic infections in the gut, we tested whether it is possible to establish persistent VDPV infections in human intestinal Caco-2 cells. Four type 3 VDPV mutants, representative of the viral evolution in the gut of a hypogammaglobulinemic patient over almost 2 years [J. Virol. 74 (2000) 3001], were used to infect both undifferentiated, dividing cells, and differentiated, polarized enterocytes. A VDPV mutant excreted 36 days postvaccination by the patient was lytic in both types of intestinal cell cultures, like the parental Sabin 3 (S3) strain. In contrast, three VDPVs excreted 136, 442, and 637 days postvaccination, established persistent infections both in undifferentiated cells and in enterocytes. Thus, viral determinants selected between day 36 and 136 conferred on VDPV mutants the capacity to infect intestinal cells persistently. The percentage of persistently VDPV-infected cultures was higher in enterocytes than in undifferentiated cells, implicating cellular determinants involved in the differentiation of enterocytes in persistent VDPV infections. The establishment of persistent infections in enterocytes was not due to poor replication of VDPVs in these cells, but was associated with reduced viral adsorption to the cell surface

  4. [Immunoreactivity of chimeric proteins carrying poliovirus epitopes on the VP6 of rotavirus as a vector].

    Science.gov (United States)

    Pan, X-X; Zhao, B-X; Teng, Y-M; Xia, W-Y; Wang, J; Li, X-F; Liao, G-Y; Yang, С; Chen, Y-D

    2016-01-01

    Rotavirus and poliovirus continue to present significant risks and burden of disease to children in developing countries. Developing a combined vaccine may effectively prevent both illnesses and may be advantageous in terms of maximizing compliance and vaccine coverage at the same visit. Recently, we sought to generate a vaccine vector by incorporating multiple epitopes into the rotavirus group antigenic protein, VP6. In the present study, a foreign epitope presenting a system using VP6 as a vector was created with six sites on the outer surface of the vector that could be used for insertion of foreign epitopes, and three VP6-based PV1 epitope chimeric proteins were constructed. The chimeric proteins were confirmed by immunoblot, immunofluorescence assay, and injected into guinea pigs to analyze the epitope-specific humoral response. Results showed that these chimeric proteins reacted with anti-VP6F and -PV1 antibodies, and elicited antibodies against both proteins in guinea pigs. Antibodies against the chimeric proteins carrying PV1 epitopes neutralized rotavirus Wa and PV1 infection in vitro. Our study contributes to a better understanding of the use of VP6-based vectors as multiple-epitope delivery vehicles and the epitopes displayed in this form could be considered for development of epitope-based vaccines against rotavirus and poliovirus.

  5. Lessons From Globally Coordinated Cessation of Serotype 2 Oral Poliovirus Vaccine for the Remaining Serotypes.

    Science.gov (United States)

    Thompson, Kimberly M; Duintjer Tebbens, Radboud J

    2017-07-01

    Comparing model expectations with the experience of oral poliovirus vaccine (OPV) containing serotype 2 (OPV2) cessation can inform risk management for the expected cessation of OPV containing serotypes 1 and 3 (OPV13). We compare the expected post-OPV2-cessation OPV2-related viruses from models with the evidence available approximately 6 months after OPV2 cessation. We also model the trade-offs of use vs nonuse of monovalent OPV (mOPV) for outbreak response considering all 3 serotypes. Although too early to tell definitively, the observed die-out of OPV2-related viruses in populations that attained sufficiently intense trivalent OPV (tOPV) use prior to OPV2 cessation appears consistent with model expectations. As expected, populations that did not intensify tOPV use prior to OPV2 cessation show continued circulation of serotype 2 vaccine-derived polioviruses (VDPVs). Failure to aggressively use mOPV to respond to circulating VDPVs results in a high risk of uncontrolled outbreaks that would require restarting OPV. Ensuring a successful endgame requires more aggressive OPV cessation risk management than has occurred to date for OPV2 cessation. This includes maintaining high population immunity to transmission up until OPV13 cessation, meeting all prerequisites for OPV cessation, and ensuring sufficient vaccine supply to prevent and respond to outbreaks. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America.

  6. Isolation and characterization of a highly evolved type 3 vaccine-derived poliovirus in China.

    Science.gov (United States)

    Zhang, Xiaowei; Qin, Chong; Li, Wei; Zheng, Zhenhua; Wang, Hanzhong; Cui, Zongqiang

    2017-06-15

    In this study, we report the identification and characterization of a highly evolved type 3 vaccine-derived poliovirus (VDPV) strain designated as WIV14, isolated in 2014 from a 4-year-old child suspected of having an enteroviral infection in China. Complete genome sequence of WIV14 revealed multiple nucleotide substitutions when compared with the attenuated poliovirus (PV) Sabin 3, including the reversion of three major attenuation sites to wild type. From the nucleotide divergence for the P1/capsid region, we estimated that the evolution time of WIV14 was more than 7 years, indicating the possible long time of replication. WIV14 strain seemed to have differences in biological characteristics compared with attenuated PV strains, such as being non-temperature-sensitive and producing large plaques. The current isolation of a highly divergent type 3 VDPV gives an idea of the risk of emergent VDPV strains, and emphasizes the importance of maintaining high vaccination coverage and herd immunity against PVs in China. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Spatial model for risk prediction and sub-national prioritization to aid poliovirus eradication in Pakistan.

    Science.gov (United States)

    Mercer, Laina D; Safdar, Rana M; Ahmed, Jamal; Mahamud, Abdirahman; Khan, M Muzaffar; Gerber, Sue; O'Leary, Aiden; Ryan, Mike; Salet, Frank; Kroiss, Steve J; Lyons, Hil; Upfill-Brown, Alexander; Chabot-Couture, Guillaume

    2017-10-11

    Pakistan is one of only three countries where poliovirus circulation remains endemic. For the Pakistan Polio Eradication Program, identifying high risk districts is essential to target interventions and allocate limited resources. Using a hierarchical Bayesian framework we developed a spatial Poisson hurdle model to jointly model the probability of one or more paralytic polio cases, and the number of cases that would be detected in the event of an outbreak. Rates of underimmunization, routine immunization, and population immunity, as well as seasonality and a history of cases were used to project future risk of cases. The expected number of cases in each district in a 6-month period was predicted using indicators from the previous 6-months and the estimated coefficients from the model. The model achieves an average of 90% predictive accuracy as measured by area under the receiver operating characteristic (ROC) curve, for the past 3 years of cases. The risk of poliovirus has decreased dramatically in many of the key reservoir areas in Pakistan. The results of this model have been used to prioritize sub-national areas in Pakistan to receive additional immunization activities, additional monitoring, or other special interventions.

  8. Ausencia de circulación de poliovirus en departamentos colombianos con coberturas vacunales inferiores a 80%

    Directory of Open Access Journals (Sweden)

    María Mercedes González

    2012-08-01

    Full Text Available El presente estudio se propuso explorar la posible circulación silente de poliovirus salvajes y derivados de la vacuna (VDPV, por sus siglas en inglés, en departamentos de Colombia con cobertura de vacunación para polio (OPV, por sus siglas en inglés menor de 80%. Se colectaron 52 muestras de aguas residuales que se concentraron mediante precipitación con polietilenglicol y cloruro de sodio. La detección viral se realizó mediante aislamiento y la identificación por neutralización del efecto citopático, así como mediante reacción en cadena de la polimerasa convencional y en tiempo real, posterior a la transcripción reversa (TR-RCP y rTR-RCP. Los poliovirus aislados se caracterizaron por secuenciación del gen VP1. En dos de las 52 muestras hubo presencia de poliovirus Sabin 2 con más de 99% de similitud de secuencia con la cepa OPV Sabin 2. Se detectó circulación de enterovirus no polio en 17,3% de las muestras. Los serotipos identificados correspondieron a coxsackievirus B1, echovirus 30 y echovirus 11. No se detectaron evidencias de circulación de VDPV ni poliovirus salvaje en los departamentos de Colombia con coberturas de OPV inferiores a 80%.

  9. Radiometric cytolysis inhibition assay, a new rapid test for neutralizing antibodies to intact and trypsin-cleaved poliovirus

    International Nuclear Information System (INIS)

    Hovi, T.; Roivainen, M.

    1989-01-01

    We have developed a new rapid test, the radiometric cytolysis inhibition assay (RACINA), for the determination of neutralizing poliovirus antibodies. HeLa cells prelabeled with 51 Cr, [ 3 H]leucine, or, preferentially, with [ 3 H]uridine are used as sensitive quantitative indicators of residual infectious virus. Both suspensions and monolayer cultures of the indicator cells can be used. Neutralization of a fraction of a high-titer virus preparation can be scored after the first replication cycle at 8 to 10 h. By lowering the incubation temperature to 30 degree C, the completion of the cytolysis due to the first replication cycle of poliovirus was delayed beyond 21 h. This makes it possible to use the RACINA, unlike the standard microneutralization assay, for measuring antibodies to trypsin-cleaved polioviruses. The RACINA was found to be as sensitive as and more reproducible than the standard microneutralization assay in the measurement of neutralizing poliovirus antibodies. The RACINA is a rapid and reliable test for neutralizing antibodies and in principle it may be applicable for quantitation of neutralizing antibodies to other cytolytic agents as well

  10. Inactivated poliovirus type 2 vaccine delivered to rat skin via high density microprojection array elicits potent neutralising antibody responses.

    Science.gov (United States)

    Muller, David A; Pearson, Frances E; Fernando, Germain J P; Agyei-Yeboah, Christiana; Owens, Nick S; Corrie, Simon R; Crichton, Michael L; Wei, Jonathan C J; Weldon, William C; Oberste, M Steven; Young, Paul R; Kendall, Mark A F

    2016-02-25

    Polio eradication is progressing rapidly, and the live attenuated Sabin strains in the oral poliovirus vaccine (OPV) are being removed sequentially, starting with type 2 in April 2016. For risk mitigation, countries are introducing inactivated poliovirus vaccine (IPV) into routine vaccination programs. After April 2016, monovalent type 2 OPV will be available for type 2 outbreak control. Because the current IPV is not suitable for house-to-house vaccination campaigns (the intramuscular injections require health professionals), we developed a high-density microprojection array, the Nanopatch, delivered monovalent type 2 IPV (IPV2) vaccine to the skin. To assess the immunogenicity of the Nanopatch, we performed a dose-matched study in rats, comparing the immunogenicity of IPV2 delivered by intramuscular injection or Nanopatch immunisation. A single dose of 0.2 D-antigen units of IPV2 elicited protective levels of poliovirus antibodies in 100% of animals. However, animals receiving IPV2 by IM required at least 3 immunisations to reach the same neutralising antibody titres. This level of dose reduction (1/40th of a full dose) is unprecedented for poliovirus vaccine delivery. The ease of administration coupled with the dose reduction observed in this study points to the Nanopatch as a potential tool for facilitating inexpensive IPV for mass vaccination campaigns.

  11. N-Terminomics TAILS Identifies Host Cell Substrates of Poliovirus and Coxsackievirus B3 3C Proteinases That Modulate Virus Infection

    Science.gov (United States)

    Jagdeo, Julienne M.; Dufour, Antoine; Klein, Theo; Solis, Nestor; Kleifeld, Oded; Kizhakkedathu, Jayachandran; Luo, Honglin; Overall, Christopher M.

    2018-01-01

    ABSTRACT Enteroviruses encode proteinases that are essential for processing of the translated viral polyprotein. In addition, viral proteinases also target host proteins to manipulate cellular processes and evade innate antiviral responses to promote replication and infection. Although some host protein substrates of enterovirus proteinases have been identified, the full repertoire of targets remains unknown. We used a novel quantitative in vitro proteomics-based approach, termed terminal amine isotopic labeling of substrates (TAILS), to identify with high confidence 72 and 34 new host protein targets of poliovirus and coxsackievirus B3 (CVB3) 3C proteinases (3Cpros) in HeLa cell and cardiomyocyte HL-1 cell lysates, respectively. We validated a subset of candidate substrates that are targets of poliovirus 3Cpro in vitro including three common protein targets, phosphoribosylformylglycinamidine synthetase (PFAS), hnRNP K, and hnRNP M, of both proteinases. 3Cpro-targeted substrates were also cleaved in virus-infected cells but not noncleavable mutant proteins designed from the TAILS-identified cleavage sites. Knockdown of TAILS-identified target proteins modulated infection both negatively and positively, suggesting that cleavage by 3Cpro promotes infection. Indeed, expression of a cleavage-resistant mutant form of the endoplasmic reticulum (ER)-Golgi vesicle-tethering protein p115 decreased viral replication and yield. As the first comprehensive study to identify and validate functional enterovirus 3Cpro substrates in vivo, we conclude that N-terminomics by TAILS is an effective strategy to identify host targets of viral proteinases in a nonbiased manner. IMPORTANCE Enteroviruses are positive-strand RNA viruses that encode proteases that cleave the viral polyprotein into the individual mature viral proteins. In addition, viral proteases target host proteins in order to modulate cellular pathways and block antiviral responses in order to facilitate virus infection

  12. Ancient and novel small RNA pathways compensate for the loss of piRNAs in multiple independent nematode lineages.

    Directory of Open Access Journals (Sweden)

    Peter Sarkies

    2015-02-01

    Full Text Available Small RNA pathways act at the front line of defence against transposable elements across the Eukaryota. In animals, Piwi interacting small RNAs (piRNAs are a crucial arm of this defence. However, the evolutionary relationships among piRNAs and other small RNA pathways targeting transposable elements are poorly resolved. To address this question we sequenced small RNAs from multiple, diverse nematode species, producing the first phylum-wide analysis of how small RNA pathways evolve. Surprisingly, despite their prominence in Caenorhabditis elegans and closely related nematodes, piRNAs are absent in all other nematode lineages. We found that there are at least two evolutionarily distinct mechanisms that compensate for the absence of piRNAs, both involving RNA-dependent RNA polymerases (RdRPs. Whilst one pathway is unique to nematodes, the second involves Dicer-dependent RNA-directed DNA methylation, hitherto unknown in animals, and bears striking similarity to transposon-control mechanisms in fungi and plants. Our results highlight the rapid, context-dependent evolution of small RNA pathways and suggest piRNAs in animals may have replaced an ancient eukaryotic RNA-dependent RNA polymerase pathway to control transposable elements.

  13. The Battle of RNA Synthesis: Virus versus Host.

    Science.gov (United States)

    Harwig, Alex; Landick, Robert; Berkhout, Ben

    2017-10-21

    Transcription control is the foundation of gene regulation. Whereas a cell is fully equipped for this task, viruses often depend on the host to supply tools for their transcription program. Over the course of evolution and adaptation, viruses have found diverse ways to optimally exploit cellular host processes such as transcription to their own benefit. Just as cells are increasingly understood to employ nascent RNAs in transcription regulation, recent discoveries are revealing how viruses use nascent RNAs to benefit their own gene expression. In this review, we first outline the two different transcription programs used by viruses, i.e., transcription (DNA-dependent) and RNA-dependent RNA synthesis. Subsequently, we use the distinct stages (initiation, elongation, termination) to describe the latest insights into nascent RNA-mediated regulation in the context of each relevant stage.

  14. Specific cross-linking of capsid proteins to virus RNA by ultraviolet irradiation of polio virus

    Energy Technology Data Exchange (ETDEWEB)

    Wetz, K.; Habermehl, K.O. (Freie Univ. Berlin (Germany, F.R.))

    1982-04-01

    Poliovirus was irradiated with u.v. light under conditions causing approx. 5% cross-linking of capsid protein to virus RNA. Cross-linked RNA-protein complexes, freed from unbound protein, were treated with nuclease, and then analysed on SDS-polyacrylamide gels. The smallest capsid polypeptide VP4 was found to be associated with the RNA to the greatest degree, followed by VP2 and VP1, while VP3 was attached only in trace amounts. Low radiation doses, which produced cross-linking of RNA to protein, did not cause breakdown of the virus particles or conformational changes of the capsid as examined physically and serologically. However, higher doses caused structural alterations of the virus capsid.

  15. Specific cross-linking of capsid proteins to virus RNA by ultraviolet irradiation of polio virus

    International Nuclear Information System (INIS)

    Wetz, K.; Habermehl, K.-O.

    1982-01-01

    Poliovirus was irradiated with u.v. light under conditions causing approx. 5% cross-linking of capsid protein to virus RNA. Cross-linked RNA-protein complexes, freed from unbound protein, were treated with nuclease, and then analysed on SDS-polyacrylamide gels. The smallest capsid polypeptide VP4 was found to be associated with the RNA to the greatest degree, followed by VP2 and VP1, while VP3 was attached only in trace amounts. Low radiation doses, which produced cross-linking of RNA to protein, did not cause breakdown of the virus particles or conformational changes of the capsid as examined physically and serologically. However, higher doses caused structural alterations of the virus capsid. (author)

  16. Environmental Surveillance of Polioviruses in Rio de Janeiro, Brazil, in Support to the Activities of Global Polio Eradication Initiative.

    Science.gov (United States)

    de Oliveira Pereira, Joseane Simone; da Silva, Lidiane Rodrigues; de Meireles Nunes, Amanda; de Souza Oliveira, Silas; da Costa, Eliane Veiga; da Silva, Edson Elias

    2016-03-01

    Wild polioviruses still remain endemic in three countries (Afghanistan, Pakistan, and Nigeria) and re-emergency of wild polio has been reported in previously polio-free countries. Environmental surveillance has been used as a supplementary tool in monitoring the circulation of wild poliovirus (PVs) and/or vaccine-derived PVs even in the absence of acute flaccid paralysis cases. This study aimed to monitor the presence of polioviruses in wastewater samples collected at one wastewater treatment plant located in the municipality of Rio de Janeiro, Brazil. From December 2011 to June 2012 and from September to December 2012, 31 samples were collected and processed. RD and L20B cell cultures were able to isolate PVs and non-polio enteroviruses in 27/31 samples. Polioviruses were isolated in eight samples (type 1 Sabin = 1, type 2 Sabin = 5, and type 3 Sabin = 2). Vaccine-derived polioviruses were not detected nor evidence of recombination with other PVs or non-polio enterovirus serotypes were observed among the isolates. The Sabin-related serotypes 2 and 3 presented nucleotide substitutions in positions associated with the neurovirulent phenotype at the 5'-UTR. Changes in important Amino acid residues at VP1 were also observed in the serotypes 2 and 3. Environmental surveillance has been used successfully in monitoring the circulation of PVs and non-polio enteroviruses and it is of crucial importance in the final stages of the WHO global polio eradication initiative. Our results show the continuous circulation of Sabin-like PVs and non-polio enteroviruses in the analyzed area during the study period.

  17. The compatibility of inactivated-Enterovirus 71 vaccination with Coxsackievirus A16 and Poliovirus immunizations in humans and animals

    Science.gov (United States)

    Mao, Qunying; Wang, Yiping; Shao, Jie; Ying, Zhifang; Gao, Fan; Yao, Xin; Li, Changgui; Ye, Qiang; Xu, Miao; Li, Rongcheng; Zhu, Fengcai; Liang, Zhenglun

    2015-01-01

    Enterovirus 71 (EV71) is the key pathogen for Hand, Foot, and Mouth Disease (HFMD) and can result in severe neurological complications and death among young children. Three inactivated-EV71 vaccines have gone through phase III clinical trials and have demonstrated good safety and efficacy. These vaccines will benefit young children under the threat of severe HFMD. However, the potential immunization-related compatibility for different enterovirus vaccines remains unclear, making it hard to include the EV71 vaccine in Expanded Program on Immunization (EPI). Here, we measured the neutralizing antibodies (NTAbs) against EV71, Coxsackievirus A16 (CA16) and Poliovirus from infants enrolled in those EV71 vaccine clinical trials. The results indicated that the levels of NTAb GMTs for EV71 increased significantly in all 3 vaccine groups (high, middle and low dosages, respectively) post-vaccination. Seroconversion ratios and Geometric mean fold increase were significantly higher in the vaccine groups (≥7/9 and 8.9~228.1) than in the placebo group (≤1/10 and 0.8~1.7, P Poliovirus. The decrease of 3 types of Poliovirus NTAb GMTs and an increase of CA16 GMTs post-EV71-vaccination were found in vaccine and placebo groups. Further animal study on CA16 and poliovirus vaccine co-immunization or pre-immunization with EV71 vaccine in mice indicated that there was no NTAb cross-activity between EV71 and CA16/Poliovirus. Our research showed that inactivated-EV71 vaccine has good specific-neutralizing capacity and can be included in EPI. PMID:25715318

  18. The compatibility of inactivated-Enterovirus 71 vaccination with Coxsackievirus A16 and Poliovirus immunizations in humans and animals.

    Science.gov (United States)

    Mao, Qunying; Wang, Yiping; Shao, Jie; Ying, Zhifang; Gao, Fan; Yao, Xin; Li, Changgui; Ye, Qiang; Xu, Miao; Li, Rongcheng; Zhu, Fengcai; Liang, Zhenglun

    2015-01-01

    Enterovirus 71 (EV71) is the key pathogen for Hand, Foot, and Mouth Disease (HFMD) and can result in severe neurological complications and death among young children. Three inactivated-EV71 vaccines have gone through phase III clinical trials and have demonstrated good safety and efficacy. These vaccines will benefit young children under the threat of severe HFMD. However, the potential immunization-related compatibility for different enterovirus vaccines remains unclear, making it hard to include the EV71 vaccine in Expanded Program on Immunization (EPI). Here, we measured the neutralizing antibodies (NTAbs) against EV71, Coxsackievirus A16 (CA16) and Poliovirus from infants enrolled in those EV71 vaccine clinical trials. The results indicated that the levels of NTAb GMTs for EV71 increased significantly in all 3 vaccine groups (high, middle and low dosages, respectively) post-vaccination. Seroconversion ratios and Geometric mean fold increase were significantly higher in the vaccine groups (≥ 7/9 and 8.9 ~ 228.1) than in the placebo group (≤ 1/10 and 0.8 ~ 1.7, P < 0.05). But no similar NTAb response trends were found in CA16 and 3 types of Poliovirus. The decrease of 3 types of Poliovirus NTAb GMTs and an increase of CA16 GMTs post-EV71-vaccination were found in vaccine and placebo groups. Further animal study on CA16 and poliovirus vaccine co-immunization or pre-immunization with EV71 vaccine in mice indicated that there was no NTAb cross-activity between EV71 and CA16/Poliovirus. Our research showed that inactivated-EV71 vaccine has good specific-neutralizing capacity and can be included in EPI.

  19. The dynamics and efficacy of antiviral RNA silencing: A model study

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    Hogeweg Paulien

    2008-03-01

    Full Text Available Abstract Background Mathematical modeling is important to provide insight in the complicated pathway of RNA silencing. RNA silencing is an RNA based mechanism that is widely used by eukaryotes to fight viruses, and to control gene expression. Results We here present the first mathematical model that combines viral growth with RNA silencing. The model involves a plus-strand RNA virus that replicates through a double-strand RNA intermediate. The model of the RNA silencing pathway consists of cleavage of viral RNA into siRNA by Dicer, target cleavage of viral RNA via the RISC complex, and a secondary response. We found that, depending on the strength of the silencing response, different viral growth patterns can occur. Silencing can decrease viral growth, cause oscillations, or clear the virus completely. Our model can explain various observed phenomena, even when they seem contradictory at first: the diverse responses to the removal of RNA dependent RNA polymerase; different viral growth curves; and the great diversity in observed siRNA ratios. Conclusion The model presented here is an important step in the understanding of the natural functioning of RNA silencing in viral infections.

  20. Sequence analysis of L RNA of Lassa virus

    International Nuclear Information System (INIS)

    Vieth, Simon; Torda, Andrew E.; Asper, Marcel; Schmitz, Herbert; Guenther, Stephan

    2004-01-01

    The L RNA of three Lassa virus strains originating from Nigeria, Ghana/Ivory Coast, and Sierra Leone was sequenced and the data subjected to structure predictions and phylogenetic analyses. The L gene products had 2218-2221 residues, diverged by 18% at the amino acid level, and contained several conserved regions. Only one region of 504 residues (positions 1043-1546) could be assigned a function, namely that of an RNA polymerase. Secondary structure predictions suggest that this domain is very similar to RNA-dependent RNA polymerases of known structure encoded by plus-strand RNA viruses, permitting a model to be built. Outside the polymerase region, there is little structural data, except for regions of strong alpha-helical content and probably a coiled-coil domain at the N terminus. No evidence for reassortment or recombination during Lassa virus evolution was found. The secondary structure-assisted alignment of the RNA polymerase region permitted a reliable reconstruction of the phylogeny of all negative-strand RNA viruses, indicating that Arenaviridae are most closely related to Nairoviruses. In conclusion, the data provide a basis for structural and functional characterization of the Lassa virus L protein and reveal new insights into the phylogeny of negative-strand RNA viruses

  1. Matrin 3 binds and stabilizes mRNA.

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    Maayan Salton

    Full Text Available Matrin 3 (MATR3 is a highly conserved, inner nuclear matrix protein with two zinc finger domains and two RNA recognition motifs (RRM, whose function is largely unknown. Recently we found MATR3 to be phosphorylated by the protein kinase ATM, which activates the cellular response to double strand breaks in the DNA. Here, we show that MATR3 interacts in an RNA-dependent manner with several proteins with established roles in RNA processing, and maintains its interaction with RNA via its RRM2 domain. Deep sequencing of the bound RNA (RIP-seq identified several small noncoding RNA species. Using microarray analysis to explore MATR3's role in transcription, we identified 77 transcripts whose amounts depended on the presence of MATR3. We validated this finding with nine transcripts which were also bound to the MATR3 complex. Finally, we demonstrated the importance of MATR3 for maintaining the stability of several of these mRNA species and conclude that it has a role in mRNA stabilization. The data suggest that the cellular level of MATR3, known to be highly regulated, modulates the stability of a group of gene transcripts.

  2. Integration of vitamin A supplementation with the expanded program on immunization does not affect seroconversion to oral poliovirus vaccine in infants.

    NARCIS (Netherlands)

    R.D. Semba; M. Muhilal; N.E. Mohgaddam (Nasrin); Z. Munasir; A. Akib; D. Permaesih; M.S. Muherdiyantiningsih; A.D.M.E. Osterhaus (Albert)

    1999-01-01

    textabstractChildhood immunization programs may provide infrastructure for delivering vitamin A supplements to infants in developing countries. The effect of giving vitamin A, an immune enhancer, on antibody responses to trivalent oral poliovirus vaccine (TOPV) is unknown. A

  3. Molecular characterization and phylogenetic relationship of wild type 1 poliovirus strains circulating across Pakistan and Afghanistan bordering areas during 2010-2012.

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    Shahzad Shaukat

    Full Text Available Pakistan and Afghanistan share a long uncontrolled border with extensive population movement on both sides. Wild poliovirus transmission has never been interrupted in this block due to war against terrorism, poor public health infrastructure, misconceptions about polio vaccines and inadequate immunization activities. All these issues complicate the eradication operations and reinforce the complexity of wiping out poliomyelitis from this region. This study illustrates the origins and routes of cross-border wild poliovirus type 1 (WPV1 transmission during 2010-2012 between Pakistan and Afghanistan. Sequence analyses were conducted based on complete VP1 capsid protein sequences for WPV1 study strains to determine the origin of poliovirus genetic lineages and their evolutionary relationships. Phylogenetic tree was constructed from VP1 gene sequences applying Maximum Likelihood method using Kimura 2- parameter model in MEGA program v 5.0. A total of 72 (14.3% out of 502 wild-type 1 polioviruses were found circulating in border areas of both countries during 2010-2012. Molecular phylogenetic analysis classified these strains in to two sub-genotypes with four clusters and 18 lineages. Genetic data confirmed that the most of WPV1 lineages (12; 66.6% were transmitted from Pakistan to Afghanistan. However, the genetic diversity was significantly reduced during 2012 as most of the lineages were completely eliminated. In conclusion, Pakistan-Afghanistan block has emerged as a single poliovirus reservoir sharing the multiple poliovirus lineages due to uncontrolled movement of people across the borders between two countries. If it is neglected, it can jeopardize the extensive global efforts done so-far to eradicate the poliovirus infection. Our data will be helpful to devise the preventive strategies for effective control of wild poliovirus transmission in this region.

  4. [Circulating vaccine-derived poliovirus type 2 outbreak in Democratic Republic of Congo 2011-2012].

    Science.gov (United States)

    Bazira, L; Coulibaly, T; Mayenga, M; Ncharre, C; Yogolelo, R; Mbule, A; Moudzeo, H; Lwamba, P; Mulumba, A W; Cabore, J

    2015-10-01

    According to the WHO records of 2013, the incidence of poliomyelitis was reduced by more than 99%, the number of endemic countries decreased from 125 in 1988 to 3 in 2013 and over 10 million cases were prevented from poliomyelitis thanks to the intensive use of Oral polio vaccine (OPV). However, the emergence of circulating vaccine-derived poliovirus strains (cVDPV), causing serious epidemics like the wild poliovirus, is a major challenge on the final straight towards the goal of eradication and OPV cessation. This paper describes the cVDPVoutbreak that occurred in the Democratic Republic of Congo (DRC) from November 2011 to April 2012. All children under 15 years of age with acute flaccid paralysis (AFP) and confirmed presence of cVDPV in the stool samples were included. Thirty (30) children, all from the administrative territories of Bukama and Malemba Nkulu in the Katanga Province (south-east DRC), were reported. The virus responsible was the cVDPV type 2 (0.7% -3.5% divergent from the reference Sabin 2 strain) in 29 children (97%) and the ambiguous vaccine-derived poliovirus strain (0.7% divergent) was confirmed in one case (3%), a boy seventeen months old and already vaccinated four times with OPV. Twentyfive children (83%) were protected by any of the routine EPI vaccines and 3 children (10%) had never received any dose of OPV. In reaction, DRC has conducted five local campaigns over a period of 10 months (from January to October 2012) and the epidemic was stopped after the second round performed in March 2012. As elsewhere in similar conditions, low immunization coverage, poor sanitation conditions and the stop of the use of OPV2 have favoured the emergence of the third cVDPV epidemic in DRC. The implementation of the Strategic Plan for Polio eradication and endgame strategic plan 2013-2018 will prevent the emergence of cVDPV and set up the conditions for a coordinated OPV phase out.

  5. Antiviral RNA silencing initiated in the absence of RDE-4, a double-stranded RNA binding protein, in Caenorhabditis elegans.

    Science.gov (United States)

    Guo, Xunyang; Zhang, Rui; Wang, Jeffrey; Lu, Rui

    2013-10-01

    Small interfering RNAs (siRNAs) processed from double-stranded RNA (dsRNA) of virus origins mediate potent antiviral defense through a process referred to as RNA interference (RNAi) or RNA silencing in diverse organisms. In the simple invertebrate Caenorhabditis elegans, the RNAi process is initiated by a single Dicer, which partners with the dsRNA binding protein RDE-4 to process dsRNA into viral siRNAs (viRNAs). Notably, in C. elegans this RNA-directed viral immunity (RDVI) also requires a number of worm-specific genes for its full antiviral potential. One such gene is rsd-2 (RNAi spreading defective 2), which was implicated in RDVI in our previous studies. In the current study, we first established an antiviral role by showing that rsd-2 null mutants permitted higher levels of viral RNA accumulation, and that this enhanced viral susceptibility was reversed by ectopic expression of RSD-2. We then examined the relationship of rsd-2 with other known components of RNAi pathways and established that rsd-2 functions in a novel pathway that is independent of rde-4 but likely requires the RNA-dependent RNA polymerase RRF-1, suggesting a critical role for RSD-2 in secondary viRNA biogenesis, likely through coordinated action with RRF-1. Together, these results suggest that RDVI in the single-Dicer organism C. elegans depends on the collective actions of both RDE-4-dependent and RDE-4-independent mechanisms to produce RNAi-inducing viRNAs. Our study reveals, for the first time, a novel siRNA-producing mechanism in C. elegans that bypasses the need for a dsRNA-binding protein.

  6. Hairpin RNA Targeting Multiple Viral Genes Confers Strong Resistance to Rice Black-Streaked Dwarf Virus

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    Fangquan Wang

    2016-05-01

    Full Text Available Rice black-streaked dwarf virus (RBSDV belongs to the genus Fijivirus in the family of Reoviridae and causes severe yield loss in rice-producing areas in Asia. RNA silencing, as a natural defence mechanism against plant viruses, has been successfully exploited for engineering virus resistance in plants, including rice. In this study, we generated transgenic rice lines harbouring a hairpin RNA (hpRNA construct targeting four RBSDV genes, S1, S2, S6 and S10, encoding the RNA-dependent RNA polymerase, the putative core protein, the RNA silencing suppressor and the outer capsid protein, respectively. Both field nursery and artificial inoculation assays of three generations of the transgenic lines showed that they had strong resistance to RBSDV infection. The RBSDV resistance in the segregating transgenic populations correlated perfectly with the presence of the hpRNA transgene. Furthermore, the hpRNA transgene was expressed in the highly resistant transgenic lines, giving rise to abundant levels of 21–24 nt small interfering RNA (siRNA. By small RNA deep sequencing, the RBSDV-resistant transgenic lines detected siRNAs from all four viral gene sequences in the hpRNA transgene, indicating that the whole chimeric fusion sequence can be efficiently processed by Dicer into siRNAs. Taken together, our results suggest that long hpRNA targeting multiple viral genes can be used to generate stable and durable virus resistance in rice, as well as other plant species.

  7. Hairpin RNA Targeting Multiple Viral Genes Confers Strong Resistance to Rice Black-Streaked Dwarf Virus.

    Science.gov (United States)

    Wang, Fangquan; Li, Wenqi; Zhu, Jinyan; Fan, Fangjun; Wang, Jun; Zhong, Weigong; Wang, Ming-Bo; Liu, Qing; Zhu, Qian-Hao; Zhou, Tong; Lan, Ying; Zhou, Yijun; Yang, Jie

    2016-05-11

    Rice black-streaked dwarf virus (RBSDV) belongs to the genus Fijivirus in the family of Reoviridae and causes severe yield loss in rice-producing areas in Asia. RNA silencing, as a natural defence mechanism against plant viruses, has been successfully exploited for engineering virus resistance in plants, including rice. In this study, we generated transgenic rice lines harbouring a hairpin RNA (hpRNA) construct targeting four RBSDV genes, S1, S2, S6 and S10, encoding the RNA-dependent RNA polymerase, the putative core protein, the RNA silencing suppressor and the outer capsid protein, respectively. Both field nursery and artificial inoculation assays of three generations of the transgenic lines showed that they had strong resistance to RBSDV infection. The RBSDV resistance in the segregating transgenic populations correlated perfectly with the presence of the hpRNA transgene. Furthermore, the hpRNA transgene was expressed in the highly resistant transgenic lines, giving rise to abundant levels of 21-24 nt small interfering RNA (siRNA). By small RNA deep sequencing, the RBSDV-resistant transgenic lines detected siRNAs from all four viral gene sequences in the hpRNA transgene, indicating that the whole chimeric fusion sequence can be efficiently processed by Dicer into siRNAs. Taken together, our results suggest that long hpRNA targeting multiple viral genes can be used to generate stable and durable virus resistance in rice, as well as other plant species.

  8. Immunodeficiency-related vaccine-derived poliovirus (iVDPV) cases: a systematic review and implications for polio eradication.

    Science.gov (United States)

    Guo, Jean; Bolivar-Wagers, Sara; Srinivas, Nivedita; Holubar, Marisa; Maldonado, Yvonne

    2015-03-03

    Vaccine-derived polioviruses (VDPVs), strains of poliovirus mutated from the oral polio vaccine, pose a challenge to global polio eradication. Immunodeficiency-related vaccine-derived polioviruses (iVDPVs) are a type of VDPV which may serve as sources of poliovirus reintroduction after the eradication of wild-type poliovirus. This review is a comprehensive update of confirmed iVDPV cases published in the scientific literature from 1962 to 2012, and describes clinically relevant trends in reported iVDPV cases worldwide. We conducted a systematic review of published iVDPV case reports from January 1960 to November 2012 from four databases. We included cases in which the patient had a primary immunodeficiency, and the vaccine virus isolated from the patient either met the sequencing definition of VDPV (>1% divergence for serotypes 1 and 3 and >0.6% for serotype 2) and/or was previously reported as an iVDPV by the World Health Organization. We identified 68 iVDPV cases in 49 manuscripts reported from 25 countries and the Palestinian territories. 62% of case patients were male, 78% presented clinically with acute flaccid paralysis, and 65% were iVDPV2. 57% of cases occurred in patients with predominantly antibody immunodeficiencies, and the overall all-cause mortality rate was greater than 60%. The median age at case detection was 1.4 years [IQR: 0.8, 4.5] and the median duration of shedding was 1.3 years [IQR: 0.7, 2.2]. We identified a poliovirus genome VP1 region mutation rate of 0.72% per year and a higher median percent divergence for iVDPV1 cases. More cases were reported from high income countries, which also had a larger age variation and different distribution of immunodeficiencies compared to upper and lower middle-income countries. Our study describes the incidence and characteristics of global iVDPV cases reported in the literature in the past five decades. It also highlights the regional and economic disparities of reported iVDPV cases. Copyright © 2015

  9. Survival of Poliovirus in Flowing Turbid Seawater Treated with Ultraviolet Light

    Science.gov (United States)

    Hill, W. F.; Hamblet, F. E.; Akin, E. W.

    1967-01-01

    The effectiveness of a model ultraviolet (UV) radiation unit for treating flowing turbid seawater contaminated with poliovirus was determined. At a turbidity of 70 ppm, the observed survival ratios ranged from 1.9 × 10-3 (99.81% reduction) to 1.5 × 10-4 (99.98% reduction) at flow rates ranging from 25 to 15 liters/min; no virus was recovered at flow rates of 10 and 5 liters/min. At a turbidity of 240 ppm, the observed survival ratios ranged from 3.2 × 10-2 (96.80% reduction) to 2.1 × 10-4 (99.98% reduction) at flow rates ranging from 25 to 5 liters/min. As expected, turbidity had an adverse influence on the effectiveness of UV radiation; however, by adjusting the flow rate of the seawater through the treatment unit, adequate disinfection was shown to be predictable. Images Fig. 1 PMID:4291955

  10. Correlation of mutations and recombination with growth kinetics of poliovirus vaccine strains.

    Science.gov (United States)

    Pliaka, V; Kyriakopoulou, Z; Tsakogiannis, D; Ruether, I G A; Gartzonika, C; Levidiotou-Stefanou, S; Krikelis, A; Markoulatos, P

    2010-12-01

    Attenuated strains of Sabin poliovirus vaccine replicate in the human gut and, in rare cases, may cause vaccine-associated paralytic poliomyelitis (VAPP). The genetic instability of Sabin strains constitutes one of the main causes of VAPP, a disease that is most frequently associated with type 3 and type 2 Sabin strains, and more rarely with type 1 Sabin strains. In the present study, the growth phenotype of eight oral poliovirus vaccine (OPV) isolates (two non-recombinants and six recombinants), as well as of Sabin vaccine strains, was evaluated using two different assays, the reproductive capacity at different temperatures (Rct) test and the one-step growth curve test in Hep-2 cells at two different temperatures (37°C and 40°C). The growth phenotype of isolates was correlated with genomic modifications in order to identify the determinants and mechanisms of reversion towards neurovirulence. All of the recombinant OPV isolates showed a thermoresistant phenotype in the Rct test. Moreover, both recombinant Sabin-3 isolates showed significantly higher viral yield than Sabin 3 vaccine strain at 37°C and 40°C in the one-step growth curve test. All of the OPV isolates displayed mutations at specific sites of the viral genome, which are associated with the attenuated and temperature-sensitive phenotype of Sabin strains. The results showed that both mutations and recombination events could affect the phenotype traits of Sabin derivatives and may lead to the reversion of vaccinal strains to neurovirulent ones. The use of phenotypic markers along with the genomic analysis may shed additional light on the molecular determinants of the reversed neurovirulent phenotype of Sabin derivatives.

  11. Phase 3 Trial of a Sabin Strain-Based Inactivated Poliovirus Vaccine.

    Science.gov (United States)

    Liao, Guoyang; Li, Rongcheng; Li, Changgui; Sun, Mingbo; Jiang, Shude; Li, Yanping; Mo, Zhaojun; Xia, Jielai; Xie, Zhongping; Che, Yanchun; Yang, Jingsi; Yin, Zhifang; Wang, Jianfeng; Chu, Jiayou; Cai, Wei; Zhou, Jian; Wang, Junzhi; Li, Qihan

    2016-12-01

     The development of a Sabin strain-based inactivated poliovirus vaccine (Sabin-IPV) is imperative to protecting against vaccine-associated paralytic poliomyelitis in developing countries.  In this double-blinded, parallel-group, noninferiority trial, eligible infants aged 60-90 days were randomly assigned in a ratio of 1:1 to receive either 3 doses of Sabin-IPV or Salk strain-based IPV (Salk-IPV) at 30-day intervals and a booster at the age of 18 months. Immunogenicity and safety were assessed on the basis of a protocol.  Of 1438 infants, 1200 eligible infants were recruited and received either Sabin-IPV or Salk-IPV. From the Sabin-IPV and Salk-IPV groups, 570 and 564 infants, respectively, completed the primary immunization and formed the per-protocol population. The seroconversion rates of the participants who received Sabin-IPV were 100%, 94.9%, and 99.0% (types I, II, and III, respectively), and those of the participants who received Salk-IPV were 94.7%, 91.3%, and 97.9% 1 month after the completion of primary immunization. An anamnestic response for poliovirus types I, II, and III was elicited by a booster in both groups. Except in the case of fever, other adverse events were similar between the 2 groups.  The immune response induced by Sabin-IPV was not inferior to that established with Salk-IPV. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

  12. Factors determining anti-poliovirus type 3 antibodies among orally immunised Indian infants.

    Science.gov (United States)

    Kaliappan, Saravanakumar Puthupalayam; Venugopal, Srinivasan; Giri, Sidhartha; Praharaj, Ira; Karthikeyan, Arun S; Babji, Sudhir; John, Jacob; Muliyil, Jayaprakash; Grassly, Nicholas; Kang, Gagandeep

    2016-09-22

    Among the three poliovirus serotypes, the lowest responses after vaccination with trivalent oral polio vaccine (tOPV) are to serotype 3. Although improvements in routine immunisation and supplementary immunisation activities have greatly increased vaccine coverage, there are limited data on antibody prevalence in Indian infants. Children aged 5-11months with a history of not having received inactivated polio vaccine were screened for serum antibodies to poliovirus serotype 3 (PV3) by a micro-neutralisation assay according to a modified World Health Organization (WHO) protocol. Limited demographic information was collected to assess risk-factors for a lack of protective antibodies. Student's t-test, logistic regression and multilevel logistic regression (MLR) model were used to estimate model parameters. Of 8454 children screened at a mean age of 8.3 (standard deviation [SD]-1.8) months, 88.1% (95% confidence interval (CI): 87.4-88.8) had protective antibodies to PV3. The number of tOPV doses received was the main determinant of seroprevalence; the maximum likelihood estimate yields a 37.7% (95% CI: 36.2-38.3) increase in seroprevalence per dose of tOPV. In multivariable logistic regression analysis increasing age, male sex, and urban residence were also independently associated with seropositivity (Odds Ratios (OR): 1.17 (95% CI: 1.12-1.23) per month of age, 1.27 (1.11-1.46) and 1.24 (1.05-1.45) respectively). Seroprevalence of antibodies to PV3 is associated with age, gender and place of residence, in addition to the number of tOPV doses received. Ensuring high coverage and monitoring of response are essential as long as oral vaccines are used in polio eradication. Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.

  13. Detection and quantification of poliovirus infection using FTIR spectroscopy and cell culture

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    Lee-Montiel Felipe T

    2011-12-01

    Full Text Available Abstract Background In a globalized word, prevention of infectious diseases is a major challenge. Rapid detection of viable virus particles in water and other environmental samples is essential to public health risk assessment, homeland security and environmental protection. Current virus detection methods, especially assessing viral infectivity, are complex and time-consuming, making point-of-care detection a challenge. Faster, more sensitive, highly specific methods are needed to quantify potentially hazardous viral pathogens and to determine if suspected materials contain viable viral particles. Fourier transform infrared (FTIR spectroscopy combined with cellular-based sensing, may offer a precise way to detect specific viruses. This approach utilizes infrared light to monitor changes in molecular components of cells by tracking changes in absorbance patterns produced following virus infection. In this work poliovirus (PV1 was used to evaluate the utility of FTIR spectroscopy with cell culture for rapid detection of infective virus particles. Results Buffalo green monkey kidney (BGMK cells infected with different virus titers were studied at 1 - 12 hours post-infection (h.p.i.. A partial least squares (PLS regression method was used to analyze and model cellular responses to different infection titers and times post-infection. The model performs best at 8 h.p.i., resulting in an estimated root mean square error of cross validation (RMSECV of 17 plaque forming units (PFU/ml when using low titers of infection of 10 and 100 PFU/ml. Higher titers, from 103 to 106 PFU/ml, could also be reliably detected. Conclusions This approach to poliovirus detection and quantification using FTIR spectroscopy and cell culture could potentially be extended to compare biochemical cell responses to infection with different viruses. This virus detection method could feasibly be adapted to an automated scheme for use in areas such as water safety monitoring and

  14. Translational repression of the Drosophila nanos mRNA involves the RNA helicase Belle and RNA coating by Me31B and Trailer hitch.

    Science.gov (United States)

    Götze, Michael; Dufourt, Jérémy; Ihling, Christian; Rammelt, Christiane; Pierson, Stephanie; Sambrani, Nagraj; Temme, Claudia; Sinz, Andrea; Simonelig, Martine; Wahle, Elmar

    2017-10-01

    Translational repression of maternal mRNAs is an essential regulatory mechanism during early embryonic development. Repression of the Drosophila nanos mRNA, required for the formation of the anterior-posterior body axis, depends on the protein Smaug binding to two Smaug recognition elements (SREs) in the nanos 3' UTR. In a comprehensive mass spectrometric analysis of the SRE-dependent repressor complex, we identified Smaug, Cup, Me31B, Trailer hitch, eIF4E, and PABPC, in agreement with earlier data. As a novel component, the RNA-dependent ATPase Belle (DDX3) was found, and its involvement in deadenylation and repression of nanos was confirmed in vivo. Smaug, Cup, and Belle bound stoichiometrically to the SREs, independently of RNA length. Binding of Me31B and Tral was also SRE-dependent, but their amounts were proportional to the length of the RNA and equimolar to each other. We suggest that "coating" of the RNA by a Me31B•Tral complex may be at the core of repression. © 2017 Götze et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  15. Atomistic mechanism of microRNA translation upregulation via molecular dynamics simulations.

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    Wei Ye

    Full Text Available MicroRNAs are endogenous 23-25 nt RNAs that play important gene-regulatory roles in animals and plants. Recently, miR369-3 was found to upregulate translation of TNFα mRNA in quiescent (G0 mammalian cell lines. Knock down and immunofluorescence experiments suggest that microRNA-protein complexes (with FXR1 and AGO2 are necessary for the translation upregulation. However the molecular mechanism of microRNA translation activation is poorly understood. In this study we constructed the microRNA-mRNA-AGO2-FXR1 quadruple complex by bioinformatics and molecular modeling, followed with all atom molecular dynamics simulations in explicit solvent to investigate the interaction mechanisms for the complex. A combined analysis of experimental and computational data suggests that AGO2-FXR1 complex relocalize microRNA:mRNA duplex to polysomes in G0. The two strands of dsRNA are then separated upon binding of AGO2 and FXR1. Finally, polysomes may improve the translation efficiency of mRNA. The mutation research confirms the stability of microRNA-mRNA-FXR1 and illustrates importance of key residue of Ile304. This possible mechanism can shed more light on the microRNA-dependent upregulation of translation.

  16. Unusual loop-sequence flexibility of the proximal RNA replication element in EMCV.

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    Jan Zoll

    Full Text Available Picornaviruses contain stable RNA structures at the 5' and 3' ends of the RNA genome, OriL and OriR involved in viral RNA replication. The OriL RNA element found at the 5' end of the enterovirus genome folds into a cloverleaf-like configuration. In vivo SELEX experiments revealed that functioning of the poliovirus cloverleaf depends on a specific structure in this RNA element. Little is known about the OriL of cardioviruses. Here, we investigated structural aspects and requirements of the apical loop of proximal stem-loop SL-A of mengovirus, a strain of EMCV. Using NMR spectroscopy, we showed that the mengovirus SL-A apical loop consists of an octaloop. In vivo SELEX experiments demonstrated that a large number of random sequences are tolerated in the apical octaloop that support virus replication. Mutants in which the SL-A loop size and the length of the upper part of the stem were varied showed that both stem-length and stability of the octaloop are important determinants for viral RNA replication and virus reproduction. Together, these data show that stem-loop A plays an important role in virus replication. The high degree of sequence flexibility and the lack of selective pressure on the octaloop argue against a role in sequence specific RNA-protein or RNA-RNA interactions in which octaloop nucleotides are involved.

  17. Seroprevalencia de anticuerpos contra el poliovirus 1 en niños mexicanos Seroprevalence of antibodies against poliovirus type 1 in Mexican children

    Directory of Open Access Journals (Sweden)

    Juan Ruiz-Gómez

    2007-01-01

    Full Text Available OBJETIVO: Analizar la frecuencia y distribución de la prevalencia de anticuerpos contra el virus de la poliomielitis tipo 1 en niños menores de 10 años en México, además de contribuir a la evaluación del programa de vacunación. MATERIAL Y MÉTODOS: Se estudió la presencia de anticuerpos contra el poliovirus tipo 1 en una muestra de la Encuesta Nacional de Salud 2000. Los sueros se recolectaron entre noviembre de 1999 y junio de 2000 a nivel nacional. La muestra constó de 6 270 niños de uno a nueve años de edad y se utilizó la técnica de neutralización. RESULTADOS: La seropositividad fue de 99.3% (IC95%99.1-99.7. Se identificaron como factores de riesgo de susceptibilidad el analfabetismo (RM= 1.5, p= 0.002 y el bajo ingreso familiar (RM= 1.4, p= 0.0487 y como factor protector el acceso a la seguridad social (RM= 0.41, p=0.04. CONCLUSIONES: Las actividades del programa de vacunación que han llevado a cabo las instituciones de salud han dado resultados en el control y eliminación de la enfermedad. Sin embargo, los programas de vacunación no deben interrumpirse, incluso si se ha registrado 99.3% de seropositividad; no puede soslayarse que al convertir el 0.7% restante, se calcula que hay 190 000 niños susceptibles de contraer la enfermedad. Estos niños se localizan sobre todo en el sur del país.OBJECTIVE: To analyze the frequency and distribution of the prevalence of antibodies against the poliomyelitis type 1 virus in children 1-9 years old in Mexico. MATERIAL AND METHODS: Antibodies against poliovirus type 1 (neutralization method were studied in 6 270 sera selected from the 24 232 sera from children one to nine years old, collected by the 2000 National Health Survey (ENSA 2000 that was conducted from November 1999 to June 2000. RESULTS: Overall seroprevalence was 99.3% (95%CI: 99.1-99.7. Using bivariate analysis, absence of antibodies was shown to be associated with illiteracy (OR= 1.5, p=0.002 and low household income (OR= 1

  18. Safety and immunogenicity of inactivated poliovirus vaccine made from Sabin strains: a phase II, randomized, positive-controlled trial.

    Science.gov (United States)

    Liao, Guoyang; Li, Rongcheng; Li, Changgui; Sun, Mingbo; Li, Yanping; Chu, Jiayou; Jiang, Shude; Li, Qihan

    2012-01-15

    The production of Sabin inactivated poliovirus vaccine (IPV) can reduce biosafety requirements in the posteradication/post-oral poliovirus vaccine (OPV) era. We conducted a phase II, randomized, positive-controlled trial to assess the safety and immunogenicity of Sabin IPV. The test groups (A, B, and C) received 3 doses of high, middle, and low D antigen (D Ag) of Sabin IPV at ages 2, 3, and 4 months, respectively. Infants in 2 control groups, group D and group E, received 3 doses of trivalent OPV and conventional IPV (cIPV), respectively, on the same schedule as that of groups A, B, and C. Serum samples were collected before and 30 days after the administration of the third dose. In total, 500 infants were randomly assigned to 5 groups, and 449 infants completed the vaccine series. No serious adverse events were associated with vaccinations. After 3 doses, the seroconversion rates in groups A, B, C, D, and E were 100%, 97.8%, 96.6%, 100%, and 90.1%, respectively, for type 1 poliovirus; 97.7%, 95.7%, 78.7%, 100%, and 90.1%, respectively, for type 2; and 98.8%, 98.9%, 93.3%, 100%, and 97.8%, respectively, for type 3. Sabin IPV has good safety characteristics. The seroconversion rates for type 1 poliovirus (most appropriate concentration, 15 D Ag units [DU]), type 2 (32 DU), and type 3 (45 DU) Sabin IPV were similar to those of the OPV and cIPV control groups. NCT01056705.

  19. Combined use of inactivated and oral poliovirus vaccines in refugee camps and surrounding communities - Kenya, December 2013.

    Science.gov (United States)

    Sheikh, Mohamed A; Makokha, Frederick; Hussein, Abdullahi M; Mohamed, Gedi; Mach, Ondrej; Humayun, Kabir; Okiror, Samuel; Abrar, Leila; Nasibov, Orkhan; Burton, John; Unshur, Ahmed; Wannemuehler, Kathleen; Estivariz, Concepcion F

    2014-03-21

    Since the launch of the Global Polio Eradication Initiative (GPEI) in 1988, circulation of indigenous wild poliovirus (WPV) has continued without interruption in only three countries: Afghanistan, Nigeria, and Pakistan. During April-December 2013, a polio outbreak caused by WPV type 1 (WPV1) of Nigerian origin resulted in 217 cases in or near the Horn of Africa, including 194 cases in Somalia, 14 cases in Kenya, and nine cases in Ethiopia (all cases were reported as of March 10, 2014). During December 14-18, 2013, Kenya conducted the first-ever campaign providing inactivated poliovirus vaccine (IPV) together with oral poliovirus vaccine (OPV) as part of its outbreak response. The campaign targeted 126,000 children aged ≤59 months who resided in Somali refugee camps and surrounding communities near the Kenya-Somalia border, where most WPV1 cases had been reported, with the aim of increasing population immunity levels to ensure interruption of any residual WPV transmission and prevent spread from potential new importations. A campaign evaluation and vaccination coverage survey demonstrated that combined administration of IPV and OPV in a mass campaign is feasible and can achieve coverage >90%, although combined IPV and OPV campaigns come at a higher cost than OPV-only campaigns and require particular attention to vaccinator training and supervision. Future operational studies could assess the impact on population immunity and the cost-effectiveness of combined IPV and OPV campaigns to accelerate interruption of poliovirus transmission during polio outbreaks and in certain areas in which WPV circulation is endemic.

  20. Chimpanzee-Human Monoclonal Antibodies for Treatment of Chronic Poliovirus Excretors and Emergency Postexposure Prophylaxis▿‡

    Science.gov (United States)

    Chen, Zhaochun; Chumakov, Konstantin; Dragunsky, Eugenia; Kouiavskaia, Diana; Makiya, Michelle; Neverov, Alexander; Rezapkin, Gennady; Sebrell, Andrew; Purcell, Robert

    2011-01-01

    Six poliovirus-neutralizing Fabs were recovered from a combinatorial Fab phage display library constructed from bone marrow-derived lymphocytes of immunized chimpanzees. The chimeric chimpanzee-human full-length IgGs (hereinafter called monoclonal antibodies [MAbs]) were generated by combining a chimpanzee IgG light chain and a variable domain of heavy chain with a human constant Fc region. The six MAbs neutralized vaccine strains and virulent strains of poliovirus. Five MAbs were serotype specific, while one MAb cross-neutralized serotypes 1 and 2. Epitope mapping performed by selecting and sequencing antibody-resistant viral variants indicated that the cross-neutralizing MAb bound between antigenic sites 1 and 2, thereby covering the canyon region containing the receptor-binding site. Another serotype 1-specific MAb recognized a region located between antigenic sites 2 and 3 that included parts of capsid proteins VP1 and VP3. Both serotype 2-specific antibodies recognized antigenic site 1. No escape mutants to serotype 3-specific MAbs could be generated. The administration of a serotype 1-specific MAb to transgenic mice susceptible to poliovirus at a dose of 5 μg/mouse completely protected them from paralysis after challenge with a lethal dose of wild-type poliovirus. Moreover, MAb injection 6 or 12 h after virus infection provided significant protection. The MAbs described here could be tested in clinical trials to determine whether they might be useful for treatment of immunocompromised chronic virus excretors and for emergency protection of contacts of a paralytic poliomyelitis case. PMID:21345966

  1. Vaccine-derived poliovirus surveillance in China during 2001-2013: the potential challenge for maintaining polio free status.

    Science.gov (United States)

    Wang, Hai-Bo; Luo, Hui-Ming; Li, Li; Fan, Chun-Xiang; Hao, Li-Xin; Ma, Chao; Su, Qi-Ru; Yang, Hong; Reilly, Kathleen H; Wang, Hua-Qing; Wen, Ning

    2017-12-02

    The goal of polio eradication is to complete elimination and containment of all wild, vaccine-related and Sabin polioviruses. Vaccine-derived poliovirus (VDPV) surveillance in China from 2001-2013 is summarized in this report, which has important implications for the global polio eradication initiative. Acute flaccid paralysis (AFP) cases and their contacts with VDPVs isolated from fecal specimens were identified in our AFP surveillance system or by field investigation. Epidemiological and laboratory information for these children were analyzed and the reasons for the VDPV outbreak was explored. VDPVs were isolated from a total of 49 children in more than two-thirds of Chinese provinces from 2001-2013, including 15 VDPV cases, 15 non-polio AFP cases and 19 contacts of AFP cases or healthy subjects. A total of 3 circulating VDPVs (cVDPVs) outbreaks were reported in China, resulting in 6 cVDPVs cases who had not been vaccinated with oral attenuated poliomyelitis vaccine. Among the 4 immunodeficiency-associated VDPVs (iVDPVs) cases, the longest duration of virus excretion was about 20 months. In addition, one imported VDPV case from Myanmar was detected in Yunnan Province. Until all wild, vaccine-related and Sabin polioviruses are eradicated in the world, high quality routine immunization and sensitive AFP surveillance should be maintained, focusing efforts on underserved populations in high risk areas.

  2. Development of oral CTL vaccine using a CTP-integrated Sabin 1 poliovirus-based vector system.

    Science.gov (United States)

    Han, Seung-Soo; Lee, Jinjoo; Jung, Yideul; Kang, Myeong-Ho; Hong, Jung-Hyub; Cha, Min-Suk; Park, Yu-Jin; Lee, Ezra; Yoon, Cheol-Hee; Bae, Yong-Soo

    2015-09-11

    We developed a CTL vaccine vector by modification of the RPS-Vax system, a mucosal vaccine vector derived from a poliovirus Sabin 1 strain, and generated an oral CTL vaccine against HIV-1. A DNA fragment encoding a cytoplasmic transduction peptide (CTP) was integrated into the RPS-Vax system to generate RPS-CTP, a CTL vaccine vector. An HIV-1 p24 cDNA fragment was introduced into the RPS-CTP vector system and a recombinant poliovirus (rec-PV) named vRPS-CTP/p24 was produced. vRPS-CTP/p24 was genetically stable and efficiently induced Th1 immunity and p24-specific CTLs in immunized poliovirus receptor-transgenic (PVR-Tg) mice. In challenge experiments, PVR-Tg mice that were pre-immunized orally with vRPS-CTP/p24 were resistant to challenge with a lethal dose of p24-expressing recombinant vaccinia virus (rMVA-p24). These results suggested that the RPS-CTP vector system had potential for developing oral CTL vaccines against infectious diseases. Copyright © 2015 Elsevier Ltd. All rights reserved.

  3. Analysis of the dose-sparing effect of adjuvanted Sabin-inactivated poliovirus vaccine (sIPV).

    Science.gov (United States)

    Li, Zhuofan; Ding, Wenting; Guo, Qi; Liu, Ze; Zhu, Zhe; Song, Shaohui; Li, Weidong; Liao, Guoyang

    2018-03-30

    Sabin-based inactivated poliovirus vaccine(sIPV) is gradually replacing live-attenuated oral polio vaccine(OPV). Sabin-inactivated poliovirus vaccine(sIPV) has played a vital role in reducing economic burden of poliomyelitis and maintaining appropriate antibody levels in the population. However, due to its high cost and limited manufacturing capacity, sIPV cannot reach its full potential for global poliovirus eradication in developing countries. Therefore, to address this situation, we designed this study to evaluate the dose-sparing effects of AS03, CpG oligodeoxynucleotides (CpG-ODN) and polyinosinic:polycytidylic acid (PolyI:C) admixed with sIPV in rats. Our results showed that a combination of 1/4-dose sIPV adjuvanted with AS03 or AS03 with BW006 provides a seroconversion rate similar to that of full-dose sIPV without adjuvant and that, this rate is 5-fold higher than that of 1/4-dose sIPV without adjuvant after the first immunization. The combination of AS03 or AS03 with BW006 as an adjuvant effectively reduced sIPV dose by at least 4-fold and induced both humoral and cellular immune responses. Therefore, our study revealed that the combination of AS03 or AS03 with BW006 is a promising adjuvant for sIPV development.

  4. Nuclear assortment of eIF4E coincides with shut-off of host protein synthesis upon poliovirus infection.

    Science.gov (United States)

    Sukarieh, R; Sonenberg, N; Pelletier, J

    2010-05-01

    Eukaryotic initiation factor (eIF) 4E is a subunit of the cap-binding protein complex, eIF4F, which recognizes the cap structure of cellular mRNAs to facilitate translation initiation. eIF4E is assembled into the eIF4F complex via its interaction with eIF4G, an event that is under Akt/mTOR regulation. The eIF4E-eIF4G interaction is regulated by the eIF4E binding partners, eIF4E-binding proteins and eIF4E-transporter. Cleavage of eIF4G occurs upon poliovirus infection and is responsible for the shut-off of host-cell protein synthesis observed early in infection. Here, we document that relocalization of eIF4E to the nucleus occurs concomitantly with cleavage of eIF4G upon poliovirus infection. This event is not dependent upon virus replication, but is dependent on eIF4G cleavage. We postulate that eIF4E nuclear relocalization may contribute to the shut-off of host protein synthesis that is a hallmark of poliovirus infection by perturbing the circular status of actively translating mRNAs.

  5. Proteolytic processing of poliovirus polypeptides: antibodies to polypeptide P3-7c inhibit cleavage at glutamine-glycine pairs

    International Nuclear Information System (INIS)

    Hanecak, R.; Semler, B.L.; Anderson, C.W.; Wimmer, E.

    1982-01-01

    Proteolytic processing of poliovirus polypeptides was examined by the addition of antibodies directed against the viral proteins P3-7c and P2-X to a cell-free translation extract prepared from infected HeLa cells. Antisera to P3-7c specifically inhibited in vitro processing at Gln-Gly pairs. Partial amino acid sequence analysis revealed a second Tyr-Gly pair that is utilized in protein processing. Neither Tyr-Gly cleavage is affected by antibody to P3-7C. Anti-P3-7c antibodies react not only with P3-7c but also with P3-6a and P3-2, two viral polypeptides NH 2 -coterminal with P3-7c. Preimmune and anti-P2-X antibodies had no effect on the processing of poliovirus proteins in vitro. The authors conclude that the activity responsible for processing poliovirus polypeptides at Gln-Gly pairs resides in the primary structure of P3-7c and not in P2-X

  6. Inactivated poliovirus vaccine given alone or in a sequential schedule with bivalent oral poliovirus vaccine in Chilean infants: a randomised, controlled, open-label, phase 4, non-inferiority study.

    Science.gov (United States)

    O'Ryan, Miguel; Bandyopadhyay, Ananda S; Villena, Rodolfo; Espinoza, Mónica; Novoa, José; Weldon, William C; Oberste, M Steven; Self, Steve; Borate, Bhavesh R; Asturias, Edwin J; Clemens, Ralf; Orenstein, Walter; Jimeno, José; Rüttimann, Ricardo; Costa Clemens, Sue Ann

    2015-11-01

    Bivalent oral poliovirus vaccine (bOPV; types 1 and 3) is expected to replace trivalent OPV (tOPV) globally by April, 2016, preceded by the introduction of at least one dose of inactivated poliovirus vaccine (IPV) in routine immunisation programmes to eliminate vaccine-associated or vaccine-derived poliomyelitis from serotype 2 poliovirus. Because data are needed on sequential IPV-bOPV schedules, we assessed the immunogenicity of two different IPV-bOPV schedules compared with an all-IPV schedule in infants. We did a randomised, controlled, open-label, non-inferiority trial with healthy, full-term (>2·5 kg birthweight) infants aged 8 weeks (± 7 days) at six well-child clinics in Santiago, Chile. We used supplied lists to randomly assign infants (1:1:1) to receive three polio vaccinations (IPV by injection or bOPV as oral drops) at age 8, 16, and 24 weeks in one of three sequential schedules: IPV-bOPV-bOPV, IPV-IPV-bOPV, or IPV-IPV-IPV. We did the randomisation with blocks of 12 stratified by study site. All analyses were done in a masked manner. Co-primary outcomes were non-inferiority of the bOPV-containing schedules compared with the all-IPV schedule for seroconversion (within a 10% margin) and antibody titres (within two-thirds log2 titres) to poliovirus serotypes 1 and 3 at age 28 weeks, analysed in the per-protocol population. Secondary outcomes were seroconversion and titres to serotype 2 and faecal shedding for 4 weeks after a monovalent OPV type 2 challenge at age 28 weeks. Safety analyses were done in the intention-to-treat population. This trial is registered with ClinicalTrials.gov, number NCT01841671, and is closed to new participants. Between April 25 and August 1, 2013, we assigned 570 infants to treatment: 190 to IPV-bOPV-bOPV, 192 to IPV-IPV-bOPV, and 188 to IPV-IPV-IPV. 564 (99%) were vaccinated and included in the intention-to-treat cohort, and 537 (94%) in the per-protocol analyses. In the IPV-bOPV-bOPV, IPV-IPV-bOPV, and IPV-IPV-IPV groups

  7. Discovery of centrosomal RNA and centrosomal hypothesis of cellular ageing and differentiation.

    Science.gov (United States)

    Chichinadze, Konstantin; Tkemaladze, Jaba; Lazarashvili, Ann

    2012-01-01

    In 2006, a group of scientists studying centrosomes of Spisula solidissima mollusc oocytes under the leadership of Alliegro (Alliegro, M.C.; Alliegro, M.A.; Palazzo, R.E. Centrosome-associated RNA in surf clam oocytes. Proc. Natl. Acad. Sci. USA 2006, 103(24), 9034-9038) reliably demonstrated the existence of specific RNA in centrosome, called centrosomal RNA (cnRNA). In their first article, five different RNAs (cnRNAs 11, 102, 113, 170, and 184) were described. During the process of full sequencing of the first transcript (cnRNA 11), it was discovered that the transcript contained a conserved structure-a reverse transcriptase domain located together with the most important centrosomal protein, γ-tubulin. In an article published in 2005, we made assumptions about several possible mechanisms for determining the most important functions of centrosomal structures and referred to one of them as a "RNA-dependent mechanism." This idea about participation of hypothetic centrosomal small interference RNA and/or microRNA in the process was made one year prior to the discovery of cnRNA by Alliegro's group. The discovery of specific RNA in a centrosome is indirect evidence of a centrosomal hypothesis of cellular ageing and differentiation. The presence of a reverse transcriptase domain in this type of RNA, together with its uniqueness and specificity, makes the centrosome a place of information storage and reproduction.

  8. Evidence for an RNA polymerization activity in axolotl and Xenopus egg extracts.

    Directory of Open Access Journals (Sweden)

    Hélène Pelczar

    2010-12-01

    Full Text Available We have previously reported a post-transcriptional RNA amplification observed in vivo following injection of in vitro synthesized transcripts into axolotl oocytes, unfertilized (UFE or fertilized eggs. To further characterize this phenomenon, low speed extracts (LSE from axolotl and Xenopus UFE were prepared and tested in an RNA polymerization assay. The major conclusions are: i the amphibian extracts catalyze the incorporation of radioactive ribonucleotide in RNase but not DNase sensitive products showing that these products correspond to RNA; ii the phenomenon is resistant to α-amanitin, an inhibitor of RNA polymerases II and III and to cordycepin (3'dAMP, but sensitive to cordycepin 5'-triphosphate, an RNA elongation inhibitor, which supports the existence of an RNA polymerase activity different from polymerases II and III; the detection of radiolabelled RNA comigrating at the same length as the exogenous transcript added to the extracts allowed us to show that iii the RNA polymerization is not a 3' end labelling and that iv the radiolabelled RNA is single rather than double stranded. In vitro cell-free systems derived from amphibian UFE therefore validate our previous in vivo results hypothesizing the existence of an evolutionary conserved enzymatic activity with the properties of an RNA dependent RNA polymerase (RdRp.

  9. Alternative inactivated poliovirus vaccines adjuvanted with Quillaja brasiliensis or Quil-a saponins are equally effective in inducing specific immune responses.

    Directory of Open Access Journals (Sweden)

    Fernanda de Costa

    Full Text Available Inactivated polio vaccines (IPV have an important role at the final stages of poliomyelitis eradication programs, reducing the risks associated with the use of attenuated polio vaccine (OPV. An affordable option to enhance vaccine immunogenicity and reduce costs of IPV may be the use of an effective and renewable adjuvant. In the present study, the adjuvant activity of aqueous extract (AE and saponin fraction QB-90 from Quillaja brasiliensis using poliovirus antigen as model were analyzed and compared to a preparation adjuvanted with Quil-A, a well-known saponin-based commercial adjuvant. Experimental vaccines were prepared with viral antigen plus saline (control, Quil-A (50 µg, AE (400 µg or QB-90 (50 µg. Sera from inoculated mice were collected at days 0, 28, 42 and 56 post-inoculation of the first dose of vaccine. Serum levels of specific IgG, IgG1 and IgG2a were significantly enhanced by AE, QB-90 and Quil-A compared to control group on day 56. The magnitude of enhancement was statistically equivalent for QB-90 and Quil-A. The cellular response was evaluated through DTH and analysis of IFN-γ and IL-2 mRNA levels using in vitro reestimulated splenocytes. Results indicated that AE and QB-90 were capable of stimulating the generation of Th1 cells against the administered antigen to the same extent as Quil-A. Mucosal immune response was enhanced by the vaccine adjuvanted with QB-90 as demonstrated by increases of specific IgA titers in bile, feces and vaginal washings, yielding comparable or higher titers than Quil-A. The results obtained indicate that saponins from Q. brasiliensis are potent adjuvants of specific cellular and humoral immune responses and represent a viable option to Quil-A.

  10. RNA oxidation

    DEFF Research Database (Denmark)

    Kjaer, L. K.; Cejvanovic, V.; Henriken, T.

    2015-01-01

    .9 significant hazard ratio for death compared with the quartile with the lowest 8oxoGuo excretion when adjusted for age, sex, BMI, smoker status, s-HbA1c, urine protein excretion and s-cholesterol. We conclude that it is now established that RNA oxidation is an independent risk factor for death in type 2...

  11. The Duration of Intestinal Immunity After an Inactivated Poliovirus Vaccine Booster Dose in Children Immunized With Oral Vaccine: A Randomized Controlled Trial.

    Science.gov (United States)

    John, Jacob; Giri, Sidhartha; Karthikeyan, Arun S; Lata, Dipti; Jeyapaul, Shalini; Rajan, Anand K; Kumar, Nirmal; Dhanapal, Pavithra; Venkatesan, Jayalakshmi; Mani, Mohanraj; Hanusha, Janardhanan; Raman, Uma; Moses, Prabhakar D; Abraham, Asha; Bahl, Sunil; Bandyopadhyay, Ananda S; Ahmad, Mohammad; Grassly, Nicholas C; Kang, Gagandeep

    2017-02-15

    In 2014, 2 studies showed that inactivated poliovirus vaccine (IPV) boosts intestinal immunity in children previously immunized with oral poliovirus vaccine (OPV). As a result, IPV was introduced in mass campaigns to help achieve polio eradication. We conducted an open-label, randomized, controlled trial to assess the duration of the boost in intestinal immunity following a dose of IPV given to OPV-immunized children. Nine hundred healthy children in Vellore, India, aged 1-4 years were randomized (1:1:1) to receive IPV at 5 months (arm A), at enrollment (arm B), or no vaccine (arm C). The primary outcome was poliovirus shedding in stool 7 days after bivalent OPV challenge at 11 months. For children in arms A, B, and C, 284 (94.7%), 297 (99.0%), and 296 (98.7%), respectively, were eligible for primary per-protocol analysis. Poliovirus shedding 7 days after challenge was less prevalent in arms A and B compared with C (24.6%, 25.6%, and 36.4%, respectively; risk ratio 0.68 [95% confidence interval: 0.53-0.87] for A versus C, and 0.70 [0.55-0.90] for B versus C). Protection against poliovirus remained elevated 6 and 11 months after an IPV boost, although at a lower level than reported at 1 month. CTRI/2014/09/004979. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America.

  12. Neurovirulent vaccine-derived polioviruses in sewage from highly immune populations.

    Directory of Open Access Journals (Sweden)

    Lester M Shulman

    Full Text Available BACKGROUND: Vaccine-derived polioviruses (VDPVs have caused poliomyelitis outbreaks in communities with sub-optimal vaccination. Israeli environmental surveillance of sewage from populations with high (>95% documented vaccine coverage of confirmed efficacy identified two separate evolutionary clusters of VDPVs: Group 1 (1998-2005, one system, population 1.6x10(6 and Group 2 (2006, 2 systems, populations 0.7x10(6 and 5x10(4. PRINCIPAL FINDINGS: Molecular analyses support evolution of nine Group 1 VDPVs along five different lineages, starting from a common ancestral type 2 vaccine-derived Sabin-2/Sabin-1 recombinant strain, and independent evolution of three Group 2 VDPVs along one lineage starting from a different recombinant strain. The primary evidence for two independent origins was based on comparison of unique recombination fingerprints, the number and distribution of identical substitutions, and evolutionary rates. Geometric mean titers of neutralizing antibodies against Group 1 VDPVs were significantly lower than against vaccine strains in all age-group cohorts tested. All individuals had neutralizing titers >1:8 against these VDPVs except 7% of the 20-50 year cohort. Group 1 VDPVs were highly neurovirulent in a transgenic mouse model. Intermediate levels of protective immunity against Group 2 VDPVs correlated with fewer (5.0+1.0 amino acid substitutions in neutralizing antigenic sites than in Group 1 VDPV's (12.1+/-1.5. SIGNIFICANCE: VDPVs that revert from live oral attenuated vaccines and reacquire characteristics of wild-type polioviruses not only threaten populations with poor immune coverage, but are also a potential source for re-introduction of poliomyelitis into highly immune populations through older individuals with waning immunity. The presence of two independently evolved groups of VDPVs in Israel and the growing number of reports of environmental VDPV elsewhere make it imperative to determine the global frequency of

  13. Phomopsis longicolla RNA virus 1 - Novel virus at the edge of myco- and plant viruses.

    Science.gov (United States)

    Hrabáková, Lenka; Koloniuk, Igor; Petrzik, Karel

    2017-06-01

    The complete nucleotide sequence of a new RNA mycovirus in the KY isolate of Phomopsis longicolla Hobbs 1985 and its protoplasts subcultures p5, p9, and ME711 was discovered. The virus, provisionally named Phomopsis longicolla RNA virus 1 (PlRV1), was localized in mitochondria and was determined to have a genome 2822 nucleotides long. A single open reading frame could be translated in silico by both standard and mitochondrial genetic codes into a product featuring conservative domains for an RNA-dependent RNA polymerase (RdRp). The RdRp of PlRV1 has no counterpart among mycoviruses, but it is about 30% identical with the RdRp of plant ourmiaviruses. Recently, new mycoviruses related to plant ourmiaviruses and forming one clade with PlRV1 have been discovered. This separate clade could represent the crucial link between plant and fungal viruses. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Hsp90 interacts specifically with viral RNA and differentially regulates replication initiation of Bamboo mosaic virus and associated satellite RNA.

    Directory of Open Access Journals (Sweden)

    Ying Wen Huang

    Full Text Available Host factors play crucial roles in the replication of plus-strand RNA viruses. In this report, a heat shock protein 90 homologue of Nicotiana benthamiana, NbHsp90, was identified in association with partially purified replicase complexes from BaMV-infected tissue, and shown to specifically interact with the 3' untranslated region (3' UTR of BaMV genomic RNA, but not with the 3' UTR of BaMV-associated satellite RNA (satBaMV RNA or that of genomic RNA of other viruses, such as Potato virus X (PVX or Cucumber mosaic virus (CMV. Mutational analyses revealed that the interaction occurs between the middle domain of NbHsp90 and domain E of the BaMV 3' UTR. The knockdown or inhibition of NbHsp90 suppressed BaMV infectivity, but not that of satBaMV RNA, PVX, or CMV in N. benthamiana. Time-course analysis further revealed that the inhibitory effect of 17-AAG is significant only during the immediate early stages of BaMV replication. Moreover, yeast two-hybrid and GST pull-down assays demonstrated the existence of an interaction between NbHsp90 and the BaMV RNA-dependent RNA polymerase. These results reveal a novel role for NbHsp90 in the selective enhancement of BaMV replication, most likely through direct interaction with the 3' UTR of BaMV RNA during the initiation of BaMV RNA replication.

  15. Current knowledge of microRNA-mediated regulation of drug metabolism in humans.

    Science.gov (United States)

    Nakano, Masataka; Nakajima, Miki

    2018-05-01

    Understanding the factors causing inter- and intra-individual differences in drug metabolism potencies is required for the practice of personalized or precision medicine, as well as for the promotion of efficient drug development. The expression of drug-metabolizing enzymes is controlled by transcriptional regulation by nuclear receptors and transcriptional factors, epigenetic regulation, such as DNA methylation and histone acetylation, and post-translational modification. In addition to such regulation mechanisms, recent studies revealed that microRNAs (miRNAs), endogenous ~22-nucleotide non-coding RNAs that regulate gene expression through the translational repression and degradation of mRNAs, significantly contribute to post-transcriptional regulation of drug-metabolizing enzymes. Areas covered: This review summarizes the current knowledge regarding miRNAs-dependent regulation of drug-metabolizing enzymes and transcriptional factors and its physiological and clinical significance. We also describe recent advances in miRNA-dependent regulation research, showing that the presence of pseudogenes, single-nucleotide polymorphisms, and RNA editing affects miRNA targeting. Expert opinion: It is unwavering fact that miRNAs are critical factors causing inter- and intra-individual differences in the expression of drug-metabolizing enzymes. Consideration of miRNA-dependent regulation would be a helpful tool for optimizing personalized and precision medicine.

  16. Yeast as a model host to study replication and recombination of defective interfering RNA of Tomato bushy stunt virus

    International Nuclear Information System (INIS)

    Panavas, Tadas; Nagy, Peter D.

    2003-01-01

    Defective interfering (DI) RNA associated with Tomato bushy stunt virus (TBSV), which is a plus-strand RNA virus, requires p33 and p92 proteins of TBSV or the related Cucumber necrosis virus (CNV), for replication in plants. To test if DI RNA can replicate in a model host, we coexpressed TBSV DI RNA and p33/p92 of CNV in yeast. We show evidence for replication of DI RNA in yeast, including (i) dependence on p33 and p92 for DI replication; (ii) presence of active CNV RNA-dependent RNA polymerase in isolated membrane-containing preparations; (iii) increasing amount of DI RNA(+) over time; (iv) accumulation of (-)stranded DI RNA; (v) presence of correct 5' and 3' ends in DI RNA; (vi) inhibition of replication by mutations in the replication enhancer; and (vii) evolution of DI RNA over time, as shown by sequence heterogeneity. We also produced evidence supporting the occurrence of DI RNA recombinants in yeast. In summary, development of yeast as a host for replication of TBSV DI RNA will facilitate studies on the roles of viral and host proteins in replication/recombination

  17. ER stress, autophagy, and RNA viruses

    Directory of Open Access Journals (Sweden)

    Jia-Rong eJheng

    2014-08-01

    Full Text Available Endoplasmic reticulum (ER stress is a general term for representing the pathway by which various stimuli affect ER functions. ER stress induces the evolutionarily conserved signaling pathways, called the unfolded protein response (UPR, which compromises the stimulus and then determines whether the cell survives or dies. In recent years, ongoing research has suggested that these pathways may be linked to the autophagic response, which plays a key role in the cell’s response to various stressors. Autophagy performs a self-digestion function, and its activation protects cells against certain pathogens. However, the link between the UPR and autophagy may be more complicated. These two systems may act dependently, or the induction of one system may interfere with the other. Experimental studies have found that different viruses modulate these mechanisms to allow them to escape the host immune response or, worse, to exploit the host’s defense to their advantage; thus, this topic is a critical area in antiviral research. In this review, we summarize the current knowledge about how RNA viruses, including influenza virus, poliovirus, coxsackievirus, enterovirus 71, Japanese encephalitis virus, hepatitis C virus, and dengue virus, regulate these processes. We also discuss recent discoveries and how these will produce novel strategies for antiviral treatment.

  18. A functional screen implicates microRNA-138-dependent regulation of the depalmitoylation enzyme APT1 in dendritic spine morphogenesis

    DEFF Research Database (Denmark)

    Siegel, Gabriele; Obernosterer, Gregor; Fiore, Roberto

    2009-01-01

    of acyl protein thioesterase 1 (APT1), an enzyme regulating the palmitoylation status of proteins that are known to function at the synapse, including the alpha(13) subunits of G proteins (Galpha(13)). RNA-interference-mediated knockdown of APT1 and the expression of membrane-localized Galpha(13) both...... suppress spine enlargement caused by inhibition of miR-138, suggesting that APT1-regulated depalmitoylation of Galpha(13) might be an important downstream event of miR-138 function. Our results uncover a previously unknown miRNA-dependent mechanism in neurons and demonstrate a previously unrecognized...

  19. Modeling the costs and benefits of temporary recommendations for poliovirus exporting countries to vaccinate international travelers.

    Science.gov (United States)

    Duintjer Tebbens, Radboud J; Thompson, Kimberly M

    2017-07-05

    Recognizing that infectious agents readily cross international borders, the International Health Regulations Emergency Committee issues Temporary Recommendations (TRs) that include vaccination of travelers from countries affected by public health emergencies, including serotype 1 wild polioviruses (WPV1s). This analysis estimates the costs and benefits of TRs implemented by countries with reported WPV1 during 2014-2016 while accounting for numerous uncertainties. We estimate the TR costs based on programmatic data and prior economic analyses and TR benefits by simulating potential WPV1 outbreaks in the absence of the TRs using the rate and extent of WPV1 importation outbreaks per reported WPV1 case during 2004-2013 and the number of reported WPV1 cases that occurred in countries with active TRs. The benefits of TRs outweigh the costs in 77% of model iterations, resulting in expected incremental net economic benefits of $210 million. Inclusion of indirect costs increases the costs by 13%, the expected savings from prevented outbreaks by 4%, and the expected incremental net benefits by 3%. Despite the considerable costs of implementing TRs, this study provides health and economic justification for these investments in the context of managing a disease in advanced stages of its global eradication. Copyright © 2017 The Auhors. Published by Elsevier Ltd.. All rights reserved.

  20. Genetically Thermo-Stabilised, Immunogenic Poliovirus Empty Capsids; a Strategy for Non-replicating Vaccines.

    Directory of Open Access Journals (Sweden)

    Helen Fox

    2017-01-01

    Full Text Available While wild type polio has been nearly eradicated there will be a need to continue immunisation programmes for some time because of the possibility of re-emergence and the existence of long term excreters of poliovirus. All vaccines in current use depend on growth of virus and most of the non-replicating (inactivated vaccines involve wild type viruses known to cause poliomyelitis. The attenuated vaccine strains involved in the eradication programme have been used to develop new inactivated vaccines as production is thought safer. However it is known that the Sabin vaccine strains are genetically unstable and can revert to a virulent transmissible form. A possible solution to the need for virus growth would be to generate empty viral capsids by recombinant technology, but hitherto such particles are so unstable as to be unusable. We report here the genetic manipulation of the virus to generate stable empty capsids for all three serotypes. The particles are shown to be extremely stable and to generate high levels of protective antibodies in animal models.

  1. Progress toward interrupting wild poliovirus circulation in countries with reestablished transmission--Africa, 2009-2010.

    Science.gov (United States)

    2011-03-18

    Through efforts of the Global Polio Eradication Initiative (GPEI), begun in 1988, indigenous transmission of wild poliovirus (WPV) had been interrupted in all but four countries (Afghanistan, Pakistan, India, and Nigeria) by 2006. Since 2002, a total of 39 previously polio-free countries experienced outbreaks following importation of WPV of Indian or Nigerian origin. Most outbreaks were stopped 12 months following importation before 2009. A key milestone of the GPEI 2010-2012 strategic plan was to interrupt WPV transmission in these African countries with reestablished transmission by the end of 2010. As of March 8, 2011, the milestone appeared to be on track only in Sudan. In Sudan, WPV type 1 (WPV1) was introduced in 2004, but no cases were detected for a 31-month period during 2005-2008. When resurgence occurred in 2008, surveillance and eradication efforts were enhanced, and no case has been detected since June 2009. In Chad, WPV type 3 (WPV3) transmission has persisted since 2007, although undetected for 7 months in 2010. In Angola, WPV1 circulation has persisted following importation in 2007, and became more widespread in 2010, with subsequent importations into DRC and Republic of the Congo (ROC). In DRC, WPV1 circulation has persisted since introduction in 2006. Achieving polio eradication depends on stopping WPV transmission in the four endemic countries and overcoming substantial, ongoing programmatic weaknesses in Chad, Angola, and DRC.

  2. Quantifying low-frequency revertants in oral poliovirus vaccine using next generation sequencing.

    Science.gov (United States)

    Sarcey, Eric; Serres, Aurélie; Tindy, Fabrice; Chareyre, Audrey; Ng, Siemon; Nicolas, Marine; Vetter, Emmanuelle; Bonnevay, Thierry; Abachin, Eric; Mallet, Laurent

    2017-08-01

    Spontaneous reversion to neurovirulence of live attenuated oral poliovirus vaccine (OPV) serotype 3 (chiefly involving the n.472U>C mutation), must be monitored during production to ensure vaccine safety and consistency. Mutant analysis by polymerase chain reaction and restriction enzyme cleavage (MAPREC) has long been endorsed by the World Health Organization as the preferred in vitro test for this purpose; however, it requires radiolabeling, which is no longer supported by many laboratories. We evaluated the performance and suitability of next generation sequencing (NGS) as an alternative to MAPREC. The linearity of NGS was demonstrated at revertant concentrations equivalent to the study range of 0.25%-1.5%. NGS repeatability and intermediate precision were comparable across all tested samples, and NGS was highly reproducible, irrespective of sequencing platform or analysis software used. NGS was performed on OPV serotype 3 working seed lots and monovalent bulks (n=21) that were previously tested using MAPREC, and which covered the representative range of vaccine production. Percentages of 472-C revertants identified by NGS and MAPREC were comparable and highly correlated (r≥0.80), with a Pearson correlation coefficient of 0.95585 (p<0.0001). NGS demonstrated statistically equivalent performance to that of MAPREC for quantifying low-frequency OPV serotype 3 revertants, and offers a valid alternative to MAPREC. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  3. An Introduction to Poliovirus: Pathogenesis, Vaccination, and the Endgame for Global Eradication.

    Science.gov (United States)

    Minor, Philip D

    2016-01-01

    Poliomyelitis is caused by poliovirus, which is a positive strand non-enveloped virus that occurs in three distinct serotypes (1, 2, and 3). Infection is mainly by the fecal-oral route and can be confined to the gut by antibodies induced either by vaccine, previous infection or maternally acquired. Vaccines include the live attenuated strains developed by Sabin and the inactivated vaccines developed by Salk; the live attenuated vaccine (Oral Polio Vaccine or OPV) has been the main tool in the Global Program of Polio eradication of the World Health Organisation. Wild type 2 virus has not caused a case since 1999 and type 3 since 2012 and eradication seems near. However most infections are entirely silent so that sophisticated environmental surveillance may be needed to ensure that the virus has been eradicated, and the live vaccine can sometimes revert to virulent circulating forms under conditions that are not wholly understood. Cessation of vaccination is therefore an increasingly important issue and inactivated polio vaccine (IPV) is playing a larger part in the end game.

  4. MS-2 and poliovirus transport in porous media: Hydrophobic effects and chemical perturbations

    Science.gov (United States)

    Bales, Roger C.; Li, Shimin; Maguire, Kimberly M.; Yahya, Moyasar T.; Gerba, Charles P.

    1993-04-01

    In a series of pH 7 continuous-flow column experiments, removal of the bacteriophage MS-2 by attachment to silica beads had a strong, systematic dependence on the amount of hydrophobic surface present on the beads. With no hydrophobic surface, removal of phage at pH 5 was much greater than at pH 7. Release of attached phage at both pH values did occur, but was slow; breakthrough curves exhibited tailing. Poliovirus attached to silica beads at pH 5.5 much more than at pH 7.0, and attachment was also slowly reversible. Time scales for phage and poliovinis attachment were of the order of hours. The sticking efficiency factor (α), reflecting microscaie physicochemical influences on virus attachment, was in the range of 0.0007-0.02. Phage release was small but measurable under steady state conditions. Release was enhanced by lowering ionic strength and by introducing beef extract, a high-ionic-strength protein solution. Results show that viruses experience reversible attachment/detachment (sometimes termed sorption), that large chemical perturbations are needed to induce rapid virus detachment, and that viruses should be quite mobile in sandy porous media. Even small amounts of hydrophobic organic material in the porous media (≥0.001%) can retard virus transport.

  5. Detection of poliovirus infection in children with acute gastroenteritis in Chiang Mai, Thailand.

    Science.gov (United States)

    Kumthip, Kattareeya; Khamrin, Pattara; Maneekarn, Niwat

    2017-05-01

    Poliovirus (PV) is typically transmitted by the fecal-oral route, which means that the risk of infection and virus distribution could be achieved by exposure to the virus contaminated in food and water. The aim of this study was to determine the occurrence of PV strains by detecting the virus in pediatric patients who admitted to the hospitals with diarrhea in Chiang Mai, Thailand during 2010-2015. By applying a reverse transcription-polymerase chain reaction and nucleotide sequencing analysis of 1,300 stool specimens collected from pediatric patients, PVs were detected at 0.61% (8 out of 1,300 specimens). Among eight PV positive samples, mixed infection with norovirus or human bocavirus was detected in one each out of eight cases. All PV strains detected in this study were characterized further by phylogenetic analysis of 343 bp of the 5' UTR and 315 bp of the partial VP1 sequences. The results revealed that eight PV strains detected in the present study two of each were PV1 and PV2, and four were PV3 serotypes of the Sabin vaccine strains. The data demonstrated the presence of PV1, PV2, and PV3 Sabin vaccine strains in children with acute gastroenteritis in Chiang Mai, Thailand. J. Med. Virol. 89:775-781, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  6. Frequency of isolation of polioviruses and non polio enteroviruses from patients with acute flaccid paralysis, enterovirus infection and children from groups at risk

    Directory of Open Access Journals (Sweden)

    N. I. Romanenkova

    2012-01-01

    Full Text Available The article describes the frequency of isolation of polioviruses and non polio enteroviruses from different categories of the investigated children. The percentage of detection of polioviruses from the patients with acute flaccid paralysis was lower than that from the children from groups at risk. Among the patients with the enterovirus infection the polioviruses were rarely revealed. The frequency of isolation of non polio enteroviruses from these patients was significantly higher than that from the other categories of investigated persons. The improvement of poliomyelitis surveillance and the reinforcement of virological surveillance of children from groups at risk and those with enterovirus infection will provide the important data for Global Polio Eradication Initiative and the maintenance of polio free status of the Russian Federation.

  7. Immunogenicity and safety of combined adsorbed low-dose diphtheria, tetanus and inactivated poliovirus vaccine (REVAXIS®) versus combined diphtheria, tetanus and inactivated poliovirus vaccine (DT Polio®) given as a booster dose at 6 years of age

    Science.gov (United States)

    Gajdos, Vincent; Soubeyrand, Benoit; Vidor, Emmanuel; Richard, Patrick; Boyer, Julie; Sadorge, Christine

    2011-01-01

    This randomized, comparative, phase-IIIb study conducted in France aimed to demonstrate whether seroprotection against diphtheria, tetanus and poliomyelitis 1 month after a single dose of REVAXIS (low-dose diphtheria) is non-inferior to seroprotection 1 month after a single dose of DT Polio (standard-dose diphtheria), both vaccines being given as a second booster to healthy children at 6 years of age. Children were randomly assigned to receive a single intramuscular dose of REVAXIS or DT Polio. Primary endpoints were the 1-month post-booster seroprotection rates for diphtheria, tetanus and poliovirus type-1, -2 and -3 antigens. Secondary endpoints were immunogenicity and safety observations. Of 788 children screened, 760 were randomized: REVAXIS group, 384 children; DT Polio group, 376 children. No relevant difference in demographic characteristics at baseline was observed between REVAXIS and DT Polio groups. Noninferiority of REVAXIS compared with DT Polio for seroprotection was demonstrated against diphtheria (respectively 98.6% and 99.3%), tetanus (respectively 99.6% and 100%) and poliovirus antigens (100% for each types in both groups). No allergic reactions to REVAXIS were reported. A benefit/risk ratio in favor of REVAXIS was suggested by the trend towards a better tolerability of REVAXIS compared with DT Polio regarding the rate of severe solicited injection-site reactions. The results support the use of REVAXIS as a booster at 6 years of age in infants who previously received a three-dose primary series within the first 6 months of life and a first booster including diphtheria, tetanus and poliovirus vaccine(s) given before 2 years of age. PMID:21441781

  8. Comparison of serum and salivary antibodies in children vaccinated with oral live or parenteral inactivated poliovirus vaccines of different antigen concentrations.

    Science.gov (United States)

    Zaman, S; Carlsson, B; Jalil, F; Mellander, L; Van Wezel, A L; Böttiger, M; Hanson, L A

    1991-12-01

    A new antigen-rich inactivated poliovirus vaccine (IPV) in ordinary (IPV1), double (IPV2) and quadruple (IPV4) antigen concentrations was given in 2 doses to 6 and 18 week old Pakistani infants. The immune responses to poliovirus types 1 and 3 were compared to those in infants given three doses of oral poliovirus vaccine (OPV) at 6, 12 and 18 weeks of age. Enzyme-linked immunosorbent assay, ELISA, was used to estimate IgG and IgA in serum and secretory IgA (SIgA) in saliva. Two to three years later, a follow-up of the serum antibody response was carried out in the same infants using a microneutralization test. Serum IgG antibody responses to poliovirus type 1 antigen after two doses of IPV1, IPV2 and IPV4 were not significantly higher than the response after three doses of OPV at 21 weeks of age (p greater than 0.05). The serum IgG responses to poliovirus type 3 were similar to those against type 1 in all the groups. Mean neutralizing antibody titres to poliovirus type 1 was significantly higher in the IPV2 group than the rest of the groups (p less than 0.01). For type 3, these titres were highest but not significantly, in the IPV4 group (p greater than 0.05). This study shows that two doses of a new antigen-rich IPV can give similar immediate serum antibody responses as OPV but higher late responses. SIgA antibodies in saliva were more efficiently induced by OPV after three doses than after 2 doses of IPV (p less than 0.05).

  9. Mycobacterium smegmatis HelY Is an RNA-Activated ATPase/dATPase and 3'-to-5' Helicase That Unwinds 3'-Tailed RNA Duplexes and RNA:DNA Hybrids.

    Science.gov (United States)

    Uson, Maria Loressa; Ordonez, Heather; Shuman, Stewart

    2015-10-01

    Mycobacteria have a large and distinctive ensemble of DNA helicases that function in DNA replication, repair, and recombination. Little is known about the roster of RNA helicases in mycobacteria or their roles in RNA transactions. The 912-amino-acid Mycobacterium smegmatis HelY (MSMEG_3885) protein is a bacterial homolog of the Mtr4 and Ski2 helicases that regulate RNA 3' processing and turnover by the eukaryal exosome. Here we characterize HelY as an RNA-stimulated ATPase/dATPase and an ATP/dATP-dependent 3'-to-5' helicase. HelY requires a 3' single-strand RNA tail (a loading RNA strand) to displace the complementary strand of a tailed RNA:RNA or RNA:DNA duplex. The findings that HelY ATPase is unresponsive to a DNA polynucleotide cofactor and that HelY is unable to unwind a 3'-tailed duplex in which the loading strand is DNA distinguish HelY from other mycobacterial nucleoside triphosphatases/helicases characterized previously. The biochemical properties of HelY, which resemble those of Mtr4/Ski2, hint at a role for HelY in mycobacterial RNA catabolism. RNA helicases play crucial roles in transcription, RNA processing, and translation by virtue of their ability to alter RNA secondary structure or remodel RNA-protein interactions. In eukarya, the RNA helicases Mtr4 and Ski2 regulate RNA 3' resection by the exosome. Mycobacterium smegmatis HelY, a bacterial homolog of Mtr4/Ski2, is characterized here as a unidirectional helicase, powered by RNA-dependent ATP/dATP hydrolysis, that tracks 3' to 5' along a loading RNA strand to displace the complementary strand of a tailed RNA:RNA or RNA:DNA duplex. The biochemical properties of HelY suggest a role in bacterial RNA transactions. HelY homologs are present in pathogenic mycobacteria (e.g., M. tuberculosis and M. leprae) and are widely prevalent in Actinobacteria and Cyanobacteria but occur sporadically elsewhere in the bacterial domain. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  10. Response to a wild poliovirus type 2 (WPV2)-shedding event following accidental exposure to WPV2, the Netherlands, April 2017.

    Science.gov (United States)

    Duizer, Erwin; Ruijs, Wilhelmina Lm; van der Weijden, Charlie P; Timen, Aura

    2017-05-25

    On 3 April 2017, a wild poliovirus type 2 (WPV2) spill occurred in a Dutch vaccine manufacturing plant. Two fully vaccinated operators with risk of exposure were advised on stringent personal hygiene and were monitored for virus shedding. Poliovirus (WPV2-MEF1) was detected in the stool of one, 4 days after exposure, later also in sewage samples. The operator was isolated at home and followed up until shedding stopped 29 days after exposure. No further transmission was detected. This article is copyright of The Authors, 2017.

  11. Multifaceted regulation of translational readthrough by RNA replication elements in a tombusvirus.

    Directory of Open Access Journals (Sweden)

    Peter A Cimino

    2011-12-01

    Full Text Available Translational readthrough of stop codons by ribosomes is a recoding event used by a variety of viruses, including plus-strand RNA tombusviruses. Translation of the viral RNA-dependent RNA polymerase (RdRp in tombusviruses is mediated using this strategy and we have investigated this process using a variety of in vitro and in vivo approaches. Our results indicate that readthrough generating the RdRp requires a novel long-range RNA-RNA interaction, spanning a distance of ∼3.5 kb, which occurs between a large RNA stem-loop located 3'-proximal to the stop codon and an RNA replication structure termed RIV at the 3'-end of the viral genome. Interestingly, this long-distance RNA-RNA interaction is modulated by mutually-exclusive RNA structures in RIV that represent a type of RNA switch. Moreover, a different long-range RNA-RNA interaction that was previously shown to be necessary for viral RNA replicase assembly was also required for efficient readthrough production of the RdRp. Accordingly, multiple replication-associated RNA elements are involved in modulating the readthrough event in tombusviruses and we propose an integrated mechanistic model to describe how this regulatory network could be advantageous by (i providing a quality control system for culling truncated viral genomes at an early stage in the replication process, (ii mediating cis-preferential replication of viral genomes, and (iii coordinating translational readthrough of the RdRp with viral genome replication. Based on comparative sequence analysis and experimental data, basic elements of this regulatory model extend to other members of Tombusviridae, as well as to viruses outside of this family.

  12. Nucleotide variation in Sabin type 3 poliovirus from an Albanian infant with agammaglobulinemia and vaccine associated poliomyelitis.

    Science.gov (United States)

    Foiadelli, Thomas; Savasta, Salvatore; Battistone, Andrea; Kota, Majlinda; Passera, Carolina; Fiore, Stefano; Bino, Silvia; Amato, Concetta; Lozza, Alessandro; Marseglia, Gian Luigi; Fiore, Lucia

    2016-06-10

    Vaccine-associated paralytic poliomyelitis (VAPP) and immunodeficient long-term polio excretors constitute a significant public health burden and are a major concern for the WHO global polio eradication endgame. Poliovirus type 3 characterized as Sabin-like was isolated from a 5-month-old Albanian child with X-linked agammaglobulinemia and VAPP after oral polio vaccine administration. Diagnostic workup and treatment were performed in Italy. Poliovirus replicated in the gut for 7 months. The 5' non coding region (NCR), VP1, VP3 capsid proteins and the 3D polymerase genomic regions of sequential isolates were sequenced. Increasing accumulation of nucleotide mutations in the VP1 region was detected over time, reaching 1.0 % of genome variation with respect to the Sabin reference strain, which is the threshold that defines a vaccine-derived poliovirus (VDPV). We identified mutations in the 5'NCR and VP3 regions that are associated with reversion to neurovirulence. Despite this, all isolates were characterized as Sabin-like. Several amino acid mutations were identified in the VP1 region, probably involved in growth adaptation and viral persistence in the human gut. Intertypic recombination with Sabin type 2 polio in the 3D polymerase region, possibly associated with increased virus transmissibility, was found in all isolates. Gamma-globulin replacement therapy led to viral clearance and neurological improvement, preventing the occurrence of persistent immunodeficiency-related VDPV. This is the first case of VAPP in an immunodeficient child detected in Albania through the Acute Flaccid Paralysis surveillance system and the first investigated case of vaccine associated poliomyelitis in Italy since the introduction of an all-Salk schedule in 2002. We discuss over the biological and clinical implications in the context of the Global Polio Eradication Program and emphasize on the importance of the Acute Flaccid Paralysis surveillance.

  13. Characteristics of an environmentally monitored prolonged type 2 vaccine derived poliovirus shedding episode that stopped without intervention.

    Directory of Open Access Journals (Sweden)

    Tapani Hovi

    Full Text Available Vaccine derived poliovirus (VDPV type 2 strains strongly divergent from the corresponding vaccine strain, Sabin 2, were repeatedly isolated from sewage in Slovakia over a period of 22 months in 2003-2005. Cell cultures of stool specimens from known immune deficient patients and from an identified putative source population of 500 people failed to identify the potential excretor(s of the virus. The occurrence of VDPV in sewage stopped without any intervention. No paralytic cases were reported in Slovakia during the episode. According to a GenBank search and similarity plotting-analysis, the closest known relative of the first isolate PV2/03/SVK/E783 through all main sections of the genome was the type 2 poliovirus Sabin strain, with nucleotide identities in 5'UTR, P1, P2, P3, and 3'UTR parts of the genome of 88.6, 85.9, 87.3, 88.5, and 94.0 percent, respectively. Phenotypic properties of selected Slovakian aVDPV strains resembled those of VDPV strains isolated from immune deficient individuals with prolonged PV infection (iVDPV, including antigenic changes and moderate neurovirulence in the transgenic mouse model. One hundred and two unique VP1 coding sequences were determined from VDPV strains isolated from 34 sewage specimens. Nucleotide differences from Sabin 2 in the VP1 coding region ranged from 12.5 to 15.6 percent, and reached a maximum of 9.6 percent between the VDPV strains under study. Most of the nucleotide substitutions were synonymous but as many as 93 amino acid positions out of 301 in VP1 showed substitutions. We conclude that (1 individuals with prolonged poliovirus infection are not as rare as suggested by the studies on immune deficient patients known to the health care systems and (2 genetic divergence of VDPV strains may remain extensive during years long replication in humans.

  14. Characteristics of an Environmentally Monitored Prolonged Type 2 Vaccine Derived Poliovirus Shedding Episode that Stopped without Intervention

    Science.gov (United States)

    Hovi, Tapani; Paananen, Anja; Blomqvist, Soile; Savolainen-Kopra, Carita; Al-Hello, Haider; Smura, Teemu; Shimizu, Hiroyuki; Nadova, Katarina; Sobotova, Zdenka; Gavrilin, Eugene; Roivainen, Merja

    2013-01-01

    Vaccine derived poliovirus (VDPV) type 2 strains strongly divergent from the corresponding vaccine strain, Sabin 2, were repeatedly isolated from sewage in Slovakia over a period of 22 months in 2003–2005. Cell cultures of stool specimens from known immune deficient patients and from an identified putative source population of 500 people failed to identify the potential excretor(s) of the virus. The occurrence of VDPV in sewage stopped without any intervention. No paralytic cases were reported in Slovakia during the episode. According to a GenBank search and similarity plotting-analysis, the closest known relative of the first isolate PV2/03/SVK/E783 through all main sections of the genome was the type 2 poliovirus Sabin strain, with nucleotide identities in 5′UTR, P1, P2, P3, and 3′UTR parts of the genome of 88.6, 85.9, 87.3, 88.5, and 94.0 percent, respectively. Phenotypic properties of selected Slovakian aVDPV strains resembled those of VDPV strains isolated from immune deficient individuals with prolonged PV infection (iVDPV), including antigenic changes and moderate neurovirulence in the transgenic mouse model. One hundred and two unique VP1 coding sequences were determined from VDPV strains isolated from 34 sewage specimens. Nucleotide differences from Sabin 2 in the VP1 coding region ranged from 12.5 to 15.6 percent, and reached a maximum of 9.6 percent between the VDPV strains under study. Most of the nucleotide substitutions were synonymous but as many as 93 amino acid positions out of 301 in VP1 showed substitutions. We conclude that (1) individuals with prolonged poliovirus infection are not as rare as suggested by the studies on immune deficient patients known to the health care systems and (2) genetic divergence of VDPV strains may remain extensive during years long replication in humans. PMID:23935826

  15. Genomic Surveillance Elucidates Persistent Wild Poliovirus Transmission During 2013-2015 in Major Reservoir Areas of Pakistan.

    Science.gov (United States)

    Alam, Muhammad Masroor; Sharif, Salmaan; Shaukat, Shahzad; Angez, Mehar; Khurshid, Adnan; Rehman, Lubna; Zaidi, Syed Sohail Zahoor

    2016-01-15

    Despite tremendous efforts in the fight against polio, Pakistan bears the highest proportion of poliomyelitis cases among the 3 endemic countries including Afghanistan and Nigeria. Apart from insecurity and inaccessibility challenges, the substantial shift of unimmunized children from North Waziristan due to recent military operations was presumed to favor the widespread poliovirus infection in Pakistan. To better understand the current epidemiological situation, we analyzed the virologic data of wild poliovirus type 1 (WPV1) strains detected in Pakistan during 2013-2015. Five genetic clusters (A-E) were identified with at least 5% nucleotide divergence in the viral protein 1 (VP1) coding region. Peshawar, Quetta, and Karachi were found to be the major endemic foci where multiple discrete genetic lineages of WPV1 were detected. Phylogenetic analysis suggests that wild poliovirus strains from endemic regions were genetically distant (with 5%-15% VP1 nucleotide divergence) from those detected in North Waziristan cases, excluding the possibility of a recent progenitor of WPV1 instigating single-source transmission across the country. Orphan lineages detected in Rawalpindi, Lahore, Hyderabad, Sukkur, and Jacobabad revealed silent transmission and the need for vigilant surveillance. Sustenance of analogous genetic lineages over a period of 3 years highlights multiple unimmunized foci present to maintain viral genetic diversity. Our findings are inconsistent with the hypothesis that impoverished populations from North Waziristan serve as a possible determinant of widespread poliomyelitis infection in Pakistan and further emphasize the need to scale-up clinical and environmental surveillance as well as immunization activities. © The Author 2015. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

  16. Cold chain and virus-free chloroplast-made booster vaccine to confer immunity against different poliovirus serotypes.

    Science.gov (United States)

    Chan, Hui-Ting; Xiao, Yuhong; Weldon, William C; Oberste, Steven M; Chumakov, Konstantin; Daniell, Henry

    2016-11-01

    The WHO recommends complete withdrawal of oral polio vaccine (OPV) type 2 by April 2016 globally and replacing with at least one dose of inactivated poliovirus vaccine (IPV). However, high-cost, limited supply of IPV, persistent circulating vaccine-derived polioviruses transmission and need for subsequent boosters remain unresolved. To meet this critical need, a novel strategy of a low-cost cold chain-free plant-made viral protein 1 (VP1) subunit oral booster vaccine after single IPV dose is reported. Codon optimization of the VP1 gene enhanced expression by 50-fold in chloroplasts. Oral boosting of VP1 expressed in plant cells with plant-derived adjuvants after single priming with IPV significantly increased VP1-IgG1 and VP1-IgA titres when compared to lower IgG1 or negligible IgA titres with IPV injections. IgA plays a pivotal role in polio eradication because of its transmission through contaminated water or sewer systems. Neutralizing antibody titres (~3.17-10.17 log 2 titre) and seropositivity (70-90%) against all three poliovirus Sabin serotypes were observed with two doses of IPV and plant-cell oral boosters but single dose of IPV resulted in poor neutralization. Lyophilized plant cells expressing VP1 stored at ambient temperature maintained efficacy and preserved antigen folding/assembly indefinitely, thereby eliminating cold chain currently required for all vaccines. Replacement of OPV with this booster vaccine and the next steps in clinical translation of FDA-approved antigens and adjuvants are discussed. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  17. Temporal association between the isolation of Sabin-related poliovirus vaccine strains and the Guillain-Barré syndrome

    OpenAIRE

    Friedrich, F.; Filippis, A. M. B.; Schatzmayr, H. G.

    1996-01-01

    Thirty eight paralysis cases classified as Guillain-Barré syndrome (GBS) in Brazil were analysed. In all these cases Sabin-related poliovirus vaccine strains were isolated. In most of the cases the last vaccine dose was given months or years before the onset of GBS, suggesting a persistent infection or the transmission of the Sabin-related strains to the patients. The isolation of Sabin-related strains from GBS cases some days or weeks after the onset of the disease, demonstrated a temporal a...

  18. Analyzed immunogenicity of fractional doses of Sabin-inactivated poliovirus vaccine (sIPV) with intradermal delivery in rats.

    Science.gov (United States)

    Ma, Lei; Cai, Wei; Sun, Mingbo; Cun, Yina; Zhou, Jian; Liu, Jing; Hu, Wenzhu; Zhang, Xinwen; Song, Shaohui; Jiang, Shude; Liao, Guoyang

    2016-12-01

    The live-attenuated oral polio vaccine (OPV) will be no longer used when wild poliovirus (WPV) eliminating in worldwide, according to GPEI (the Global Polio Eradication Initiative) Reports. It is planning to replace OPV by Sabin-based inactivated poliovirus vaccine (sIPV) in developing countries, with purpose of reducing of the economic burden and maintaining of the appropriate antibody levels in population. It studied serial fractional doses immunized by intradermal injection (ID) in rats, to reduce consume of antigen and financial burden, maintaining sufficient immunogenicity; Methods: Study groups were divided in 4 groups of dose gradient, which were one-tenth (1/10), one-fifth (1/5), one-third (1/3) and one-full dose (1/1), according to the volume of distribution taken from the same batch of vaccine (sIPV). Wistar rats were injected intradermally with the needle and syringe sing the mantoux technique taken once month for 3 times. It was used as positive control that intramuscular inoculation (IM) was injected with one-full dose (1/1) with same batch of sIPV. PBS was used as negative control. Blood samples were collected via tail vein. After 30 d with 3 round of immunization, it analyzed the changes of neutralization antibody titers in the each group by each immunization program end; Results: The results of seroconversion had positive correlation with different doses in ID groups. The higher concentration of D-antigen (D-Ag) could conduct higher seroconversion. Furthermore, different types of viruses had different seroconversion trend. It showed that the geometric mean titers (GMTs) of each fractional-dose ID groups increased by higher concentration of D-Ag, and it got significant lower than the full-dose IM group. At 90 th days of immunization, the GMTs for each poliovirus subtypes of fractional doses were almost higher than 1:8, implied that it could be meaning positive seroprotection titer for polio vaccine types, according to WHO suggestion; Conclusions

  19. Implementation of coordinated global serotype 2 oral poliovirus vaccine cessation: risks of potential non-synchronous cessation.

    Science.gov (United States)

    Duintjer Tebbens, Radboud J; Hampton, Lee M; Thompson, Kimberly M

    2016-05-26

    The endgame for polio eradication involves coordinated global cessation of oral poliovirus vaccine (OPV) with cessation of serotype 2 OPV (OPV2 cessation) implemented in late April and early May 2016 and cessation of serotypes 1 and 3 OPV (OPV13 cessation) currently planned for after 2018. The logistics associated with globally switching all use of trivalent OPV (tOPV) to bivalent OPV (bOPV) represent a significant undertaking, which may cause some complications, including delays that lead to different timing of the switch across shared borders. Building on an integrated global model for long-term poliovirus risk management, we consider the expected vulnerability of different populations to transmission of OPV2-related polioviruses as a function of time following the switch. We explore the relationship between the net reproduction number (Rn) of OPV2 at the time of the switch and the time until OPV2-related viruses imported from countries still using OPV2 can establish transmission. We also analyze some specific situations modeled after populations at high potential risk of circulating serotype 2 vaccine-derived poliovirus (cVDPV2) outbreaks in the event of a non-synchronous switch. Well-implemented tOPV immunization activities prior to the tOPV to bOPV switch (i.e., tOPV intensification sufficient to prevent the creation of indigenous cVDPV2 outbreaks) lead to sufficient population immunity to transmission to cause die-out of any imported OPV2-related viruses for over 6 months after the switch in all populations in the global model. Higher Rn of OPV2 at the time of the switch reduces the time until imported OPV2-related viruses can establish transmission and increases the time during which indigenous OPV2-related viruses circulate. Modeling specific connected populations suggests a relatively low vulnerability to importations of OPV2-related viruses that could establish transmission in the context of a non-synchronous switch from tOPV to bOPV, unless the gap

  20. Enhancement of RNA synthesis by promoter duplication in tombusviruses

    International Nuclear Information System (INIS)

    Panavas, T.; Panaviene, Z.; Pogany, J.; Nagy, P.D.

    2003-01-01

    Replication of tombusviruses, small plus-strand RNA viruses of plants, is regulated by cis-acting elements present in the viral RNA. The role of cis-acting elements can be studied in vitro by using a partially purified RNA-dependent RNA polymerase (RdRp) preparation obtained from tombusvirus-infected plants , Virology 276, 279- 288). Here, we demonstrate that the minus-strand RNA of tombusviruses contains, in addition to the 3'-terminal minimal plus-strand initiation promoter, a second cis-acting element, termed the promoter proximal enhancer (PPE). The PPE element enhanced RNA synthesis by almost threefold from the adjacent minimal promoter in the in vitro assay. The sequence of the PPE element is 70% similar to the minimal promoter, suggesting that sequence duplication of the minimal promoter may have been the mechanism leading to the generation of the PPE. Consistent with this proposal, replacement of the PPE element with the minimal promoter, which resulted in a perfectly duplicated promoter region, preserved its enhancer-like function. In contrast, mutagenesis of the PPE element or its replacement with an artificial G/C-rich sequence abolished its stimulative effect on initiation of RNA synthesis in vitro. In vivo experiments are also consistent with the role of the PPE element in enhancement of tombusvirus replication. Sequence comparison of several tombusviruses and related carmoviruses further supports the finding that duplication of minimal promoter sequences may have been an important mechanism during the evolution of cis-acting elements in tombusviruses and related RNA viruses

  1. MicroRNA Related Polymorphisms and Breast Cancer Risk

    Science.gov (United States)

    Khan, Sofia; Greco, Dario; Michailidou, Kyriaki; Milne, Roger L.; Muranen, Taru A.; Heikkinen, Tuomas; Aaltonen, Kirsimari; Dennis, Joe; Bolla, Manjeet K.; Liu, Jianjun; Hall, Per; Irwanto, Astrid; Humphreys, Keith; Li, Jingmei; Czene, Kamila; Chang-Claude, Jenny; Hein, Rebecca; Rudolph, Anja; Seibold, Petra; Flesch-Janys, Dieter; Fletcher, Olivia; Peto, Julian; dos Santos Silva, Isabel; Johnson, Nichola; Gibson, Lorna; Aitken, Zoe; Hopper, John L.; Tsimiklis, Helen; Bui, Minh; Makalic, Enes; Schmidt, Daniel F.; Southey, Melissa C.; Apicella, Carmel; Stone, Jennifer; Waisfisz, Quinten; Meijers-Heijboer, Hanne; Adank, Muriel A.; van der Luijt, Rob B.; Meindl, Alfons; Schmutzler, Rita K.; Müller-Myhsok, Bertram; Lichtner, Peter; Turnbull, Clare; Rahman, Nazneen; Chanock, Stephen J.; Hunter, David J.; Cox, Angela; Cross, Simon S.; Reed, Malcolm W. R.; Schmidt, Marjanka K.; Broeks, Annegien; Veer, Laura J. V. a. n't.; Hogervorst, Frans B.; Fasching, Peter A.; Schrauder, Michael G.; Ekici, Arif B.; Beckmann, Matthias W.; Bojesen, Stig E.; Nordestgaard, Børge G.; Nielsen, Sune F.; Flyger, Henrik; Benitez, Javier; Zamora, Pilar M.; Perez, Jose I. A.; Haiman, Christopher A.; Henderson, Brian E.; Schumacher, Fredrick; Le Marchand, Loic; Pharoah, Paul D. P.; Dunning, Alison M.; Shah, Mitul; Luben, Robert; Brown, Judith; Couch, Fergus J.; Wang, Xianshu; Vachon, Celine; Olson, Janet E.; Lambrechts, Diether; Moisse, Matthieu; Paridaens, Robert; Christiaens, Marie-Rose; Guénel, Pascal; Truong, Thérèse; Laurent-Puig, Pierre; Mulot, Claire; Marme, Frederick; Burwinkel, Barbara; Schneeweiss, Andreas; Sohn, Christof; Sawyer, Elinor J.; Tomlinson, Ian; Kerin, Michael J.; Miller, Nicola; Andrulis, Irene L.; Knight, Julia A.; Tchatchou, Sandrine; Mulligan, Anna Marie; Dörk, Thilo; Bogdanova, Natalia V.; Antonenkova, Natalia N.; Anton-Culver, Hoda; Darabi, Hatef; Eriksson, Mikael; Garcia-Closas, Montserrat; Figueroa, Jonine; Lissowska, Jolanta; Brinton, Louise; Devilee, Peter; Tollenaar, Robert A. E. M.; Seynaeve, Caroline; van Asperen, Christi J.; Kristensen, Vessela N.; Slager, Susan; Toland, Amanda E.; Ambrosone, Christine B.; Yannoukakos, Drakoulis; Lindblom, Annika; Margolin, Sara; Radice, Paolo; Peterlongo, Paolo; Barile, Monica; Mariani, Paolo; Hooning, Maartje J.; Martens, John W. M.; Collée, J. Margriet; Jager, Agnes; Jakubowska, Anna; Lubinski, Jan; Jaworska-Bieniek, Katarzyna; Durda, Katarzyna; Giles, Graham G.; McLean, Catriona; Brauch, Hiltrud; Brüning, Thomas; Ko, Yon-Dschun; Brenner, Hermann; Dieffenbach, Aida Karina; Arndt, Volker; Stegmaier, Christa; Swerdlow, Anthony; Ashworth, Alan; Orr, Nick; Jones, Michael; Simard, Jacques; Goldberg, Mark S.; Labrèche, France; Dumont, Martine; Winqvist, Robert; Pylkäs, Katri; Jukkola-Vuorinen, Arja; Grip, Mervi; Kataja, Vesa; Kosma, Veli-Matti; Hartikainen, Jaana M.; Mannermaa, Arto; Hamann, Ute; Chenevix-Trench, Georgia; Blomqvist, Carl; Aittomäki, Kristiina; Easton, Douglas F.; Nevanlinna, Heli

    2014-01-01

    Genetic variations, such as single nucleotide polymorphisms (SNPs) in microRNAs (miRNA) or in the miRNA binding sites may affect the miRNA dependent gene expression regulation, which has been implicated in various cancers, including breast cancer, and may alter individual susceptibility to cancer. We investigated associations between miRNA related SNPs and breast cancer risk. First we evaluated 2,196 SNPs in a case-control study combining nine genome wide association studies (GWAS). Second, we further investigated 42 SNPs with suggestive evidence for association using 41,785 cases and 41,880 controls from 41 studies included in the Breast Cancer Association Consortium (BCAC). Combining the GWAS and BCAC data within a meta-analysis, we estimated main effects on breast cancer risk as well as risks for estrogen receptor (ER) and age defined subgroups. Five miRNA binding site SNPs associated significantly with breast cancer risk: rs1045494 (odds ratio (OR) 0.92; 95% confidence interval (CI): 0.88–0.96), rs1052532 (OR 0.97; 95% CI: 0.95–0.99), rs10719 (OR 0.97; 95% CI: 0.94–0.99), rs4687554 (OR 0.97; 95% CI: 0.95–0.99, and rs3134615 (OR 1.03; 95% CI: 1.01–1.05) located in the 3′ UTR of CASP8, HDDC3, DROSHA, MUSTN1, and MYCL1, respectively. DROSHA belongs to miRNA machinery genes and has a central role in initial miRNA processing. The remaining genes are involved in different molecular functions, including apoptosis and gene expression regulation. Further studies are warranted to elucidate whether the miRNA binding site SNPs are the causative variants for the observed risk effects. PMID:25390939

  2. MicroRNA related polymorphisms and breast cancer risk.

    Science.gov (United States)

    Khan, Sofia; Greco, Dario; Michailidou, Kyriaki; Milne, Roger L; Muranen, Taru A; Heikkinen, Tuomas; Aaltonen, Kirsimari; Dennis, Joe; Bolla, Manjeet K; Liu, Jianjun; Hall, Per; Irwanto, Astrid; Humphreys, Keith; Li, Jingmei; Czene, Kamila; Chang-Claude, Jenny; Hein, Rebecca; Rudolph, Anja; Seibold, Petra; Flesch-Janys, Dieter; Fletcher, Olivia; Peto, Julian; dos Santos Silva, Isabel; Johnson, Nichola; Gibson, Lorna; Aitken, Zoe; Hopper, John L; Tsimiklis, Helen; Bui, Minh; Makalic, Enes; Schmidt, Daniel F; Southey, Melissa C; Apicella, Carmel; Stone, Jennifer; Waisfisz, Quinten; Meijers-Heijboer, Hanne; Adank, Muriel A; van der Luijt, Rob B; Meindl, Alfons; Schmutzler, Rita K; Müller-Myhsok, Bertram; Lichtner, Peter; Turnbull, Clare; Rahman, Nazneen; Chanock, Stephen J; Hunter, David J; Cox, Angela; Cross, Simon S; Reed, Malcolm W R; Schmidt, Marjanka K; Broeks, Annegien; Van't Veer, Laura J; Hogervorst, Frans B; Fasching, Peter A; Schrauder, Michael G; Ekici, Arif B; Beckmann, Matthias W; Bojesen, Stig E; Nordestgaard, Børge G; Nielsen, Sune F; Flyger, Henrik; Benitez, Javier; Zamora, Pilar M; Perez, Jose I A; Haiman, Christopher A; Henderson, Brian E; Schumacher, Fredrick; Le Marchand, Loic; Pharoah, Paul D P; Dunning, Alison M; Shah, Mitul; Luben, Robert; Brown, Judith; Couch, Fergus J; Wang, Xianshu; Vachon, Celine; Olson, Janet E; Lambrechts, Diether; Moisse, Matthieu; Paridaens, Robert; Christiaens, Marie-Rose; Guénel, Pascal; Truong, Thérèse; Laurent-Puig, Pierre; Mulot, Claire; Marme, Frederick; Burwinkel, Barbara; Schneeweiss, Andreas; Sohn, Christof; Sawyer, Elinor J; Tomlinson, Ian; Kerin, Michael J; Miller, Nicola; Andrulis, Irene L; Knight, Julia A; Tchatchou, Sandrine; Mulligan, Anna Marie; Dörk, Thilo; Bogdanova, Natalia V; Antonenkova, Natalia N; Anton-Culver, Hoda; Darabi, Hatef; Eriksson, Mikael; Garcia-Closas, Montserrat; Figueroa, Jonine; Lissowska, Jolanta; Brinton, Louise; Devilee, Peter; Tollenaar, Robert A E M; Seynaeve, Caroline; van Asperen, Christi J; Kristensen, Vessela N; Slager, Susan; Toland, Amanda E; Ambrosone, Christine B; Yannoukakos, Drakoulis; Lindblom, Annika; Margolin, Sara; Radice, Paolo; Peterlongo, Paolo; Barile, Monica; Mariani, Paolo; Hooning, Maartje J; Martens, John W M; Collée, J Margriet; Jager, Agnes; Jakubowska, Anna; Lubinski, Jan; Jaworska-Bieniek, Katarzyna; Durda, Katarzyna; Giles, Graham G; McLean, Catriona; Brauch, Hiltrud; Brüning, Thomas; Ko, Yon-Dschun; Brenner, Hermann; Dieffenbach, Aida Karina; Arndt, Volker; Stegmaier, Christa; Swerdlow, Anthony; Ashworth, Alan; Orr, Nick; Jones, Michael; Simard, Jacques; Goldberg, Mark S; Labrèche, France; Dumont, Martine; Winqvist, Robert; Pylkäs, Katri; Jukkola-Vuorinen, Arja; Grip, Mervi; Kataja, Vesa; Kosma, Veli-Matti; Hartikainen, Jaana M; Mannermaa, Arto; Hamann, Ute; Chenevix-Trench, Georgia; Blomqvist, Carl; Aittomäki, Kristiina; Easton, Douglas F; Nevanlinna, Heli

    2014-01-01

    Genetic variations, such as single nucleotide polymorphisms (SNPs) in microRNAs (miRNA) or in the miRNA binding sites may affect the miRNA dependent gene expression regulation, which has been implicated in various cancers, including breast cancer, and may alter individual susceptibility to cancer. We investigated associations between miRNA related SNPs and breast cancer risk. First we evaluated 2,196 SNPs in a case-control study combining nine genome wide association studies (GWAS). Second, we further investigated 42 SNPs with suggestive evidence for association using 41,785 cases and 41,880 controls from 41 studies included in the Breast Cancer Association Consortium (BCAC). Combining the GWAS and BCAC data within a meta-analysis, we estimated main effects on breast cancer risk as well as risks for estrogen receptor (ER) and age defined subgroups. Five miRNA binding site SNPs associated significantly with breast cancer risk: rs1045494 (odds ratio (OR) 0.92; 95% confidence interval (CI): 0.88-0.96), rs1052532 (OR 0.97; 95% CI: 0.95-0.99), rs10719 (OR 0.97; 95% CI: 0.94-0.99), rs4687554 (OR 0.97; 95% CI: 0.95-0.99, and rs3134615 (OR 1.03; 95% CI: 1.01-1.05) located in the 3' UTR of CASP8, HDDC3, DROSHA, MUSTN1, and MYCL1, respectively. DROSHA belongs to miRNA machinery genes and has a central role in initial miRNA processing. The remaining genes are involved in different molecular functions, including apoptosis and gene expression regulation. Further studies are warranted to elucidate whether the miRNA binding site SNPs are the causative variants for the observed risk effects.

  3. MicroRNA related polymorphisms and breast cancer risk.

    Directory of Open Access Journals (Sweden)

    Sofia Khan

    Full Text Available Genetic variations, such as single nucleotide polymorphisms (SNPs in microRNAs (miRNA or in the miRNA binding sites may affect the miRNA dependent gene expression regulation, which has been implicated in various cancers, including breast cancer, and may alter individual susceptibility to cancer. We investigated associations between miRNA related SNPs and breast cancer risk. First we evaluated 2,196 SNPs in a case-control study combining nine genome wide association studies (GWAS. Second, we further investigated 42 SNPs with suggestive evidence for association using 41,785 cases and 41,880 controls from 41 studies included in the Breast Cancer Association Consortium (BCAC. Combining the GWAS and BCAC data within a meta-analysis, we estimated main effects on breast cancer risk as well as risks for estrogen receptor (ER and age defined subgroups. Five miRNA binding site SNPs associated significantly with breast cancer risk: rs1045494 (odds ratio (OR 0.92; 95% confidence interval (CI: 0.88-0.96, rs1052532 (OR 0.97; 95% CI: 0.95-0.99, rs10719 (OR 0.97; 95% CI: 0.94-0.99, rs4687554 (OR 0.97; 95% CI: 0.95-0.99, and rs3134615 (OR 1.03; 95% CI: 1.01-1.05 located in the 3' UTR of CASP8, HDDC3, DROSHA, MUSTN1, and MYCL1, respectively. DROSHA belongs to miRNA machinery genes and has a central role in initial miRNA processing. The remaining genes are involved in different molecular functions, including apoptosis and gene expression regulation. Further studies are warranted to elucidate whether the miRNA binding site SNPs are the causative variants for the observed risk effects.

  4. Identification of RNA Binding Proteins Associated with Dengue Virus RNA in Infected Cells Reveals Temporally Distinct Host Factor Requirements.

    Directory of Open Access Journals (Sweden)

    Olga V Viktorovskaya

    2016-08-01

    Full Text Available There are currently no vaccines or antivirals available for dengue virus infection, which can cause dengue hemorrhagic fever and death. A better understanding of the host pathogen interaction is required to develop effective therapies to treat DENV. In particular, very little is known about how cellular RNA binding proteins interact with viral RNAs. RNAs within cells are not naked; rather they are coated with proteins that affect localization, stability, translation and (for viruses replication.Seventy-nine novel RNA binding proteins for dengue virus (DENV were identified by cross-linking proteins to dengue viral RNA during a live infection in human cells. These cellular proteins were specific and distinct from those previously identified for poliovirus, suggesting a specialized role for these factors in DENV amplification. Knockdown of these proteins demonstrated their function as viral host factors, with evidence for some factors acting early, while others late in infection. Their requirement by DENV for efficient amplification is likely specific, since protein knockdown did not impair the cell fitness for viral amplification of an unrelated virus. The protein abundances of these host factors were not significantly altered during DENV infection, suggesting their interaction with DENV RNA was due to specific recruitment mechanisms. However, at the global proteome level, DENV altered the abundances of proteins in particular classes, including transporter proteins, which were down regulated, and proteins in the ubiquitin proteasome pathway, which were up regulated.The method for identification of host factors described here is robust and broadly applicable to all RNA viruses, providing an avenue to determine the conserved or distinct mechanisms through which diverse viruses manage the viral RNA within cells. This study significantly increases the number of cellular factors known to interact with DENV and reveals how DENV modulates and usurps

  5. Comparison of poliovirus recombinants: accumulation of point mutations provides further advantages.

    Science.gov (United States)

    Savolainen-Kopra, Carita; Samoilovich, Elena; Kahelin, Heidi; Hiekka, Anna-Kaisa; Hovi, Tapani; Roivainen, Merja

    2009-08-01

    The roles of recombination and accumulation of point mutations in the origin of new poliovirus (PV) characteristics have been hypothesized, but it is not known which are essential to evolution. We studied phenotypic differences between recombinant PV strains isolated from successive stool specimens of an oral PV vaccine recipient. The studied strains included three PV2/PV1 recombinants with increasing numbers of mutations in the VP1 gene, two of the three with an amino acid change I-->T in the DE-loop of VP1, their putative PV1 parent and strains Sabin 1 and 2. Growth of these viruses was examined in three cell lines: colorectal adenocarcinoma, neuroblastoma and HeLa. The main observation was a higher growth rate between 4 and 6 h post-infection of the two recombinants with the I-->T substitution. All recombinants grew at a higher rate than parental strains in the exponential phase of the replication cycle. In a temperature sensitivity test, the I-->T-substituted recombinants replicated equally well at an elevated temperature. Complete genome sequencing of the three recombinants revealed 12 (3), 19 (3) and 27 (3) nucleotide (amino acid) differences from Sabin. Mutations were located in regions defining attenuation, temperature sensitivity, antigenicity and the cis-acting replicating element. The recombination site was in the 5' end of 3D. In a competition assay, the most mutated recombinant beat parental Sabin in all three cell lines, strongly suggesting that this virus has an advantage. Two independent intertypic recombinants, PV3/PV1 and PV3/PV2, also showed similar growth advantages, but they also contained several point mutations. Thus, our data defend the hypothesis that accumulation of certain advantageous mutations plays a key role in gaining increased fitness.

  6. Progress toward interruption of wild poliovirus transmission--worldwide, January 2011-March 2012.

    Science.gov (United States)

    2012-05-18

    In January 2012, completion of polio eradication was declared a programmatic emergency for global public health by the Executive Board of the World Health Organization (WHO). Despite major progress since the launch of the Global Polio Eradication Initiative (GPEI) in 1988, circulation of indigenous wild poliovirus (WPV) continues in three countries (Afghanistan, Nigeria, and Pakistan). India has not reported a polio case since January 2011, and is considered polio-free since February 2012. This report highlights progress toward global polio eradication during January 2011-March 2012. The number of polio cases reported globally decreased by 52%, from 1,352 in 2010 to 650 in 2011. Those 650 cases included 341 (53%) reported from the four polio-endemic countries (Afghanistan, India, Nigeria, and Pakistan), 230 (35%) from previously polio-free countries in which WPV importations led to reestablished transmission for ≥12 months (Angola, Chad, and Democratic Republic of the Congo [DRC]), and 79 (12%) from nine countries affected by outbreaks. Compared with 2010, WPV cases increased in 2011 in Afghanistan (69%), Nigeria (66%), and Pakistan (27%), but decreased in India (98%). During January-March 2012, 59% fewer cases were reported worldwide (as of May 15) compared with the same period in 2011, and all cases in 2012 have been reported from Afghanistan, Chad, Nigeria, and Pakistan. Although progress toward polio eradication was substantial in 2011, persistent WPV circulation in 2012, particularly in Nigeria and Pakistan, poses an ongoing threat to eradication efforts, underscoring the need for emergency measures by polio-affected countries and those at risk for outbreaks after importation.

  7. Outbreaks following wild poliovirus importations --- Europe, Africa, and Asia, January 2009-September 2010.

    Science.gov (United States)

    2010-11-05

    The Global Polio Eradication Initiative (GPEI) began in 1988. By 2006, indigenous transmission of wild poliovirus (WPV) had been interrupted in all but four countries (Afghanistan, India, Nigeria, and Pakistan). However, outbreaks following WPV importations into previously polio-free countries remain an ongoing risk until polio is eradicated. The GPEI Strategic Plan for 2010-2012 set the following two goals for outbreak control: 1) end outbreaks occurring in 2009 by mid-2010 and 2) end outbreaks occurring during 2010 to mid-2012 within 6 months of confirmation. This report describes new outbreaks that have occurred in the World Health Organization (WHO) European Region and updates previous reports on the status of outbreaks in Africa and Asia. In 2010, the first WPV importation into the European Region since the region was declared polio-free in 2002 resulted in 476 confirmed cases: 458 in Tajikistan, 14 in Russia, three in Turkmenistan, and one in Kazakhstan. In Africa and Asia, 11 new importations into six countries were observed in 2010; 30 WPV importations that occurred during 2008-2009 resulted in 215 cases in 15 African countries during 2009-2010. An outbreak is considered interrupted if 6 months have elapsed since the latest confirmed case and surveillance performance indicators meet WHO standards. All 2009 outbreaks in Africa appear to have been interrupted, and 2010 outbreaks in three countries appear to have been interrupted. Maintaining high routine vaccination coverage and sensitive surveillance at all times and rapidly instituting additional immunization programs to control outbreaks are key to limiting and stopping the spread of WPV.

  8. Use of Preservative Agents and Antibiotics for Increased Poliovirus Survival on Positively Charged Filters.

    Science.gov (United States)

    Fagnant, Christine Susan; Kossik, Alexandra Lynn; Zhou, Nicolette Angela; Sánchez-Gonzalez, Liliana; Falman, Jill Christin; Keim, Erika Karen; Linden, Yarrow; Scheibe, Alana; Barnes, Kilala Sayisha; Beck, Nicola Koren; Boyle, David S; Meschke, John Scott

    2017-12-01

    Environmental surveillance of poliovirus (PV) and other non-enveloped viruses can help identify silent circulation and is necessary to certify eradication. The bag-mediated filtration system is an efficient method to filter large volumes of environmental waters at field sites for monitoring the presence of viruses. As filters may require long transit times to off-site laboratories for processing, viral inactivation or overgrowth of bacteria and fungi can interfere with virus detection and quantification (Miki and Jacquet in Aquatic Microb Ecol 51(2):195-208, 2008). To evaluate virus survival over time on ViroCap ™ filters, the filters were seeded with PV type 1 (PV1) and/or MS2 and then dosed with preservatives or antibiotics prior to storage and elution. These filters were stored at various temperatures and time periods, and then eluted for PV1 and MS2 recovery quantification. Filters dosed with the preservative combination of 2% sodium benzoate and 0.2% calcium propionate had increased virus survival over time when stored at 25 °C, compared to samples stored at 25 °C with no preservatives. While elution within 24 h of filtration is recommended, if storage or shipping is required then this preservative mixture can help preserve sample integrity. Addition of an antibiotic cocktail containing cephapirin, gentamicin, and Proclin ™ 300 increased recovery after storage at 4 and 25 °C, when compared to storage with no antibiotics. The antibiotic cocktail can aid sample preservation if access to appropriate antibiotics storage is available and sample cold chain is unreliable. This study demonstrated that the use of preservatives or antibiotics is a simple, cost-effective method to improve virus detection from ViroCap cartridge filters over time.

  9. [Genetic Characteristics of Type 2 Vaccine-derived Poliovirus in Shanxi Province (China) in 2014].

    Science.gov (United States)

    Yan, Dongrei; Li, Xiaolei; Zhang, Yong; Yang, Jianfang; Zhu, Shuangli; Wang, Dongyan; Zhang, Chuangye; Zhu, Hui; Xu, Wenbo

    2015-03-01

    The World Health Organization redefined the type 2 vaccine-derived poliovirus (VDPV) in 2010. To study the genetic characteristics and evolution of type 2 VDPV under this new definition, we conducted genome sequencing and analyses of type 2 VDPVs isolated from one patient with acute flaccid paralysis in Shanxi province (China) in 2014. Nucleotide sequencing revealed that the full-length of type 2 VDPV is 7439 bases encoding 2207 amino acids with no insertion or deletion of nucleotides compared with Sabin2. One nucleotide substitution identified as a key determinant of the attenuated phenotype of the Sabin 2 strain (A-G reversion at nucleotide nt 481 in the 5-end of the untranslated region) had reverted in the Shanxi type 2 VDPV. The other known key determinant of the attenuated phenotype of the Sabin 2 strain (U-->C reversion at nt2909 in the VP1 coding region that caused a Ile143Thr substitution in VP1) had not reverted in the Shanxi VDPV. The Shanxi type 2 VDPV was S2/S1 recombinant, the crossover site of which mapped to the 3-end of the 3D region (between nt 6247 and nt 6281). A phylogentic tree based on the VP1 coding region showed that evolution of the Shanxi type 2 VDPV was independent of other type 2 VDPVs detected worldwide. We estimated that the strain circulated for approximately = 11 months in the population according to the known evolution rate. The present study confirmed that the Chinese Polio Laboratory Network could discover the VDPV promptly and that it played an important part in maintenance of a polio-free China.

  10. The recent outbreaks and reemergence of poliovirus in war and conflict-affected areas.

    Science.gov (United States)

    Akil, Luma; Ahmad, H Anwar

    2016-08-01

    Poliomyelitis is a highly infectious disease caused by poliovirus, which becomes difficult to manage/eradicate in politically unstable areas. The objectives of this study were to determine the movement and management of such polio outbreaks in endemic countries and countries with reoccurring cases of polio and to determine the effect of political instability on polio eradication. In this study, the extent of polio outbreaks was examined and modeled using statistical methodologies and mapped with GIS software. Data on polio cases and immunization were collected for countries with polio cases for the period 2011 to 2014. Weekly data from the Global Polio Eradication Initiative were collected for selected countries. The recent virus origin and current movement was mapped using GIS. Correlations between immunization rates, the Global Peace Index (GPI), and other indicators of a country's political stability with polio outbreaks were determined. Data were analyzed using SAS 9.4 and ArcGIS 10. For several reasons, Pakistan remains highly vulnerable