WorldWideScience

Sample records for virus polymerase gene

  1. Hepatitis B virus DNA polymerase gene polymorphism based ...

    African Journals Online (AJOL)

    Conclusion: A method for determination of HBV genotypes using pol gene sequencing which simultaneously detects major drug resistance mutations has been established. HBV genetic diversity may play an important role in treatment decision. Keywords: Hepatitis B virus, nested PCR, genotype, sub-genotypes, YMDD ...

  2. Polymerase chain reaction (PCR) amplification of a nucleoprotein gene sequence of infectious hematopoietic necrosis virus

    Science.gov (United States)

    Arakawa, C.K.; Deering, R.E.; Higman, K.H.; Oshima, K.H.; O'Hara, P.J.; Winton, J.R.

    1990-01-01

    The polymerase chain reaction [PCR) was used to amplify a portion of the nucleoprotein [NI gene of infectious hematopoietic necrosis virus (IHNV). Using a published sequence for the Round Butte isolate of IHNV, a pair of PCR pnmers was synthesized that spanned a 252 nucleotide region of the N gene from residue 319 to residue 570 of the open reading frame. This region included a 30 nucleotide target sequence for a synthetic oligonucleotide probe developed for detection of IHNV N gene messenger RNA. After 25 cycles of amplification of either messenger or genomic RNA, the PCR product (DNA) of the expected size was easily visible on agarose gels stained with ethidium bromide. The specificity of the amplified DNA was confirmed by Southern and dot-blot analysis using the biotinylated oligonucleotide probe. The PCR was able to amplify the N gene sequence of purified genomic RNA from isolates of IHNV representing 5 different electropherotypes. Using the IHNV primer set, no PCR product was obtained from viral hemorrhagic septicemia virus RNA, but 2 higher molecular weight products were synthesized from hirame rhabdovirus RNA that did not hybridize with the biotinylated probe. The PCR could be efficiently performed with all IHNV genomic RNA template concentrations tested (1 ng to 1 pg). The lowest level of sensitivity was not determined. The PCR was used to amplify RNA extracted from infected cell cultures and selected tissues of Infected rainbow trout. The combination of PCR and nucleic acid probe promises to provide a detection method for IHNV that is rapid, h~ghly specific, and sensitive.

  3. Molecular Function Analysis of Rabies Virus RNA Polymerase L Protein by Using an L Gene-Deficient Virus.

    Science.gov (United States)

    Nakagawa, Kento; Kobayashi, Yuki; Ito, Naoto; Suzuki, Yoshiyuki; Okada, Kazuma; Makino, Machiko; Goto, Hideo; Takahashi, Tatsuki; Sugiyama, Makoto

    2017-10-15

    While the RNA-dependent RNA polymerase L protein of rabies virus (RABV), a member of the genus Lyssavirus of the family Rhabdoviridae, has potential to be a therapeutic target for rabies, the molecular functions of this protein have remained largely unknown. In this study, to obtain a novel experimental tool for molecular function analysis of the RABV L protein, we established by using a reverse genetics approach an L gene-deficient RABV (Nishi-ΔL/Nluc), which infects, propagates, and correspondingly produces NanoLuc luciferase in cultured neuroblastoma cells transfected to express the L protein. trans-Complementation with wild-type L protein, but not that with a functionally defective L protein mutant, efficiently supported luciferase production by Nishi-ΔL/Nluc, confirming its potential for function analysis of the L protein. Based on the findings obtained from comprehensive genetic analyses of L genes from various RABV and other lyssavirus species, we examined the functional importance of a highly conserved L protein region at positions 1914 to 1933 by a trans-complementation assay with Nishi-ΔL/Nluc and a series of L protein mutants. The results revealed that the amino acid sequence at positions 1929 to 1933 (NPYNE) is functionally important, and this was supported by other findings that this sequence is critical for binding of the L protein with its essential cofactor, P protein, and thus also for L protein's RNA polymerase activity. Our findings provide useful information for the development of an anti-RABV drug targeting the L-P protein interaction.IMPORTANCE To the best of our knowledge, this is the first report on the establishment of an L gene-deficient, reporter gene-expressing virus in all species of the order Mononegavirales, also highlighting its applicability to a trans-complementation assay, which is useful for molecular function analyses of their L proteins. Moreover, this study revealed for the first time that the NPYNE sequence at positions

  4. A broadly applicable method to characterize large DNA viruses and adenoviruses based on the DNA polymerase gene

    Directory of Open Access Journals (Sweden)

    Montgomery Roy D

    2006-04-01

    Full Text Available Abstract Background Many viral pathogens are poorly characterized, are difficult to culture or reagents are lacking for confirmatory diagnoses. We have developed and tested a robust assay for detecting and characterizing large DNA viruses and adenoviruses. The assay is based on the use of degenerate PCR to target a gene common to these viruses, the DNA polymerase, and sequencing the products. Results We evaluated our method by applying it to fowl adenovirus isolates, catfish herpesvirus isolates, and largemouth bass ranavirus (iridovirus from cell culture and lymphocystis disease virus (iridovirus and avian poxvirus from tissue. All viruses with the exception of avian poxvirus produced the expected product. After optimization of extraction procedures, and after designing and applying an additional primer we were able to produce polymerase gene product from the avian poxvirus genome. The sequence data that we obtained demonstrated the simplicity and potential of the method for routine use in characterizing large DNA viruses. The adenovirus samples were demonstrated to represent 2 types of fowl adenovirus, fowl adenovirus 1 and an uncharacterized avian adenovirus most similar to fowl adenovirus 9. The herpesvirus isolate from blue catfish was shown to be similar to channel catfish virus (Ictalurid herpesvirus 1. The case isolate of largemouth bass ranavirus was shown to exactly match the type specimen and both were similar to tiger frog virus and frog virus 3. The lymphocystis disease virus isolate from largemouth bass was shown to be related but distinct from the two previously characterized lymphocystis disease virus isolates suggesting that it may represent a distinct lymphocystis disease virus species. Conclusion The method developed is rapid and broadly applicable to cell culture isolates and infected tissues. Targeting a specific gene for in the large DNA viruses and adenoviruses provide a common reference for grouping the newly identified

  5. Polymerase read-through at the first transcription termination site contributes to regulation of borna disease virus gene expression.

    Science.gov (United States)

    Poenisch, Marion; Wille, Sandra; Staeheli, Peter; Schneider, Urs

    2008-10-01

    An unusually long noncoding sequence is located between the N gene of Borna disease virus (BDV) and the genes for regulatory factor X and polymerase cofactor P. Most of these nucleotides are transcribed and seem to control translation of the bicistronic X/P mRNA. We report here that Vero cells persistently infected with mutant viruses containing minor alterations in this control region showed almost normal levels of N, X, and P proteins but exhibited greatly reduced levels of mRNAs coding for these viral gene products. Surprisingly, cells infected with these BDV mutants accumulated a viral transcript 1.9 kb in length that represents a capped and polyadenylated mRNA containing the coding regions of the N, X, and P genes. Cells infected with wild-type BDV also contained substantial amounts of this read-through mRNA, which yielded both N and P protein when translated in vitro. Viruses carrying mutations that promoted read-through transcription at the first gene junction failed to replicate in the brain of adult rats. In the brains of newborn rats, these mutant viruses were able to replicate after acquiring second-site mutations in or near the termination signal located downstream of the N gene. Thus, sequence elements adjacent to the core termination signal seem to regulate the frequency by which the polymerase terminates transcription after the N gene. We conclude from these observations that BDV uses read-through transcription for fine-tuning the expression of the N, X, and P genes which, in turn, influence viral polymerase activity.

  6. Polymerase Read-Through at the First Transcription Termination Site Contributes to Regulation of Borna Disease Virus Gene Expression ▿

    Science.gov (United States)

    Poenisch, Marion; Wille, Sandra; Staeheli, Peter; Schneider, Urs

    2008-01-01

    An unusually long noncoding sequence is located between the N gene of Borna disease virus (BDV) and the genes for regulatory factor X and polymerase cofactor P. Most of these nucleotides are transcribed and seem to control translation of the bicistronic X/P mRNA. We report here that Vero cells persistently infected with mutant viruses containing minor alterations in this control region showed almost normal levels of N, X, and P proteins but exhibited greatly reduced levels of mRNAs coding for these viral gene products. Surprisingly, cells infected with these BDV mutants accumulated a viral transcript 1.9 kb in length that represents a capped and polyadenylated mRNA containing the coding regions of the N, X, and P genes. Cells infected with wild-type BDV also contained substantial amounts of this read-through mRNA, which yielded both N and P protein when translated in vitro. Viruses carrying mutations that promoted read-through transcription at the first gene junction failed to replicate in the brain of adult rats. In the brains of newborn rats, these mutant viruses were able to replicate after acquiring second-site mutations in or near the termination signal located downstream of the N gene. Thus, sequence elements adjacent to the core termination signal seem to regulate the frequency by which the polymerase terminates transcription after the N gene. We conclude from these observations that BDV uses read-through transcription for fine-tuning the expression of the N, X, and P genes which, in turn, influence viral polymerase activity. PMID:18653450

  7. Subgenotypes and Mutations in the S and Polymerase Genes of Hepatitis B Virus Carriers in the West Bank, Palestine

    Science.gov (United States)

    Abdelnabi, Zakeih; Saleh, Niveen; Baraghithi, Sabri; Glebe, Dieter; Azzeh, Maysa

    2014-01-01

    The mutation rate and genetic variability of hepatitis B virus (HBV) are crucial factors for efficient treatment and successful vaccination against HBV. Until today, genetic properties of this virus among the Palestinian population remain unknown. Therefore, we performed genetic analysis of the overlapping S and polymerase genes of HBV, isolated from 40 Palestinian patients' sera. All patients were HBsAg positive and presented with a viral load above 105 HBV genome copies/ml. The genotyping results of the S gene demonstrated that HBV D1 was detected in 90% of the samples representing the most prominent subgenotype among Palestinians carrying HBV. Various mutations existed within the S gene; in five patients four known escape mutations including the common G145R and D144E were found. Furthermore, a ratio of 4.25 of non-synonymous to synonymous mutations in the S gene indicated a strong selection pressure on the HBs antigen loops of HBV strains circulating in those Palestinian patients. Although all patients were treatment-naïve, with the exception of one, several mutations were found in the HBV polymerase gene, but none pointed to drug resistance. The study presented here is the first report to address subgenotypes and mutation analyses of HBV S and polymerase genes in Palestine. PMID:25503289

  8. Genotyping the hepatitis B virus with a fragment of the HBV DNA polymerase gene in Shenyang, China

    Directory of Open Access Journals (Sweden)

    Juan Feng

    2011-06-01

    Full Text Available Abstract The hepatitis B virus (HBV has been classified into eight genotypes (A-H based on intergenotypic divergence of at least 8% in the complete nucleotide sequence or more than 4% in the S gene. To facilitate the investigation of the relationship between the efficacy of drug treatment and the mutation with specific genotype of HBV, we have established a new genotyping strategy based on a fragment of the HBV DNA polymerase gene. Pairwise sequence and phylogenetic analyses were performed using CLUSTAL V (DNASTAR on the eight (A-H standard full-length nucleotide sequences of HBV DNA from GenBank (NCBI and the corresponding semi-nested PCR products from the HBV DNA polymerase gene. The differences in the semi-nested PCR fragments of the polymerase genes among genotypes A through F were greater than 4%, which is consistent with the intergenotypic divergence of at least 4% in HBV DNA S gene sequences. Genotyping using the semi-nested PCR products of the DNA polymerase genes revealed that only genotypes B, C, and D were present in the 50 cases, from Shenyang, China, with a distribution of 11 cases (22%, 25 cases (50%, and 14 cases (28% respectively. These results demonstrate that our new genotyping method utilizing a fragment of the HBV DNA polymerase gene is valid and can be employed as a general genotyping strategy in areas with prevalent HBV genotypes A through F. In Shenyang, China, genotypes C, B, and D were identified with this new genotyping method, and genotype C was demonstrated to be the dominant genotype.

  9. Mutations in polymerase genes enhanced the virulence of 2009 pandemic H1N1 influenza virus in mice.

    Directory of Open Access Journals (Sweden)

    Wenfei Zhu

    Full Text Available Influenza A virus can infect a wide variety of animal species with illness ranging from mild to severe, and is a continual cause for concern. Genetic mutations that occur either naturally or during viral adaptation in a poorly susceptible host are key mechanisms underlying the evolution and virulence of influenza A virus. Here, the variants containing PA-A36T or PB2-H357N observed in the mouse-adapted descendants of 2009 pandemic H1N1 virus (pH1N1, A/Sichuan/1/2009 (SC, were characterized. Both mutations enhanced polymerase activity in mammalian cells. These effects were confirmed using recombinant SC virus containing polymerase genes with wild type (WT or mutant PA or PB2. The PA-A36T mutant showed enhanced growth property compared to the WT in both human A549 cells and porcine PK15 cells in vitro, without significant effect on viral propagation in murine LA-4 cells and pathogenicity in mice; however, it did enhance the lung virus titer. PB2-H357N variant demonstrated growth ability comparable to the WT in A549 cells, but replicated well in PK15, LA-4 cells and in mice with an enhanced pathogenic phenotype. Despite such mutations are rare in nature, they could be observed in avian H5 and H7 subtype viruses which were currently recognized to pose potential threat to human. Our findings indicated that pH1N1 may adapt well in mammals when acquiring these mutations. Therefore, future molecular epidemiological surveillance should include scrutiny of both markers because of their potential impact on pathogenesis.

  10. Antiviral resistance due to deletion in the neuraminidase gene and defective interfering-like viral polymerase basic 2 RNA of influenza A virus subtype H3N2

    DEFF Research Database (Denmark)

    Trebbien, Ramona; Christiansen, Claus Bohn; Fischer, Thea Kølsen

    2018-01-01

    two major out-of-frame deletions in the polymerase basic 2 (PB2) gene, indicating defective interfering-like viral RNA. Conclusions: The viruses harboring the 245–248 deletion in the neuraminidase gene were still present after discontinuation of oseltamivir treatment and passages in cell cultures...... to zanamivir. Nine days after discontinuation of oseltamivir treatment the deleted H3N2 virus was still present in the patient. After three passages of the deleted virus in cell culture, the deletion was retained. Several variant mutations appeared in the other genes of the H3N2 virus, where most striking were...

  11. Occupancy of RNA Polymerase II Phosphorylated on Serine 5 (RNAP S5P) and RNAP S2P on Varicella-Zoster Virus Genes 9, 51, and 66 Is Independent of Transcript Abundance and Polymerase Location within the Gene.

    Science.gov (United States)

    Henderson, Heather H; Timberlake, Kensey B; Austin, Zoe A; Badani, Hussain; Sanford, Bridget; Tremblay, Keriann; Baird, Nicholas L; Jones, Kenneth; Rovnak, Joel; Frietze, Seth; Gilden, Don; Cohrs, Randall J

    2015-11-11

    Regulation of gene transcription in varicella-zoster virus (VZV), a ubiquitous human neurotropic alphaherpesvirus, requires coordinated binding of multiple host and virus proteins onto specific regions of the virus genome. Chromatin immunoprecipitation (ChIP) is widely used to determine the location of specific proteins along a genomic region. Since the size range of sheared virus DNA fragments governs the limit of accurate protein localization, particularly for compact herpesvirus genomes, we used a quantitative PCR (qPCR)-based assay to determine the efficiency of VZV DNA shearing before ChIP, after which the assay was used to determine the relationship between transcript abundance and the occupancy of phosphorylated RNA polymerase II (RNAP) on the gene promoter, body, and terminus of VZV genes 9, 51, and 66. The abundance of VZV gene 9, 51, and 66 transcripts in VZV-infected human fetal lung fibroblasts was determined by reverse transcription-linked quantitative PCR. Our results showed that the C-terminal domain of RNAP is hyperphosphorylated at serine 5 (S5(P)) on VZV genes 9, 51, and 66 independently of transcript abundance and the location within the virus gene at both 1 and 3 days postinfection (dpi). In contrast, phosphorylated serine 2 (S2(P))-modified RNAP was not detected at any virus gene location at 3 dpi and was detected at levels only slightly above background levels at 1 dpi. Regulation of herpesvirus gene transcription is an elaborate choreography between proteins and DNA that is revealed by chromatin immunoprecipitation (ChIP). We used a quantitative PCR-based assay to determine fragment size after DNA shearing, a critical parameter in ChIP assays, and exposed a basic difference in the mechanism of transcription between mammalian cells and VZV. We found that hyperphosphorylation at serine 5 of the C-terminal domain of RNAP along the lengths of VZV genes (the promoter, body, and transcription termination site) was independent of mRNA abundance. In

  12. A rapid assay for detection of Rose rosette virus using reverse transcription-recombinase polymerase amplification using multiple gene targets.

    Science.gov (United States)

    Babu, Binoy; Washburn, Brian K; Miller, Steven H; Poduch, Kristina; Sarigul, Tulin; Knox, Gary W; Ochoa-Corona, Francisco M; Paret, Mathews L

    2017-02-01

    Rose rosette disease caused by Rose rosette virus (RRV; genus Emaravirus) is the most economically relevant disease of Knock Out® series roses in the U.S. As there are no effective chemical control options for the disease, the most critical disease management strategies include the use of virus free clean plants for propagation and early detection and destruction of infected plants. The current diagnostic techniques for RRV including end-point reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR (RT-qPCR) are highly sensitive, but limited to diagnostic labs with the equipment and expertise; and is time consuming. To address this limitation, an isothermal reverse transcription-recombinase polymerase amplification (RT-RPA) assay based on multiple gene targets for specific detection of RRV was developed. The assay is highly specific and did not cross react with other viruses belonging to the inclusive and exclusive genus. Dilution assays using the in vitro transcripts showed that the primer sets designed (RPA-267, RPA-131, and RPA-321) are highly sensitive, consistently detecting RRV with a detection limit of 1fg/μL. Testing of the infected plants using the primer sets indicated that the virus could be detected from leaves, stems and petals of roses. The primer pair RPA-267 produced 100% positive detection of the virus from infected leaf tissues, while primer set RPA-131 produced 100% detection from stems and petals. The primer set RPA-321 produced 83%, 87.5% and 75% positive detection from leaves, petals and stem tissues, respectively. In addition, the assay has been efficiently used in the detection of RRV infecting Knock Out® roses, collected from different states in the U.S. The assay can be completed in 20min as compared to the end-point RT-PCR assay (3-4h) and RT-qPCR (1.5h). The RT-RPA assay is reliable, rapid, highly sensitive, and can be easily used in diagnostic laboratories for detection of RRV with no need for any special equipment

  13. 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...

  14. The Tomato Yellow Leaf Curl Virus resistance genes Ty-1 and Ty-3 are allelic and code for DFDGD-class RNA-dependent RNA polymerases.

    Directory of Open Access Journals (Sweden)

    Maarten G Verlaan

    2013-03-01

    Full Text Available Tomato Yellow Leaf Curl Virus Disease incited by Tomato yellow leaf curl virus (TYLCV causes huge losses in tomato production worldwide and is caused by different related begomovirus species. Breeding for TYLCV resistance has been based on the introgression of multiple resistance genes originating from several wild tomato species. In this study we have fine-mapped the widely used Solanum chilense-derived Ty-1 and Ty-3 genes by screening nearly 12,000 plants for recombination events and generating recombinant inbred lines. Multiple molecular markers were developed and used in combination with disease tests to fine-map the genes to a small genomic region (approximately 70 kb. Using a Tobacco Rattle Virus-Virus Induced Gene Silencing approach, the resistance gene was identified. It is shown that Ty-1 and Ty-3 are allelic and that they code for a RNA-dependent RNA polymerase (RDR belonging to the RDRγ type, which has an atypical DFDGD motif in the catalytic domain. In contrast to the RDRα type, characterized by a catalytic DLDGD motif, no clear function has yet been described for the RDRγ type, and thus the Ty-1/Ty-3 gene unveils a completely new class of resistance gene. Although speculative, the resistance mechanism of Ty-1/Ty-3 and its specificity towards TYLCV are discussed in light of the function of the related RDRα class in the amplification of the RNAi response in plants and transcriptional silencing of geminiviruses in plants.

  15. Different patterns of codon usage in the overlapping polymerase and surface genes of hepatitis B virus suggest a de novo origin by modular evolution.

    Science.gov (United States)

    Pavesi, Angelo

    2015-12-01

    The polymerase (P) and surface (S) genes of hepatitis B virus (HBV) show the longest gene overlap in animal viruses. Gene overlaps originate by the overprinting of a novel frame onto an ancestral pre-existing frame. Identifying which frame is ancestral and which frame is de novo (the genealogy of the overlap) is an appealing topic. However, the P/S overlap of HBV is an intriguing paradox, because both genes are indispensable for virus survival. Thus, the hypothesis of a primordial virus without the surface protein or without the polymerase makes no biological sense. With the aim to determine the genealogy of the overlap, the codon usage of the overlapping frames P and S was compared to that of the non-overlapping region. It was found that the overlap of human HBV had two patterns of codon usage. One was localized in the 59 one-third of the overlap and the other in the 39 two-thirds. By extending the analysis to non-human HBVs, it was found that this feature occurred in all hepadnaviruses. Under the assumption that the ancestral frame has a codon usage significantly closer to that of the non-overlapping region than the de novo frame, the ancestral frames in the 59 and 39 region of the overlap could be predicted. They were, respectively, frame S and frame P. These results suggest that the spacer domain of the polymerase and the S domain of the surface protein originated de novo by overprinting. They support a modular evolution hypothesis for the origin of the overlap.

  16. Oligonucleotide microarray analysis of gene expression profiles followed by real-time reverse-transcriptase polymerase chain reaction assay in chronic active Epstein-Barr virus infection.

    Science.gov (United States)

    Ito, Yoshinori; Shibata-Watanabe, Yukiko; Ushijima, Yoko; Kawada, Jun-Ichi; Nishiyama, Yukihiro; Kojima, Seiji; Kimura, Hiroshi

    2008-03-01

    Chronic active Epstein-Barr virus infection (CAEBV) is characterized by recurrent infectious mononucleosis-like symptoms and has high mortality and morbidity. To clarify the mechanisms of CAEBV, the gene-expression profiles of peripheral blood obtained from patients with CAEBV were investigated. Twenty genes were differentially expressed in 4 patients with CAEBV. This microarray result was verified using a real-time reverse-transcriptase polymerase chain reaction assay in a larger group of patients with CAEBV. Eventually, 3 genes were found to be significantly upregulated: guanylate binding protein 1, tumor necrosis factor-induced protein 6, and guanylate binding protein 5. These genes may be associated with the inflammatory reaction or with cell proliferation.

  17. Development of nested polymerase chain reaction-based diagnosis of duck enteritis virus and detection of DNA polymerase gene from non-descriptive duck breeds of West Bengal, India

    Directory of Open Access Journals (Sweden)

    Partha Sarathi Mandal

    2017-03-01

    Full Text Available Aim: The study was undertaken to detect the clinical signs, postmortem lesions of embryonated duck plague (DP infected eggs, and histopathological changes of chorioallantoic membrane (CAM in non-descriptive ducks of West Bengal with special reference to standardize nested polymerase chain reaction (PCR. Materials and Methods: After postmortem of suspected carcasses, samples were collected for virus isolation and identification through specific pathogen free (Khaki Campbell embryonated duck eggs. PCR was also done as confirmatory test after doing postmortem of duck embryos. DP specific nested PCR was standardized for better confirmation of the disease. Sensitivity of nested primers was also tested for DP virus. Results: Gross, postmortem and histopathological changes were prominent in dead embryos. First set of primer was able to detect 602 bp fragments of DNA polymerase gene of duck enteritis virus from infected CAM. Subsequently, a DP specific nested PCR which was very much sensitive for very small amount of viral genome was successfully standardized. After NCBI blast nucleotide sequence of nested PCR product (Accession No. HG425076 showed homology with the sequences data available in GenBank. Conclusion: The study concludes that PCR assay is very much helpful to diagnose DP disease and developed nested PCR is a double confirmatory diagnostic tool for DP.

  18. Establishment of real-time quantitative reverse transcription polymerase chain reaction assay for transcriptional analysis of duck enteritis virus UL55 gene

    Directory of Open Access Journals (Sweden)

    Zhang Shunchuan

    2011-06-01

    Full Text Available Abstract Background Real-time quantitative reverse transcription polymerase chain reaction assay (qRT-PCR has become the benchmark for detection and quantification of target gene expression level and been utilized increasingly in detection of viral load and therapy monitoring. The dynamic transcription variation of duck enteritis virus UL55 gene during the life cycle of duck enteritis virus in infected cells has not been reported yet. Results The newly identified duck enteritis virus UL55 gene was amplified and cloned into pMD18-T vector after digestion to generate a recombinant plasmid pMD18-T/UL55 for the establishment of qRT-PCR as standard DNA. The results of agarose gel electrophoresis and melting curve analysis demonstrated the primers we designed for qRT-PCR were specific and available. We used β-actin as a reference gene for normalization and established two standard curves based on pMD18-T/UL55 and pMD18-T/β-actin successfully. Based on that, the transcriptional analysis of DEV UL55 gene was performed, and the result suggested the expression of UL55 mRNA was at a low level from 0 to 8 h post-infection(p.i., then accumulated quickly since 12 h p.i. and peaked at 36 h p.i., it can be detected till 60 h p.i.. Nucleic acid inhibition test was carried out for analyzing a temporal regulation condition of DEV UL55 gene, result revealed that it was sensitive to ganciclovir. Synthesis procedures of DEV UL55 gene can be inhibited by ganciclovir. Conclusions The method we established in this paper can provide quantitative values reflecting the amounts of measured mRNA in samples. It's available for detection and quantification, also can be used in DEV diagnosis. The DEV UL55 gene was produced most abundantly during the late phase of replication in DEV-infected cells and the transcription of it depended on the synthesized DNA. DEV UL55 gene is a γ2 gene which occurs last and have a strict requirement for viral DNA synthesis.

  19. The Tomato Yellow Leaf Curl Virus Resistance Genes Ty-1 and Ty-3 Are Allelic and Code for DFDGD-Class RNA–Dependent RNA Polymerases

    NARCIS (Netherlands)

    Verlaan, M.G.; Hutton, S.F.; Ibrahem, R.M.; Kormelink, R.J.M.; Visser, R.G.F.; Scott, J.W.; Edwards, J.D.; Bai, Y.

    2013-01-01

    Tomato Yellow Leaf Curl Virus Disease incited by Tomato yellow leaf curl virus (TYLCV) causes huge losses in tomato production worldwide and is caused by different related begomovirus species. Breeding for TYLCV resistance has been based on the introgression of multiple resistance genes originating

  20. RNA polymerase activity of Ustilago maydis virus

    Energy Technology Data Exchange (ETDEWEB)

    Yie, S.W.

    1986-01-01

    Ustilago maydis virus has an RNA polymerase enzyme which is associated with virion capsids. In the presence of Mg/sup 2 +/ ion and ribonucleotide triphosphate, the enzyme catalyzes the in vitro synthesis of mRNA by using dsRNA as a template. The products of the UmV RNA polymerase were both ssRNA and dsRNA. The dsRNA was determined by characteristic mobilities in gel electrophoresis, lack of sensitivity to RNase, and specific hybridization tests. The ssRNAs were identified by elution from a CF-11 column and by their RNase sensitivity. On the basis of the size of ssRNAs, it was concluded that partial transcripts were produced from H dsRNA segments, and full length transcripts were produced from M and L dsRNA segments. The following observations indicates that transcription occurs by strand displacement; (1) Only the positive strand of M2 dsRNA was labeled by the in vitro reaction. (2) The M2 dsRNA which had been labeled with /sup 32/''P-UTP in vitro could be chased from dsRNA with unlabeled UTP. The transcription products of three UmV strains were compared, and the overall pattern of transcription was very similar among them.

  1. Identification of duck plague virus by polymerase chain reaction

    Science.gov (United States)

    Hansen, W.R.; Brown, Sean E.; Nashold, S.W.; Knudson, D.L.

    1999-01-01

    A polymerase chain reaction (PCR) assay was developed for detecting duck plague virus. A 765-bp EcoRI fragment cloned from the genome of the duck plague vaccine (DP-VAC) virus was sequenced for PCR primer development. The fragment sequence was found by GenBank alignment searches to be similar to the 3a?? ends of an undefined open reading frame and the gene for DNA polymerase protein in other herpesviruses. Three of four primer sets were found to be specific for the DP-VAC virus and 100% (7/7) of field isolates but did not amplify DNA from inclusion body disease of cranes virus. The specificity of one primer set was tested with genome templates from other avian herpesviruses, including those from a golden eagle, bald eagle, great horned owl, snowy owl, peregrine falcon, prairie falcon, pigeon, psittacine, and chicken (infectious laryngotracheitis), but amplicons were not produced. Hence, this PCR test is highly specific for duck plague virus DNA. Two primer sets were able to detect 1 fg of DNA from the duck plague vaccine strain, equivalent to five genome copies. In addition, the ratio of tissue culture infectious doses to genome copies of duck plague vaccine virus from infected duck embryo cells was determined to be 1:100, making the PCR assay 20 times more sensitive than tissue culture for detecting duck plague virus. The speed, sensitivity, and specificity of this PCR provide a greatly improved diagnostic and research tool for studying the epizootiology of duck plague. /// Se desarroll?? una prueba de reacci??n en cadena por la polimerasa para detectar el virus de la peste del pato. Un fragmento EcoRI de 765 pares de bases clonado del genoma del virus vacunal de la peste del pato fue secuenciado para la obtenci??n de los iniciadores de la prueba de la reacci??n en cadena por la polimerasa. En investigaciones de alineaci??n en el banco de genes ('GenBank') se encontr?? que la secuencia del fragmento era similar a los extremos 3a?? de un marco de lectura abierto

  2. Cloning the Horse RNA Polymerase I Promoter and Its Application to Studying Influenza Virus Polymerase Activity

    Directory of Open Access Journals (Sweden)

    Gang Lu

    2016-05-01

    Full Text Available An influenza virus polymerase reconstitution assay based on the human, dog, or chicken RNA polymerase I (PolI promoter has been developed and widely used to study the polymerase activity of the influenza virus in corresponding cell types. Although it is an important member of the influenza virus family and has been known for sixty years, no studies have been performed to clone the horse PolI promoter or to study the polymerase activity of equine influenza virus (EIV in horse cells. In our study, the horse RNA PolI promoter was cloned from fetal equine lung cells. Using the luciferase assay, it was found that a 500 bp horse RNA PolI promoter sequence was required for efficient transcription. Then, using the developed polymerase reconstitution assay based on the horse RNA PolI promoter, the polymerase activity of two EIV strains was compared, and equine myxovirus resistance A protein was identified as having the inhibiting EIV polymerase activity function in horse cells. Our study enriches our knowledge of the RNA PolI promoter of eukaryotic species and provides a useful tool for the study of influenza virus polymerase activity in horse cells.

  3. Investigation of Influenza Virus Polymerase Activity in Pig Cells

    Science.gov (United States)

    Moncorgé, Olivier; Long, Jason S.; Cauldwell, Anna V.; Zhou, Hongbo; Lycett, Samantha J.

    2013-01-01

    Reassortant influenza viruses with combinations of avian, human, and/or swine genomic segments have been detected frequently in pigs. As a consequence, pigs have been accused of being a “mixing vessel” for influenza viruses. This implies that pig cells support transcription and replication of avian influenza viruses, in contrast to human cells, in which most avian influenza virus polymerases display limited activity. Although influenza virus polymerase activity has been studied in human and avian cells for many years by use of a minigenome assay, similar investigations in pig cells have not been reported. We developed the first minigenome assay for pig cells and compared the activities of polymerases of avian or human influenza virus origin in pig, human, and avian cells. We also investigated in pig cells the consequences of some known mammalian host range determinants that enhance influenza virus polymerase activity in human cells, such as PB2 mutations E627K, D701N, G590S/Q591R, and T271A. The two typical avian influenza virus polymerases used in this study were poorly active in pig cells, similar to what is seen in human cells, and mutations that adapt the avian influenza virus polymerase for human cells also increased activity in pig cells. In contrast, a different pattern was observed in avian cells. Finally, highly pathogenic avian influenza virus H5N1 polymerase activity was tested because this subtype has been reported to replicate only poorly in pigs. H5N1 polymerase was active in swine cells, suggesting that other barriers restrict these viruses from becoming endemic in pigs. PMID:23077313

  4. Redistributive properties of the vesicular stomatitis virus polymerase

    Energy Technology Data Exchange (ETDEWEB)

    Helfman, W.B.; Perrault, J. (San Diego State Univ., CA (USA))

    1989-08-01

    The template for transcription of the vesicular stomatitis virus (VSV) genome consists of a negative-strand RNA (approximately 11 kb) tightly associated with approximately 1250 copies of the nucleocapsid or N protein (N-RNA template). The interaction between the virion-associated polymerase and this template was probed with a novel assay using purified N-RNA complexes added to detergent-disrupted uv-irradiated standard virions or unirradiated defective interfering (DI) particles. In contrast to the well-known stability of assembled cellular transcription complexes, the VSV polymerase copied exogenously added templates efficiently and yielded products indistinguishable from control virus transcription. Addition of uv-irradiated N-RNA templates to unirradiated virus effectively competed for transcription of endogenous template indicating that most or all of the polymerase can freely redistribute. Furthermore preincubation of virus and added templates at high ionic strength to solubilize L and NS polymerase proteins did not release additional active enzyme for redistribution. Pretranscription of virus also had little or no effect on redistributed activity indicating that polymerase complexes are capable of multiple rounds of synthesis beginning at the 3' end promoter. Unexpectedly, titration with saturating amounts of added N-RNA showed that active polymerase complexes are only in slight excess relative to template in standard or DI particles despite the large surplus of packaged L and NS polypeptides. Moreover, added standard virus templates competed equally well for the redistributing polymerase from DI particles or standard virus indicating no significant polymerase-binding preference for interfering templates. These findings bear important implications regarding mechanisms of VSV transcription and replication.

  5. The polymerase of negative-stranded RNA viruses.

    Science.gov (United States)

    Morin, Benjamin; Kranzusch, Philip J; Rahmeh, Amal A; Whelan, Sean P J

    2013-04-01

    Negative-sense (NS) RNA viruses deliver into cells a mega-dalton RNA-protein complex competent for transcription. Within this complex, the RNA is protected in a nucleocapsid protein (NP) sheath which the viral polymerase negotiates during RNA synthesis. The NP-RNA templates come as nonsegmented (NNS) or segmented (SNS), necessitating distinct strategies for transcription by their polymerases. Atomic-level understanding of the NP-RNA of both NNS and SNS RNA viruses show that the RNA must be transiently dissociated from NP during RNA synthesis. Here we summarize and compare the polymerases of NNS and SNS RNA viruses, and the current structural data on the polymerases. Those comparisons inform us on the evolution of related RNA synthesis machines which use two distinct mechanisms for mRNA cap formation. Copyright © 2013. Published by Elsevier B.V.

  6. [Relationship between hepatitis B virus polymerase gene mutation patterns of rtM204I/V and pre-core/basal core promoter mutations].

    Science.gov (United States)

    Yan, Li; Wang, Jie-Fei; Wang, Zhan-Hui; Sun, Jian; Zhou, Bin; Hou, Jinlin

    2014-12-01

    To investigate the relationship between mutations of rtM204V/I (methionine to valine or isoleucine at position rt204 of reverse transcriptase domain) in the hepatitis B virus (HBV) polymerase gene and the G1896A and G1899A single mutations in the pre-eore (PC) region and the A1762T and G1764A double-mutations in the basal core promoter (BCP) region. A total of 2,849 hepatitis B complete genome sequences were retrieved from the GenBank/EMBL/DDBJ. The amino acid sequence of the of reverse transcriptase domain and genome sequences of the PC region and the BCP region were aligned using MEGA4 software. Data were calculated using Microsoft Excel and evaluated using SPSS 13.0 statistical software. Among the 2, 849 HBV complete genome sequences, 217 (8%) strains were identified with Y(I/V) DD and 120 of those had the YIDD mutation and 97 had the YVDD mutation. Of the 1543 strains (54.2%) with PC-BCP mutations, seven mutation patterns of G 1896A-G 1899A-G 1896A-G 1899A-A 1762T/G 1764A, A 1762T/G 1764AG 1896A, A 1762T/G 1764A-G 1899A, and A 1762T/G 1764A-G 1896A-G 1899A were identified. of YMDD and PC-BCP had a higher incidence than the single YMDD mutation (76% vs 24.0%, x2=45.283, P=0.000). The double-mutations of YIDD and PC-BCP had a higher incidence than the double-mutation of YVDD and PC-BCP (85% vs 64.9%, x2=11.836, P=0.000). The double-mutation for lamivudine resistance of YMDD and PC-BCP had a higher incidence than the double pre-existent YMDD and PC-BCP mutations (89.3% vs 58.9%, x2=27.084, P=0.000). The three mutation patterns of G1896A-G1899A (P=0.000, OR=7.573), A1762T/G1764A-G1899A (P=0.000, OR=6.539) and A1762T/G1764A-G1896A-G1899A (P=0.000, OR=6.596) were associated with a greater risk of developing the YIDD mutation, according to binary logistic analysis. There is a relationship between the HBV YI/VDD mutation and PC-BCP mutations. Different PC-BCP mutation patterns have different effects on the YI/VDD mutation.

  7. The polymerase of negative-stranded RNA viruses

    OpenAIRE

    Morin, Benjamin; Kranzusch, Philip J.; Rahmeh, Amal A.; Whelan, Sean P. J.

    2013-01-01

    Negative-sense (NS) RNA viruses deliver into cells a mega-dalton RNA-protein complex competent for transcription. Within this complex, the RNA is protected in a nucleocapsid protein (NP) sheath which the viral polymerase negotiates during RNA synthesis. The NP-RNA templates come as nonsegemented (NNS) or segmented (SNS), necessitating distinct strategies for transcription by their polymerases. Atomic-level understanding of the NP-RNA of both NNS and SNS RNA viruses show that the RNA must be t...

  8. Establishment of recombinase polymerase amplification assay for five hemorrhagic fever-related viruses

    Directory of Open Access Journals (Sweden)

    Xue-feng CAO

    2017-08-01

    Full Text Available Objective To establish a one-step recombinase polymerase amplification (RPA method for pathogen screening and rapid detection in the field targeting for five hemorrhagic fever related viruses (Zaire ebola virus, Sudan ebola virus, Marburg virus, Lassa virus and Yellow fever virus. Methods The specific nucleic acid (NA fragments of each virus were selected as target genes by genome sequence analysis, and the primers and probes for RPA assays were designed according to the sequence. A series of diluted template genes were used for RPA detection to determine the sensitivity. The hemorrhagic fever-related viral nucleic acids were used for RPA detection to determine the specificity. The amplification experiments were carried out at different temperature ranging from 37℃ to 42℃ to validate the reaction temperature range. Results The RPA reaction systems of the five hemorrhagic fever viruses could effectively amplify the target genes, the sensitivities were between 1.5×102 and 1.5×103 copies. No cross reactions existed with the other hemorrhagic fever-related viral genes. Meanwhile, RPA assay could effectively amplify the target genes at 37-42℃. Conclusion The isothermal RPA assays of five hemorrhagic fever viruses are established, which may amply target genes fast and react at a wide temperature range, and be potentially useful for in field pathogens detection. DOI: 10.11855/j.issn.0577-7402.2017.06.09

  9. Asymmetric packaging of polymerases within vesicular stomatitis virus

    Energy Technology Data Exchange (ETDEWEB)

    Hodges, Jeffery; Tang, Xiaolin; Landesman, Michael B. [Dept. of Physics and Astronomy, University of Utah (United States); Center for Cell and Genome Science, University of Utah (United States); Ruedas, John B. [Dept. of Biology, San Diego State University (United States); Ghimire, Anil [Dept. of Physics and Astronomy, University of Utah (United States); Gudheti, Manasa V. [Vutara, Inc., Salt Lake City, UT (United States); Dept. of Biology, University of Utah (United States); Perrault, Jacques [Dept. of Biology, San Diego State University (United States); Jorgensen, Erik M. [Howard Hughes Medical Institute (United States); Dept. of Biology, University of Utah (United States); Gerton, Jordan M. [Dept. of Physics and Astronomy, University of Utah (United States); Dept. of Bioengineering, University of Utah (United States); Saffarian, Saveez, E-mail: saffarian@physics.utah.edu [Dept. of Physics and Astronomy, University of Utah (United States); Center for Cell and Genome Science, University of Utah (United States); Dept. of Biology, University of Utah (United States)

    2013-10-18

    Highlights: •The VSV polymerases (L proteins) are localized to the blunt end of the virus. •The VSV phosphoproteins (P proteins) are localized to the blunt end of the virus. •Each VSV virion packages a variable number of P and L proteins. -- Abstract: Vesicular stomatitis virus (VSV) is a prototypic negative sense single-stranded RNA virus. The bullet-shape appearance of the virion results from tightly wound helical turns of the nucleoprotein encapsidated RNA template (N-RNA) around a central cavity. Transcription and replication require polymerase complexes, which include a catalytic subunit L and a template-binding subunit P. L and P are inferred to be in the cavity, however lacking direct observation, their exact position has remained unclear. Using super-resolution fluorescence imaging and atomic force microscopy (AFM) on single VSV virions, we show that L and P are packaged asymmetrically towards the blunt end of the virus. The number of L and P proteins varies between individual virions and they occupy 57 ± 12 nm of the 150 nm central cavity of the virus. Our finding positions the polymerases at the opposite end of the genome with respect to the only transcriptional promoter.

  10. Assembly of a functional Machupo virus polymerase complex.

    Science.gov (United States)

    Kranzusch, Philip J; Schenk, Andreas D; Rahmeh, Amal A; Radoshitzky, Sheli R; Bavari, Sina; Walz, Thomas; Whelan, Sean P J

    2010-11-16

    Segmented negative-sense viruses of the family Arenaviridae encode a large polymerase (L) protein that contains all of the enzymatic activities required for RNA synthesis. These activities include an RNA-dependent RNA polymerase (RdRP) and an RNA endonuclease that cleaves capped primers from cellular mRNAs to prime transcription. Using purified catalytically active Machupo virus L, we provide a view of the overall architecture of this multifunctional polymerase and reconstitute complex formation with an RNA template in vitro. The L protein contains a central ring domain that is similar in appearance to the RdRP of dsRNA viruses and multiple accessory appendages that may be responsible for 5' cap formation. RNA template recognition by L requires a sequence-specific motif located at positions 2-5 in the 3' terminus of the viral genome. Moreover, L-RNA complex formation depends on single-stranded RNA, indicating that inter-termini dsRNA interactions must be partially broken for complex assembly to occur. Our results provide a model for arenavirus polymerase-template interactions and reveal the structural organization of a negative-strand RNA virus L protein.

  11. Use of the Capripoxvirus homologue of Vaccinia virus 30 kDa RNA polymerase subunit (RPO30) gene as a novel diagnostic and genotyping target: development of a classical PCR method to differentiate Goat poxvirus from Sheep poxvirus.

    Science.gov (United States)

    Lamien, Charles Euloge; Le Goff, Christian; Silber, Roland; Wallace, David B; Gulyaz, Velý; Tuppurainen, Eeva; Madani, Hafsa; Caufour, Philippe; Adam, Tajelser; El Harrak, Mehdi; Luckins, Antony George; Albina, Emmanuel; Diallo, Adama

    2011-04-21

    Sheep poxvirus (SPPV), Goat poxvirus (GTPV) and Lumpy skin disease virus (LSDV) are Capripoxviruses (CaPVs) responsible for causing severe poxvirus disease in sheep, goats and cattle, respectively. Serological differentiation of CaPVs is not possible and strain identification has relied on the implicitly accepted hypothesis that the viruses show well defined host specificity. However, it is now known that cross infections can occur and authentication of identity based on the host animal species from which the strain was first isolated, is not valid and should be replaced with molecular techniques to allow unequivocal strain differentiation. To identify a diagnostic target for strain genotyping, the CaPV homologue of the Vaccinia virus E4L gene which encodes the 30 kDa DNA-dependent RNA polymerase subunit, RPO30 was analyzed. Forty-six isolates from different hosts and geographical origins were included. Most CaPVs fit into one of the three different groups according to their host origins: the SPPV, the GTPV and the LSDV group. A unique 21-nucleotide deletion was found in all SPPV isolates which was exploited to develop a RPO30-based classical PCR test to differentiate SPPV from GTPV that will allow rapid differential diagnosis of disease during CaPV outbreaks in small ruminants. Copyright © 2010 Elsevier B.V. All rights reserved.

  12. Development and validation of a Myxoma virus real-time polymerase chain reaction assay.

    Science.gov (United States)

    Albini, Sarah; Sigrist, Brigitte; Güttinger, Regula; Schelling, Claude; Hoop, Richard K; Vögtlin, Andrea

    2012-01-01

    To aid in the rapid diagnosis of myxomatosis in rabbits, a real-time polymerase chain reaction (PCR) for the specific detection of Myxoma virus is described. Primers and probe were designed to amplify a 147-bp fragment within the Serp2 gene. The assay was able to detect 23 copies of a synthesized oligo indicating a reliable sensitivity. In addition, the real-time PCR did not detect the Rabbit fibroma virus used in myxomatosis vaccines. The novel PCR was shown to be able to detect Myxoma virus in fresh and paraffin-embedded rabbit tissues originating from myxomatosis cases from various regions in Switzerland.

  13. Molecular architecture of the vesicular stomatitis virus RNA polymerase.

    Science.gov (United States)

    Rahmeh, Amal A; Schenk, Andreas D; Danek, Eric I; Kranzusch, Philip J; Liang, Bo; Walz, Thomas; Whelan, Sean P J

    2010-11-16

    Nonsegmented negative-strand (NNS) RNA viruses initiate infection by delivering into the host cell a highly specialized RNA synthesis machine comprising the genomic RNA completely encapsidated by the viral nucleocapsid protein and associated with the viral polymerase. The catalytic core of this protein-RNA complex is a 250-kDa multifunctional large (L) polymerase protein that contains enzymatic activities for nucleotide polymerization as well as for each step of mRNA cap formation. Working with vesicular stomatitis virus (VSV), a prototype of NNS RNA viruses, we used negative stain electron microscopy (EM) to obtain a molecular view of L, alone and in complex with the viral phosphoprotein (P) cofactor. EM analysis, combined with proteolytic digestion and deletion mapping, revealed the organization of L into a ring domain containing the RNA polymerase and an appendage of three globular domains containing the cap-forming activities. The capping enzyme maps to a globular domain, which is juxtaposed to the ring, and the cap methyltransferase maps to a more distal and flexibly connected globule. Upon P binding, L undergoes a significant rearrangement that may reflect an optimal positioning of its functional domains for transcription. The structural map of L provides new insights into the interrelationship of its various domains, and their rearrangement on P binding that is likely important for RNA synthesis. Because the arrangement of conserved regions involved in catalysis is homologous, the structural insights obtained for VSV L likely extend to all NNS RNA viruses.

  14. Polymerase chain reaction to search for Herpes viruses in uveitic ...

    African Journals Online (AJOL)

    Cite as: Laaks D, Smit DP, Harvey J. Polymerase chain reaction to search for Herpes viruses in uveitic and healthy eyes: a South African perspective. Afri Health Sci. 2015;15(3):748-54. doi: http://dx.doi.org/10.4314/ahs.v15i3.7. Introduction. Uveitis is internationally classified according to the an- atomical locus of infection: ...

  15. Genotypic frequency of calpastatin gene in lori sheep by polymerase ...

    African Journals Online (AJOL)

    Genomic DNA was extracted from 100 sheep blood sample. Polymerase chain reaction was performed to amplify a 622 bp fragment of this gene. Restriction reaction of polymerase chain reaction (PCR) products was done using MspI enzyme. The MspI digestion of the PCR products produced digestion fragments of 336 and ...

  16. Viruses Infecting a Freshwater Filamentous Cyanobacterium (Nostoc sp.) Encode a Functional CRISPR Array and a Proteobacterial DNA Polymerase B.

    Science.gov (United States)

    Chénard, Caroline; Wirth, Jennifer F; Suttle, Curtis A

    2016-06-14

    Here we present the first genomic characterization of viruses infecting Nostoc, a genus of ecologically important cyanobacteria that are widespread in freshwater. Cyanophages A-1 and N-1 were isolated in the 1970s and infect Nostoc sp. strain PCC 7210 but remained genomically uncharacterized. Their 68,304- and 64,960-bp genomes are strikingly different from those of other sequenced cyanophages. Many putative genes that code for proteins with known functions are similar to those found in filamentous cyanobacteria, showing a long evolutionary history in their host. Cyanophage N-1 encodes a CRISPR array that is transcribed during infection and is similar to the DR5 family of CRISPRs commonly found in cyanobacteria. The presence of a host-related CRISPR array in a cyanophage suggests that the phage can transfer the CRISPR among related cyanobacteria and thereby provide resistance to infection with competing phages. Both viruses also encode a distinct DNA polymerase B that is closely related to those found in plasmids of Cyanothece sp. strain PCC 7424, Nostoc sp. strain PCC 7120, and Anabaena variabilis ATCC 29413. These polymerases form a distinct evolutionary group that is more closely related to DNA polymerases of proteobacteria than to those of other viruses. This suggests that the polymerase was acquired from a proteobacterium by an ancestral virus and transferred to the cyanobacterial plasmid. Many other open reading frames are similar to a prophage-like element in the genome of Nostoc sp. strain PCC 7524. The Nostoc cyanophages reveal a history of gene transfers between filamentous cyanobacteria and their viruses that have helped to forge the evolutionary trajectory of this previously unrecognized group of phages. Filamentous cyanobacteria belonging to the genus Nostoc are widespread and ecologically important in freshwater, yet little is known about the genomic content of their viruses. Here we report the first genomic analysis of cyanophages infecting

  17. A Simple Reverse Transcription-Polymerase Chain Reaction for Dengue Type 2 Virus Identification

    Directory of Open Access Journals (Sweden)

    Figueiredo Luiz Tadeu M

    1997-01-01

    Full Text Available We show here a simplified reverse transcription-polymerase chain reaction (RT-PCR for identification of dengue type 2 virus. Three dengue type 2 virus strains, isolated from Brazilian patients, and yellow fever vaccine 17DD, as a negative control, were used in this study. C6/36 cells were infected with the virus, and tissue culture fluids were collected after 7 days of infection period. The RT-PCR, a combination of RT and PCR done after a single addition of reagents in a single reaction vessel was carried out following a digestion of virus with 1% Nonidet P-40. The 50ml assay reaction mixture included 50 pmol of a dengue type 2 specific primer pair amplifying a 210 base pair sequence of the envelope protein gene, 0.1 mM of the four deoxynucleoside triphosphates, 7.5U of reverse transcriptase, and 1U of thermostable Taq DNA polymerase. The reagent mixture was incubated for 15 min at 37oC for RT followed by a variable amount of cycles of two-step PCR amplification (92oC for 60 sec, 53oC for 60 sec with slow temperature increment. The PCR products were subjected to 1.7% agarose gel electrophoresis and visualized with UV light after gel incubation in ethidium bromide solution. DNA bands were observed after 25 and 30 cycles of PCR. Virus amount as low as 102.8 TCID50/ml was detected by RT-PCR. Specific DNA amplification was observed with the three dengue type 2 strains. This assay has advantages compared to other RT-PCRs: it avoids laborious extraction of virus RNA; the combination of RT and PCR reduces assay time, facilitates the performance and reduces risk of contamination; the two-step PCR cycle produces a clear DNA amplification, saves assay time and simplifies the technique

  18. Rapid Detection/pathotyping of Newcastle disease virus isolates in clinical samples using real time polymerase chain reaction assay

    OpenAIRE

    sprotocols

    2014-01-01

    Authors: Abdul Wajid, Muhammad Wasim, Tahir Yaqub, Shafqat F Rehmani, Tasra Bibi, Nadia Mukhtar, Javed Muhammad, Umar Bacha, Suliman Qadir Afridi, Muhammad Nauman Zahid, Zia u ddin, Muhammad Zubair Shabbir, Kamran Abbas & Muneer Ahmad ### Abstract In the present protocol we describe the real time reverse transcription polymerase chain reaction (rRT-PCR) assay for the rapid detection/pathotyping of Newcastle disease virus (NDV) isoaltes in clinical samples. Fusion gene and matrix gene...

  19. Genotypic frequency of calpastatin gene in lori sheep by polymerase ...

    African Journals Online (AJOL)

    SAM

    2014-05-07

    May 7, 2014 ... (PCR) products was done using MspI enzyme. The MspI digestion of the PCR ... Key words: Calpastatin gene, polymorphism, lori sheep, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). .... J. Anim. Sci. 67: 3313-3321. Goll DE, Thompson VF, Taylor RG, Ouali A (1998).

  20. Genotypic frequency of calpastatin gene in lori sheep by polymerase ...

    African Journals Online (AJOL)

    The results show that in the population, genotypes AA, AB and BB, respectively, had frequencies 32.2, 63.2 and 4.6, and that this locus was not at Hardy - Weinberg equilibrium in the lori sheep strain (P<0.05). Keywords: Calpastatin gene, polymorphism, lori sheep, polymerase chain reaction-restriction fragment length ...

  1. RNA polymerase III promoter elements enhance transcription of RNA polymerase II genes

    Energy Technology Data Exchange (ETDEWEB)

    Oliviero, S.; Monaci, P.

    1988-02-25

    Using transient expression assays in cultured human cells the authors have observed that RNA Polymerase III promoter sequences exert a positive cis-acting enhancer effect on RNA Polymerase II transcription. A DNA segment containing a copy of the Alu repeated element enhances transcription of the liver specific Haptoglobin related (Hpr) promoter in Hepatoma cell lines but not in HeLa cells. A tRNA/sup pro/ gene acts as enhancer of the SV40 promoter both in Hepatoma and in HeLa cell lines. Transcription from the SV40 promoter is also enhanced by DNA segments containing only the box A or the box B of the tRNA/sup pro/ promoter.

  2. [Effect of Point Mutations in the Polymerase Genes of the Influenza A/PR/8/34 (H1N1) Virus on the Immune Response in a Mouse Model].

    Science.gov (United States)

    Kuznetsova, S A; Isakova-Sivak, I N; Kuznetcova, V A; Petukhova, G D; Losev, I V; Donina, S A; Rudenko, L G; Naikhin, A N

    2015-01-01

    The vaccine strains for live attenuated influenza vaccines (LAIVs) have cold-adapted, temperature-sensitive, and attenuated phenotypes, which are guaranteed by the presence of specific mutations from the master donor virus in their internal genes. In this study, we used mutant viruses of the pathogenic A/Puerto Rico/8/34 (H1N1) that contained ts-mutations in PB1 (K265N, V591I), PB2 (V478L), and PA (L28P, V341L) genes along and/or in different combinations to evaluate the impact of these mutations in the immune responses. Sequential addition of tested mutations resulted in the stepwise decrease in virus-specific serum and, to a lesser extent, mucosal antibody levels. We demonstrated strong positive correlation between virus attenuation (virus titer in lung) and antibody titers. The ts-mutations in PB1, PB2, and PA genes are mostly involved in the modulation of the humoral immunity, but also have a moderate effect on the cellular adaptive immune response.

  3. Aquarius, a novel gene isolated by gene trapping with an RNA-dependent RNA polymerase motif.

    Science.gov (United States)

    Sam, M; Wurst, W; Klüppel, M; Jin, O; Heng, H; Bernstein, A

    1998-06-01

    In a retinoic acid (RA) gene trap screen of mouse embryonic stem (ES) cells, a novel gene, named Aquarius (Aqr), was identified and characterized. The promoterless lacZ marker was used to trap the genomic locus and to determine the expression pattern of the gene. Aqr transcripts are strongly induced in response to RA in vitro. During embryogenesis, Aqr is expressed in mesoderm, in the neural crest and its target tissues, and in neuroepithelium. Expression was first detected at 8.5 days postcoitum, when neural crest cells are visible at the lateral ridges of the neural plate. The gene-trapped Aqr locus was transmitted through the mouse germ line in three genetic backgrounds. In the F2 generation, the expected mendelian ratio of 1:2:1 was observed in all backgrounds, indicating that homozygous mice are viable. Homozygotes are normal in size and weight and breed normally. The gene trap insertion, however, does not seem to generate a null mutation, because Aqr transcripts are still present in the homozygous mutant animals. The Aqr open reading frame has weak homology to RNA-dependent RNA polymerases (RRPs) of the murine hepatitis viruses and contains an RRP motif. Aqr was mapped to mouse chromosome 2 between regions E5 through F2 by using fluorescence in situ hybridization analysis.

  4. Enhanced polymerase activity confers replication competence of Borna disease virus in mice.

    Science.gov (United States)

    Ackermann, Andreas; Kugel, Daniela; Schneider, Urs; Staeheli, Peter

    2007-11-01

    We previously showed that mouse adaptation of cDNA-derived Borna disease virus (BDV) strain He/80(FR) was associated exclusively with mutations in the viral polymerase complex. Interestingly, independent mouse adaptation of non-recombinant He/80 was correlated with different alterations in the polymerase and mutations in the viral glycoprotein. We used reverse genetics to demonstrate that changes in the polymerase which improve enzymatic activity represent the decisive host range mutations. The glycoprotein mutations did not confer replication competence in mice, although they slightly improved viral performance if combined with polymerase mutations. Our findings suggest that the viral polymerase restricts the host range of BDV.

  5. Characterization of the catalytic center of the Ebola virus L polymerase.

    Directory of Open Access Journals (Sweden)

    Marie Luisa Schmidt

    2017-10-01

    Full Text Available Ebola virus (EBOV causes a severe hemorrhagic fever in humans and non-human primates. While no licensed therapeutics are available, recently there has been tremendous progress in developing antivirals. Targeting the ribonucleoprotein complex (RNP proteins, which facilitate genome replication and transcription, and particularly the polymerase L, is a promising antiviral approach since these processes are essential for the virus life cycle. However, until now little is known about L in terms of its structure and function, and in particular the catalytic center of the RNA-dependent RNA polymerase (RdRp of L, which is one of the most promising molecular targets, has never been experimentally characterized.Using multiple sequence alignments with other negative sense single-stranded RNA viruses we identified the putative catalytic center of the EBOV RdRp. An L protein with mutations in this center was then generated and characterized using various life cycle modelling systems. These systems are based on minigenomes, i.e. miniature versions of the viral genome, in which the viral genes are exchanged against a reporter gene. When such minigenomes are coexpressed with RNP proteins in mammalian cells, the RNP proteins recognize them as authentic templates for replication and transcription, resulting in reporter activity reflecting these processes. Replication-competent minigenome systems indicated that our L catalytic domain mutant was impaired in genome replication and/or transcription, and by using replication-deficient minigenome systems, as well as a novel RT-qPCR-based genome replication assay, we showed that it indeed no longer supported either of these processes. However, it still showed similar expression to wild-type L, and retained its ability to be incorporated into inclusion bodies, which are the sites of EBOV genome replication.We have experimentally defined the catalytic center of the EBOV RdRp, and thus a promising antiviral target

  6. 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

  7. Vesicular stomatitis virus polymerase's strong affinity to its template suggests exotic transcription models.

    Directory of Open Access Journals (Sweden)

    Xiaolin Tang

    2014-12-01

    Full Text Available Vesicular stomatitis virus (VSV is the prototype for negative sense non segmented (NNS RNA viruses which include potent human and animal pathogens such as Rabies, Ebola and measles. The polymerases of NNS RNA viruses only initiate transcription at or near the 3' end of their genome template. We measured the dissociation constant of VSV polymerases from their whole genome template to be 20 pM. Given this low dissociation constant, initiation and sustainability of transcription becomes nontrivial. To explore possible mechanisms, we simulated the first hour of transcription using Monte Carlo methods and show that a one-time initial dissociation of all polymerases during entry is not sufficient to sustain transcription. We further show that efficient transcription requires a sliding mechanism for non-transcribing polymerases and can be realized with different polymerase-polymerase interactions and distinct template topologies. In conclusion, we highlight a model in which collisions between transcribing and sliding non-transcribing polymerases result in release of the non-transcribing polymerases allowing for redistribution of polymerases between separate templates during transcription and suggest specific experiments to further test these mechanisms.

  8. Mutations conferring resistance to viral DNA polymerase inhibitors in camelpox virus give different drug-susceptibility profiles in vaccinia virus.

    Science.gov (United States)

    Duraffour, Sophie; Andrei, Graciela; Topalis, Dimitri; Krečmerová, Marcela; Crance, Jean-Marc; Garin, Daniel; Snoeck, Robert

    2012-07-01

    Cidofovir or (S)-HPMPC is one of the three antiviral drugs that might be used for the treatment of orthopoxvirus infections. (S)-HPMPC and its 2,6-diaminopurine counterpart, (S)-HPMPDAP, have been described to select, in vitro, for drug resistance mutations in the viral DNA polymerase (E9L) gene of vaccinia virus (VACV). Here, to extend our knowledge of drug resistance development among orthopoxviruses, we selected, in vitro, camelpox viruses (CMLV) resistant to (S)-HPMPDAP and identified a single amino acid change, T831I, and a double mutation, A314V+A684V, within E9L. The production of recombinant CMLV and VACV carrying these amino acid substitutions (T831I, A314V, or A314V+A684V) demonstrated clearly their involvement in conferring reduced sensitivity to viral DNA polymerase inhibitors, including (S)-HPMPDAP. Both CMLV and VACV harboring the A314V change showed comparable drug-susceptibility profiles to various antivirals and similar impairments in viral growth. In contrast, the single change T831I and the double change A314V+A684V in VACV were responsible for increased levels of drug resistance and for cross-resistance to viral DNA polymerase antivirals that were not observed with their CMLV counterparts. Each amino acid change accounted for an attenuated phenotype of VACV in vivo. Modeling of E9L suggested that the T→I change at position 831 might abolish hydrogen bonds between E9L and the DNA backbone and have a direct impact on the incorporation of the acyclic nucleoside phosphonates. Our findings demonstrate that drug-resistance development in two related orthopoxvirus species may impact drug-susceptibility profiles and viral fitness differently.

  9. Viruses Infecting a Freshwater Filamentous Cyanobacterium (Nostoc sp. Encode a Functional CRISPR Array and a Proteobacterial DNA Polymerase B

    Directory of Open Access Journals (Sweden)

    Caroline Chénard

    2016-06-01

    Full Text Available Here we present the first genomic characterization of viruses infecting Nostoc, a genus of ecologically important cyanobacteria that are widespread in freshwater. Cyanophages A-1 and N-1 were isolated in the 1970s and infect Nostoc sp. strain PCC 7210 but remained genomically uncharacterized. Their 68,304- and 64,960-bp genomes are strikingly different from those of other sequenced cyanophages. Many putative genes that code for proteins with known functions are similar to those found in filamentous cyanobacteria, showing a long evolutionary history in their host. Cyanophage N-1 encodes a CRISPR array that is transcribed during infection and is similar to the DR5 family of CRISPRs commonly found in cyanobacteria. The presence of a host-related CRISPR array in a cyanophage suggests that the phage can transfer the CRISPR among related cyanobacteria and thereby provide resistance to infection with competing phages. Both viruses also encode a distinct DNA polymerase B that is closely related to those found in plasmids of Cyanothece sp. strain PCC 7424, Nostoc sp. strain PCC 7120, and Anabaena variabilis ATCC 29413. These polymerases form a distinct evolutionary group that is more closely related to DNA polymerases of proteobacteria than to those of other viruses. This suggests that the polymerase was acquired from a proteobacterium by an ancestral virus and transferred to the cyanobacterial plasmid. Many other open reading frames are similar to a prophage-like element in the genome of Nostoc sp. strain PCC 7524. The Nostoc cyanophages reveal a history of gene transfers between filamentous cyanobacteria and their viruses that have helped to forge the evolutionary trajectory of this previously unrecognized group of phages.

  10. Effects of different polymerases of avian influenza viruses on the growth and pathogenicity of A/Puerto Rico/8/1934 (H1N1)-derived reassorted viruses.

    Science.gov (United States)

    Kim, Il-Hwan; Choi, Jun-Gu; Lee, Youn-Jeong; Kwon, Hyuk-Joon; Kim, Jae-Hong

    2014-01-10

    We generated reassorted PR8 viruses containing six different combinations of avian influenza virus (AIV) polymerase genes from A/chicken/Korea/01310/2001 (H9N2) (01310) and A/chicken/Korea/KBNP-0028/2000 (H9N2) (0028) to examine the effects of the AIV polymerase genes PB1, PB2, and PA on replication efficiency in different host cells and pathogenicity in mice. The virus titers of the reassorted viruses possessing 01310 [rPR8-PB2(01310)] and 0028 [rPR8-PB2(0028)] PB2 genes were significantly higher than those of the others except the rPR8 virus in embryonated chicken eggs at 37°C, and those of avian polymerase reassorted viruses were significantly less than rPR8 in MDCK cells at 32 and 37°C. rPR8-PB2(01310), rPR8-PB2(0028), and rPR8-PA(0028) caused no body weight loss in BALB/c mice but rPR8-PA(01310), rPR8-PB1(01310), and rPR8-PB1(0028) caused mortality and significantly different body weight loss compared to those in the mock treatment. In contrast to rPR8-PB2(0028) and rPR8-PA(0028), rPR8-PB2(01310) was not isolated from infected mice, and rPR8-PB1(0028) was less pathogenic than rPR8-PB1(01310). We determined the amino acid residues that were specific to the less pathogenic polymerases. A comparison with those of pandemic 2009 H1N1, human fatal H5N1 and H7N9, and pathogenic AIVs to mice without adaptation revealed that they possessed the mammalian pathogenic constellation of polymerases. Thus, the novel polymerase genes and amino acid residues may be useful to understand the host-barrier overcome of AIVs in mice and to develop safer and efficacious vaccines. Copyright © 2013 Elsevier B.V. All rights reserved.

  11. [DNA-dependent DNA polymerase induced by herpes virus papio (HVP) in producing cells].

    Science.gov (United States)

    D'iachenko, A G; Beriia, L Ia; Matsenko, L D; Kakubava, V V; Kokosh, L V

    1980-11-01

    A new DNA polymerase was found in the cells of suspension lymphoblastoid cultures, which produce lymphotropic baboon herpes virus (HVP). The enzyme was isolated in a partially purified form. In some properties the enzyme differs from other cellular DNA polymerases. The HVP-induced DNA polymerase has the molecular weight of 1,6 x 10(5) and sedimentation coefficient of about 8S. The enzyme is resistant to high salt concentrations and N-ethylmaleimide, but shows a pronounced sensitivity to phosphonoacetate. The enzyme effectively copies "activated" DNA and synthetic deoxyribohomopolymers. The attempts to detect the DNA polymerase activity in HVP virions were unsuccessful.

  12. Attenuation of foot-and-mouth disease virus by engineered viral polymerase fidelity

    Science.gov (United States)

    The foot-and-mouth disease virus (FMDV) RNA dependent RNA polymerase (RdRp or 3Dpol) catalyzes viral RNA synthesis. The 3Dpol is a low fidelity enzyme incapable of proofreading which results in a high mutation frequencies that allow the virus to rapidly adapt to different environments. In this study...

  13. The Chilo iridescent virus DNA polymerase promoter contains an essential AAAAT motif

    NARCIS (Netherlands)

    Nalcacioglu, R.; Ince, I.A.; Vlak, J.M.; Demirbag, Z.; Oers, van M.M.

    2007-01-01

    The delayed-early DNA polymerase promoter of Chilo iridescent virus (CIV), officially known as Invertebrate iridescent virus, was fine mapped by constructing a series of increasing deletions and by introducing point mutations. The effects of these mutations were examined in a luciferase reporter

  14. Characterization of genes encoding poly(A polymerases in plants: evidence for duplication and functional specialization.

    Directory of Open Access Journals (Sweden)

    Lisa R Meeks

    Full Text Available BACKGROUND: Poly(A polymerase is a key enzyme in the machinery that mediates mRNA 3' end formation in eukaryotes. In plants, poly(A polymerases are encoded by modest gene families. To better understand this multiplicity of genes, poly(A polymerase-encoding genes from several other plants, as well as from Selaginella, Physcomitrella, and Chlamydomonas, were studied. METHODOLOGY/PRINCIPAL FINDINGS: Using bioinformatics tools, poly(A polymerase-encoding genes were identified in the genomes of eight species in the plant lineage. Whereas Chlamydomonas reinhardtii was found to possess a single poly(A polymerase gene, other species possessed between two and six possible poly(A polymerase genes. With the exception of four intron-lacking genes, all of the plant poly(A polymerase genes (but not the C. reinhardtii gene possessed almost identical intron positions within the poly(A polymerase coding sequences, suggesting that all plant poly(A polymerase genes derive from a single ancestral gene. The four Arabidopsis poly(A polymerase genes were found to be essential, based on genetic analysis of T-DNA insertion mutants. GFP fusion proteins containing three of the four Arabidopsis poly(A polymerases localized to the nucleus, while one such fusion protein was localized in the cytoplasm. The fact that this latter protein is largely pollen-specific suggests that it has important roles in male gametogenesis. CONCLUSIONS/SIGNIFICANCE: Our results indicate that poly(A polymerase genes have expanded from a single ancestral gene by a series of duplication events during the evolution of higher plants, and that individual members have undergone sorts of functional specialization so as to render them essential for plant growth and development. Perhaps the most interesting of the plant poly(A polymerases is a novel cytoplasmic poly(A polymerase that is expressed in pollen in Arabidopsis; this is reminiscent of spermatocyte-specific cytoplasmic poly(A polymerases in

  15. Targeting CTCF to Control Virus Gene Expression: A Common Theme amongst Diverse DNA Viruses.

    Science.gov (United States)

    Pentland, Ieisha; Parish, Joanna L

    2015-07-06

    All viruses target host cell factors for successful life cycle completion. Transcriptional control of DNA viruses by host cell factors is important in the temporal and spatial regulation of virus gene expression. Many of these factors are recruited to enhance virus gene expression and thereby increase virus production, but host cell factors can also restrict virus gene expression and productivity of infection. CCCTC binding factor (CTCF) is a host cell DNA binding protein important for the regulation of genomic chromatin boundaries, transcriptional control and enhancer element usage. CTCF also functions in RNA polymerase II regulation and in doing so can influence co-transcriptional splicing events. Several DNA viruses, including Kaposi's sarcoma-associated herpesvirus (KSHV), Epstein-Barr virus (EBV) and human papillomavirus (HPV) utilize CTCF to control virus gene expression and many studies have highlighted a role for CTCF in the persistence of these diverse oncogenic viruses. CTCF can both enhance and repress virus gene expression and in some cases CTCF increases the complexity of alternatively spliced transcripts. This review article will discuss the function of CTCF in the life cycle of DNA viruses in the context of known host cell CTCF functions.

  16. Polymerase chain reaction to search for Herpes viruses in uveitic ...

    African Journals Online (AJOL)

    Objective: To analyse aqueous polymerase chain reaction (PCR) results in patients diagnosed with undifferentiated uveitis and determine prevalence of herpesviridae in non-uveitic patients undergoing routine cataract extraction. Design: Retrospective comparative case series and prospective cross-sectional study.

  17. Polymerase chain reaction-based gene removal from plasmids

    Directory of Open Access Journals (Sweden)

    Vishnu Vardhan Krishnamurthy

    2015-09-01

    Full Text Available This data article contains supplementary figures and methods to the research article entitled, “Multiplex gene removal by two-step polymerase chain reactions” (Krishnamurthy et al., Anal. Biochem., 2015, doi:http://dx.doi.org/10.1016/j.ab.2015.03.033, which presents a restriction-enzyme free method to remove multiple DNA segments from plasmids. Restriction-free cloning methods have dramatically improved the flexibility and speed of genetic manipulation compared to conventional assays based on restriction enzyme digestion (Lale and Valla, 2014. DNA Cloning and Assembly Methods, vol. 1116. Here, we show the basic scheme and characterize the success rate for single and multiplex gene removal from plasmids. In addition, we optimize experimental conditions, including the amount of template, multiple primers mixing, and buffers for DpnI treatment, used in the one-pot reaction for multiplex gene removal.

  18. In Silico Screening Hepatitis B Virus DNA Polymerase Inhibitors from Medicinal Plants

    Directory of Open Access Journals (Sweden)

    Mokhtar Nosrati

    2017-08-01

    Full Text Available Abstract Background: Hepatitis B virus infection (HBV is a significant global health problem and is a major cause of morbidity and mortality worldwide. Therefore, currently, introducing novel anti Hepatitis B drugs is taken into consideration. This study was planned to in silico screening novel Hepatitis B virus DNA polymerase inhibitors from two medicinal plants Terminalis chebula and Caesalpinia sappan. Materials and Methods: This is a descriptive-analytic study. In the study, three-dimensional structure of the Hepatitis B virus DNA polymerase was predicted using homology modeling method. A set of phytochemicals from mentioned plants were retrieved from Pubchem database in SDF format. In silico screening was carried out using molecular docking between mentioned phytochemicals and modeled polymerase by iGemdock 2.1 software. Results: Results of the study confirmed that all evaluated ligands have appropriate interactions to the polymerase with least toxicity and without genotoxicity potential. Results also showed that most interactions occur in reverse transcriptase domain which located in 354-694 area in the amino acid sequence of tested polymerase. Analysis of energy and amino acids involved in ligand-polymerase interaction revealed that Terchebin, Chebulinic Acid and Terflavin A have more effective interaction with the polymerase in compared to other ligands. Conclusion: Based on the results it can be concluded that evaluated compounds could be good candidates for in vitro and in vivo research in order to develop novel anti- Hepatitis B drugs.

  19. Adaptation of Borna disease virus to new host species attributed to altered regulation of viral polymerase activity.

    Science.gov (United States)

    Ackermann, Andreas; Staeheli, Peter; Schneider, Urs

    2007-08-01

    Borna disease virus (BDV) can persistently infect the central nervous system of a broad range of mammalian species. Mice are resistant to infections with primary BDV isolates, but certain laboratory strains can be adapted to replicate in mice. We determined the molecular basis of adaptation by studying mutations acquired by a cDNA-derived BDV strain during one brain passage in rats and three passages in mice. The adapted virus propagated efficiently in mouse brains and induced neurological disease. Its genome contained seven point mutations, three of which caused amino acid changes in the L polymerase (L1116R and N1398D) and in the polymerase cofactor P (R66K). Recombinant BDV carrying these mutations either alone or in combination all showed enhanced multiplication speed in Vero cells, indicating improved intrinsic viral polymerase activity rather than adaptation to a mouse-specific factor. Mutations R66K and L1116R, but not N1398D, conferred replication competence of recombinant BDV in mice if introduced individually. Virus propagation in mouse brains was substantially enhanced if both L mutations were present simultaneously, but infection remained mostly nonsymptomatic. Only if all three amino acid substitutions were combined did BDV replicate vigorously and induce early disease in mice. Interestingly, the virulence-enhancing effect of the R66K mutation in P could be attributed to reduced negative regulation of polymerase activity by the viral X protein. Our data demonstrate that BDV replication competence in mice is mediated by the polymerase complex rather than the viral envelope and suggest that altered regulation of viral gene expression can favor adaptation to new host species.

  20. Detection and strain differentiation of infectious bronchitis virus in tracheal tissues from experimentally infected chickens by reverse transcription-polymerase chain reaction. Comparison with an immunohistochemical technique

    DEFF Research Database (Denmark)

    Handberg, Kurt; Nielsen, O.L.; Pedersen, M.W.

    1999-01-01

    Oligonucleotide pairs were constructed for priming the amplification of fragments of nucleocapsid (N) protein and spike glycoprotein (S) genes of avian infectious bronchitis virus (IBV) by reverse transcription-polymerase chain reaction (RT-PCR). One oligonucleotide pair amplified a common segment...

  1. Cloning and analysis of the DNA polymerase-encoding gene from Thermus caldophilus GK24.

    Science.gov (United States)

    Kwon, S T; Kim, J S; Park, J H; Kim, H K; Lee, D S

    1997-04-30

    The gene encoding Thermus caldophilus GK24 (Tca) DNA polymerase was cloned into Escherichia coli using the structural gene coding for Thermus aquaticus YT-1 (Taq) DNA polymerase as a hybridization probe. The nucleotide sequence of the cloned DNA was determined. The primary structure of the Tca DNA polymerase was deduced from the nucleotide sequence. The Tca DNA polymerase comprised 834 amino acid residues and its molecular mass was determined to be 93,810. On alignment of the whole amino acid sequence, Tca DNA polymerase showed a high sequence homology with the E. coli DNA polymerase I-like DNA polymerases, and 86% identity with Taq DNA polymerase, 38% with E. coli and Streptococcus pneumoniae (Spn) DNA polymerase I. An extremely high sequence identity was observed in the region containing the polymerase activity. The codon usage in the Tca DNA polymerase gene was in fact similar to the characteristic usages in the genes for proteins from bacteria of genus Thermus: the G+C content in the third position of the codons was as high as 93%. The Tca DNA polymerase gene was expressed under the control of tac promoter on a high copy plasmid, pTCA in E. coli.

  2. New tools to study RNA interference to fish viruses: Fish cell lines permanently expressing siRNAs targeting the viral polymerase of viral hemorrhagic septicemia virus

    DEFF Research Database (Denmark)

    Ruiz, S.; Schyth, Brian Dall; Encinas, P.

    2009-01-01

    Previous studies have indicated that low transfection efficiency can be a major problem when gene inhibition by the use of small interfering RNAs (siRNAs) is attempted in fish cells. This may especially be true when targeting genes of viruses which are fast replicating and which can still infect...... cells that have not been transfected with the antiviral siRNAs. To increase the amount of antiviral siRNAs per cell a different strategy than transfection was taken here. Thus, we describe carp epithelioma papulosum cyprinid (EPC) cell clones expressing siRNAs designed to target the L polymerase gene...... of the viral hemorrhagic septicemia virus (VHSV), a rhabdovirus affecting fish. Eight siRNA sequences were first designed, synthesized and screened for inhibition of in vitro VHSV infectivity. Small hairpin (sh) DNAs corresponding to three selected siRNAs were then cloned into pRNA-CMV3.1/puro plasmids...

  3. Multisubunit RNA Polymerases IV and V: Purveyors of Non-Coding RNA for Plant Gene Silencing

    Energy Technology Data Exchange (ETDEWEB)

    Haag, Jeremy R.; Pikaard, Craig S.

    2011-08-01

    In all eukaryotes, nuclear DNA-dependent RNA polymerases I, II and III synthesize the myriad RNAs that are essential for life. Remarkably, plants have evolved two additional multisubunit RNA polymerases, RNA polymerases IV and V, which orchestrate non-coding RNA-mediated gene silencing processes affecting development, transposon taming, antiviral defence and allelic crosstalk. Biochemical details concerning the templates and products of RNA polymerases IV and V are lacking. However, their subunit compositions reveal that they evolved as specialized forms of RNA polymerase II, which provides the unique opportunity to study the functional diversification of a eukaryotic RNA polymerase family.

  4. Multiple, non-allelic, intein-coding sequences in eukaryotic RNA polymerase genes

    Directory of Open Access Journals (Sweden)

    Butler Margaret I

    2006-10-01

    Full Text Available Abstract Background Inteins are self-splicing protein elements. They are translated as inserts within host proteins that excise themselves and ligate the flanking portions of the host protein (exteins with a peptide bond. They are encoded as in-frame insertions within the genes for the host proteins. Inteins are found in all three domains of life and in viruses, but have a very sporadic distribution. Only a small number of intein coding sequences have been identified in eukaryotic nuclear genes, and all of these are from ascomycete or basidiomycete fungi. Results We identified seven intein coding sequences within nuclear genes coding for the second largest subunits of RNA polymerase. These sequences were found in diverse eukaryotes: one is in the second largest subunit of RNA polymerase I (RPA2 from the ascomycete fungus Phaeosphaeria nodorum, one is in the RNA polymerase III (RPC2 of the slime mould Dictyostelium discoideum and four intein coding sequences are in RNA polymerase II genes (RPB2, one each from the green alga Chlamydomonas reinhardtii, the zygomycete fungus Spiromyces aspiralis and the chytrid fungi Batrachochytrium dendrobatidis and Coelomomyces stegomyiae. The remaining intein coding sequence is in a viral relic embedded within the genome of the oomycete Phytophthora ramorum. The Chlamydomonas and Dictyostelium inteins are the first nuclear-encoded inteins found outside of the fungi. These new inteins represent a unique dataset: they are found in homologous proteins that form a paralogous group. Although these paralogues diverged early in eukaryotic evolution, their sequences can be aligned over most of their length. The inteins are inserted at multiple distinct sites, each of which corresponds to a highly conserved region of RNA polymerase. This dataset supports earlier work suggesting that inteins preferentially occur in highly conserved regions of their host proteins. Conclusion The identification of these new inteins

  5. Attenuation of Foot-and-Mouth Disease Virus by Engineered Viral Polymerase Fidelity.

    Science.gov (United States)

    Rai, Devendra K; Diaz-San Segundo, Fayna; Campagnola, Grace; Keith, Anna; Schafer, Elizabeth A; Kloc, Anna; de Los Santos, Teresa; Peersen, Olve; Rieder, Elizabeth

    2017-08-01

    Foot-and-mouth disease virus (FMDV) RNA-dependent RNA polymerase (RdRp) (3Dpol) catalyzes viral RNA synthesis. Its characteristic low fidelity and absence of proofreading activity allow FMDV to rapidly mutate and adapt to dynamic environments. In this study, we used the structure of FMDV 3Dpol in combination with previously reported results from similar picornaviral polymerases to design point mutations that would alter replication fidelity. In particular, we targeted Trp237 within conserved polymerase motif A because of the low reversion potential inherent in the single UGG codon. Using biochemical and genetic tools, we show that the replacement of tryptophan 237 with phenylalanine imparts higher fidelity, but replacements with isoleucine and leucine resulted in lower-fidelity phenotypes. Viruses containing these W237 substitutions show in vitro growth kinetics and plaque morphologies similar to those of the wild-type (WT) A24 Cruzeiro strain in BHK cells, and both high- and low-fidelity variants retained fitness during coinfection with the wild-type virus. The higher-fidelity W237F (W237FHF) mutant virus was more resistant to the mutagenic nucleoside analogs ribavirin and 5-fluorouracil than the WT virus, whereas the lower-fidelity W237I (W237ILF) and W237LLF mutant viruses exhibited lower ribavirin resistance. Interestingly, the variant viruses showed heterogeneous and slightly delayed growth kinetics in primary porcine kidney cells, and they were significantly attenuated in mouse infection experiments. These data demonstrate, for a single virus, that either increased or decreased RdRp fidelity attenuates virus growth in animals, which is a desirable feature for the development of safer and genetically more stable vaccine candidates.IMPORTANCE Foot-and-mouth disease (FMD) is the most devastating disease affecting livestock worldwide. Here, using structural and biochemical analyses, we have identified FMDV 3Dpol mutations that affect polymerase fidelity

  6. 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

  7. Application of polymerase chain reaction to differentiate herpes simplex virus 1 and 2 serotypes in culture negative intraocular aspirates.

    Science.gov (United States)

    Shyamal, G; Sowmya, P; Sudha, B; Malathi, J; Therese, L K; Madhavan, H N

    2005-10-01

    To standardize and apply a polymerase chain reaction (PCR) on the glycoprotein D gene to differentiate Herpes simplex virus (HSV) 1 & 2 serotypes in culture negative intraocular specimens. Twenty-one intraocular fluids collected from 19 patients were subjected to cultures for HSV and uniplex PCR (uPCR) for DNA polymerase gene. To differentiate HSV serotypes, as 1 & 2, a seminested PCR (snPCR) targeting the glycoprotein D gene was standardised and applied onto 21 intraocular fluids. The specificity of the snPCR was verified by application onto ATCC strains of HSV 1 and 2, clinical isolates and DNA sequencing of the amplified products. All specimens were also tested for the presence of cytomegalovirus (CMV) and varicella zoster virus (VZV) by nucleic acid amplification methods. Four of the 21 intraocular fluids were positive for HSV by uPCR. snPCR detected HSV in three additional specimens (total of seven specimens), and identified three as HSV 1 and four as HSV 2. DNA sequencing of PCR products showed 100% homology with the standard strains of HSV 1 and 2 respectively. None of the samples were positive in culture. Among the other patients, CMV DNA was detected in two and VZV DNA in five others. The standardized snPCR can be applied directly onto the culture negative specimens for rapid differentiation of HSV serotypes.

  8. Application of polymerase chain reaction to differentiate herpes simplex virus 1 and 2 serotypes in culture negative intraocular aspirates

    Directory of Open Access Journals (Sweden)

    Shyamal G

    2005-01-01

    Full Text Available Purpose: To standardize and apply a polymerase chain reaction (PCR on the glycoprotein D gene to differentiate Herpes simplex virus (HSV 1 & 2 serotypes in culture negative intraocular specimens. Methods: Twenty-one intraocular fluids collected from 19 patients were subjected to cultures for HSV and uniplex PCR (uPCR for DNA polymerase gene. To differentiate HSV serotypes, as 1 & 2, a seminested PCR (snPCR targeting the glycoprotein D gene was standardised and applied onto 21 intraocular fluids. The specificity of the snPCR was verified by application onto ATCC strains of HSV 1 and 2, clinical isolates and DNA sequencing of the amplified products. All specimens were also tested for the presence of cytomegalovirus (CMV and varicella zoster virus (VZV by nucleic acid amplification methods. Results: Four of the 21 intraocular fluids were positive for HSV by uPCR. snPCR detected HSV in three additional specimens (total of seven specimens, and identified three as HSV 1 and four as HSV 2. DNA sequencing of PCR products showed 100% homology with the standard strains of HSV 1 and 2 respectively. None of the samples were positive in culture. Among the other patients, CMV DNA was detected in two and VZV DNA in five others. Conclusions: The standardized snPCR can be applied directly onto the culture negative specimens for rapid differentiation of HSV serotypes.

  9. Expression of RNA virus proteins by RNA polymerase II dependent expression plasmids is hindered at multiple steps

    Directory of Open Access Journals (Sweden)

    Überla Klaus

    2007-06-01

    Full Text Available Abstract Background Proteins of human and animal viruses are frequently expressed from RNA polymerase II dependent expression cassettes to study protein function and to develop gene-based vaccines. Initial attempts to express the G protein of vesicular stomatitis virus (VSV and the F protein of respiratory syncytial virus (RSV by eukaryotic promoters revealed restrictions at several steps of gene expression. Results Insertion of an intron flanked by exonic sequences 5'-terminal to the open reading frames (ORF of VSV-G and RSV-F led to detectable cytoplasmic mRNA levels of both genes. While the exonic sequences were sufficient to stabilise the VSV-G mRNA, cytoplasmic mRNA levels of RSV-F were dependent on the presence of a functional intron. Cytoplasmic VSV-G mRNA levels led to readily detectable levels of VSV-G protein, whereas RSV-F protein expression remained undetectable. However, RSV-F expression was observed after mutating two of four consensus sites for polyadenylation present in the RSV-F ORF. Expression levels could be further enhanced by codon optimisation. Conclusion Insufficient cytoplasmic mRNA levels and premature polyadenylation prevent expression of RSV-F by RNA polymerase II dependent expression plasmids. Since RSV replicates in the cytoplasm, the presence of premature polyadenylation sites and elements leading to nuclear instability should not interfere with RSV-F expression during virus replication. The molecular mechanisms responsible for the destabilisation of the RSV-F and VSV-G mRNAs and the different requirements for their rescue by insertion of an intron remain to be defined.

  10. Biochemical characterization of enzyme fidelity of influenza A virus RNA polymerase complex.

    Directory of Open Access Journals (Sweden)

    Shilpa Aggarwal

    2010-04-01

    Full Text Available It is widely accepted that the highly error prone replication process of influenza A virus (IAV, together with viral genome assortment, facilitates the efficient evolutionary capacity of IAV. Therefore, it has been logically assumed that the enzyme responsible for viral RNA replication process, influenza virus type A RNA polymerase (IAV Pol, is a highly error-prone polymerase which provides the genomic mutations necessary for viral evolution and host adaptation. Importantly, however, the actual enzyme fidelity of IAV RNA polymerase has never been characterized.Here we established new biochemical assay conditions that enabled us to assess both polymerase activity with physiological NTP pools and enzyme fidelity of IAV Pol. We report that IAV Pol displays highly active RNA-dependent RNA polymerase activity at unbiased physiological NTP substrate concentrations. With this robust enzyme activity, for the first time, we were able to compare the enzyme fidelity of IAV Pol complex with that of bacterial phage T7 RNA polymerase and the reverse transcriptases (RT of human immunodeficiency virus (HIV-1 and murine leukemia virus (MuLV, which are known to be low and high fidelity enzymes, respectively. We observed that IAV Pol displayed significantly higher fidelity than HIV-1 RT and T7 RNA polymerase and equivalent or higher fidelity than MuLV RT. In addition, the IAV Pol complex showed increased fidelity at lower temperatures. Moreover, upon replacement of Mg(++ with Mn(++, IAV Pol displayed increased polymerase activity, but with significantly reduced processivity, and misincorporation was slightly elevated in the presence of Mn(++. Finally, when the IAV nucleoprotein (NP was included in the reactions, the IAV Pol complex exhibited enhanced polymerase activity with increased fidelity.Our study indicates that IAV Pol is a high fidelity enzyme. We envision that the high fidelity nature of IAV Pol may be important to counter-balance the multiple rounds of

  11. Cloning and analysis of the DNA polymerase-encoding gene from Thermus filiformis.

    Science.gov (United States)

    Jung, S E; Choi, J J; Kim, H K; Kwon, S T

    1997-12-31

    The gene encoding Thermus filiformis (Tfi) DNA polymerase was cloned and its nucleotide sequence was determined. The primary structure of Tfi DNA polymerase was deduced from its nucleotide sequence. Tfi DNA polymerase is comprised of 833 amino acid residues and its molecular mass was determined to be 93,890 Da. The deduced amino acid sequence of Tfi DNA polymerase showed a high sequence homology to E. coli DNA polymerase I-like DNA polymerases: 78.5% homology to Taq DNA polymerase, 78.4% to Tca DNA polymerase, and 41.8% to E. coli DNA polymerase I. An extremely high sequence identity was observed in the region containing polymerase activity. The G + C content of the coding region for the Tfi DNA polymerase gene was 68.5%, which was higher than that of the chromosomal DNA (65%). The G + C contents in the first, second, and third positions of the codons used were 71.8%, 40.9%, and 92.7% respectively. Codon usage in Tfi DNA polymerase was heavily biased towards the use of G + C in the third position. Rare codons with U or A as the third base were sometimes used to avoid using GA(A/T) TC and TCGA sequences, as they are recognition sites for the restriction endonucleases TfiI and TaqI.

  12. Multiplex Reverse Transcription-Polymerase Chain Reaction untuk Deteksi Cepat Virus Flu Burung H5N1 (MULTIPLEX REVERSE TRANSCRIPTION-POLYMERASE CHAIN REACTION FOR RAPID DETECTION OF H5N1 AVIAN INFLUENZA VIRUS

    Directory of Open Access Journals (Sweden)

    Raden Wasito

    2015-05-01

    Full Text Available Avian influenza virus subtype H5N1 (AIV H5N1 is highly pathogenic and fatal in poultry. The virusis still endemic with low virulence rate, although it may play a critical role in causing high morbidity andmortality rates in poultry in Indonesia. In general, diagnostic approach for AIV H5N1 is based onconventional serological and viral isolation methods that have the potential to produce consumings oftime and relatively expensive cost within the laboratory without compromising test utility. Thus, amolecular approach of multiplex reverse transcription-polymerase chain reaction (mRT-PCR was developedand applied for the detection of matrix gene type A influenza viruses, AIV subtype subtype H5hemagglutinin gene with simultaneous detection of N1 nucleoprotein gene. Thirty sera specimens fromthe diseased commercial chickens that were specifically amplified positive-RT-PCR for AIV H5N1 wereselected for mRT-PCR. The mRT-PCR products were visualized by agarose gel electrophoresis and consistedof DNA fragments of AIV of 245 bp, 545 bp and 343 bp for M, H5 and N1 genes, respectively. Thus, themRT-PCR that can rapidly differentiate simultaneously between these genes is very important for thecontrol and even eradication of AIV transmission in poultry in Indonesia.

  13. Targeting the pseudorabies virus DNA polymerase processivity factor UL42 by RNA interference efficiently inhibits viral replication.

    Science.gov (United States)

    Wang, Yi-Ping; Huang, Li-Ping; Du, Wen-Juan; Wei, Yan-Wu; Wu, Hong-Li; Feng, Li; Liu, Chang-Ming

    2016-08-01

    RNA interference (RNAi) is a conserved gene-silencing mechanism in which small interfering RNAs (siRNAs) induce the sequence-specific degradation of homologous RNAs. It has been shown to be a novel and effective antiviral therapy against a wide range of viruses. The pseudorabies virus (PRV) processivity factor UL42 can enhance the catalytic activity of the DNA polymerase and is essential for viral replication, thus it may represent a potential drug target of antiviral therapy against PRV infection. Here, we synthesized three siRNAs (siR-386, siR-517, and siR-849) directed against UL42 and determined their antiviral activities in cell culture. We first examined the kinetics of UL42 expression and found it was expressed with early kinetics during PRV replication. We verified that siR-386, siR-517, and siR-849 efficiently inhibited UL42 expression in an in vitro transfection system, thereby validating their inhibitory effects. Furthermore, we confirmed that these three siRNAs induced potent inhibitory effects on UL42 expression after PRV infection, comparable to the positive control siRNA, siR-1046, directed against the PRV DNA polymerase, the UL30 gene product, which is essential for virus replication. In addition, PRV replication was markedly reduced upon downregulation of UL42 expression. These results indicate that UL42-targeted RNAi efficiently inhibits target gene expression and impairs viral replication. This study provides a new clue for the design of an intervention strategy against herpesviruses by targeting their processivity factors. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. Detection of measles virus by reverse-transcriptase polymerase chain reaction in a placenta.

    Science.gov (United States)

    Bar-On, Shikma; Ochshorn, Yifat; Halutz, Ora; Aboudy, Yair; Many, Ariel

    2010-08-01

    Measles virus (MV) during pregnancy is associated with maternal morbidity and mortality and can put the fetus and newborn at risk of a wide range of complications. Reverse-transcriptase polymerase chain reaction (RT-PCR) for detecting MV in the placenta has not been reported. A case of RT-PCR detection of MV in the placenta of a 38-year-old woman who presented with premature rupture of membranes at 16 weeks' gestation is presented.

  15. Specific effect of zinc ions on DNA polymerase activity of avian myeloblastosis virus.

    Science.gov (United States)

    Palan, P R; Eidinoff, M L

    1978-11-01

    The effect of selected cations on DNA synthesis by DNA-polymerase of avian myeloblastosis virus (AMV) was studied. Zinc ions at low concentration (0.2mM) in the assay system enhanced the activity about 2 x fold and at higher concentration (2.0 mM) inhibited the activity completely. In contrast, addition of lithium and potassium salts produced inhibitory effects in this ionic concentration range. Replacement of K+ ion had an inhibitory effect on the activity.

  16. Development of a recombinase polymerase amplification combined with lateral-flow dipstick assay for detection of bovine ephemeral fever virus.

    Science.gov (United States)

    Hou, Peili; Zhao, Guimin; Wang, Hongmei; He, Chengqiang; Huan, Yanjun; He, Hongbin

    2017-12-26

    Bovine ephemeral fever virus (BEFV), identified as the causative pathogen of bovine ephemeral fever (BEF), is responsible for increasing numbers of epidemics/outbreaks and has a significant harmful effect on the livestock industry. Therefore, a rapid detection assay is imperative for BEFV diagnosis. In this study, we described the development of lateral-flow dipstick isothermal recombinase polymerase amplification (LFD-RPA) assays for detection of BEFV. RPA primers and LF probes were designed by targeting the specific G gene, and the amplification product can be visualized on a simple lateral flow dipstick with the naked eyes. The amplification reaction was performed at 38 °C for 20 min and LFD incubation time within 5 min. The detection limit of this assay was 8 copies per reaction, and there was no cross-reactivity with other bovine infectious viruses such as bovine viral diarrhea virus, infectious bovine rhinotracheitis virus, bovine respiratory syncytial virus, bovine coronavirus, bovine parainfluenza virus type 3, bovine vesicular stomatitis virus. In addition, the assay was performed with total 128 clinical specimens and the diagnostic results were compared with conventional RT-PCR, real-time quantative(q) PCR. The result showed that the coincidence rate of BEFV LFD-RPA and real-time qPCR was 96.09% (123/128), which was higher than conventional RT-PCR. The RPA combined with LFD assay probably provides a rapid and sensitive alternative for diagnosis of BEFV infections outbreak. Copyright © 2018 Elsevier Ltd. All rights reserved.

  17. Internal control for real-time polymerase chain reaction based on MS2 bacteriophage for RNA viruses diagnostics

    Directory of Open Access Journals (Sweden)

    Miriam Ribas Zambenedetti

    Full Text Available BACKGROUND Real-time reverse transcription polymerase chain reaction (RT-PCR is routinely used to detect viral infections. In Brazil, it is mandatory the use of nucleic acid tests to detect hepatitis C virus (HCV, hepatitis B virus and human immunodeficiency virus in blood banks because of the immunological window. The use of an internal control (IC is necessary to differentiate the true negative results from those consequent from a failure in some step of the nucleic acid test. OBJECTIVES The aim of this study was the construction of virus-modified particles, based on MS2 bacteriophage, to be used as IC for the diagnosis of RNA viruses. METHODS The MS2 genome was cloned into the pET47b(+ plasmid, generating pET47b(+-MS2. MS2-like particles were produced through the synthesis of MS2 RNA genome by T7 RNA polymerase. These particles were used as non-competitive IC in assays for RNA virus diagnostics. In addition, a competitive control for HCV diagnosis was developed by cloning a mutated HCV sequence into the MS2 replicase gene of pET47b(+-MS2, which produces a non-propagating MS2 particle. The utility of MS2-like particles as IC was evaluated in a one-step format multiplex real-time RT-PCR for HCV detection. FINDINGS We demonstrated that both competitive and non-competitive IC could be successfully used to monitor the HCV amplification performance, including the extraction, reverse transcription, amplification and detection steps, without compromising the detection of samples with low target concentrations. In conclusion, MS2-like particles generated by this strategy proved to be useful IC for RNA virus diagnosis, with advantage that they are produced by a low cost protocol. An attractive feature of this system is that it allows the construction of a multicontrol by the insertion of sequences from more than one pathogen, increasing its applicability for diagnosing different RNA viruses.

  18. Structure of Hepatitis C Virus Polymerase in Complex with Primer-Template RNA

    Energy Technology Data Exchange (ETDEWEB)

    Mosley, Ralph T.; Edwards, Thomas E.; Murakami, Eisuke; Lam, Angela M.; Grice, Rena L.; Du, Jinfa; Sofia, Michael J.; Furman, Philip A.; Otto, Michael J. (Pharmasset); (Emerald)

    2012-08-01

    The replication of the hepatitis C viral (HCV) genome is accomplished by the NS5B RNA-dependent RNA polymerase (RdRp), for which mechanistic understanding and structure-guided drug design efforts have been hampered by its propensity to crystallize in a closed, polymerization-incompetent state. The removal of an autoinhibitory {beta}-hairpin loop from genotype 2a HCV NS5B increases de novo RNA synthesis by >100-fold, promotes RNA binding, and facilitated the determination of the first crystallographic structures of HCV polymerase in complex with RNA primer-template pairs. These crystal structures demonstrate the structural realignment required for primer-template recognition and elongation, provide new insights into HCV RNA synthesis at the molecular level, and may prove useful in the structure-based design of novel antiviral compounds. Additionally, our approach for obtaining the RNA primer-template-bound structure of HCV polymerase may be generally applicable to solving RNA-bound complexes for other viral RdRps that contain similar regulatory {beta}-hairpin loops, including bovine viral diarrhea virus, dengue virus, and West Nile virus.

  19. Interactions between the Dengue Virus Polymerase NS5 and Stem-Loop A.

    Science.gov (United States)

    Bujalowski, Paul J; Bujalowski, Wlodzimierz; Choi, Kyung H

    2017-06-01

    The process of RNA replication by dengue virus is still not completely understood despite the significant progress made in the last few years. Stem-loop A (SLA), a part of the viral 5' untranslated region (UTR), is critical for the initiation of dengue virus replication, but quantitative analysis of the interactions between the dengue virus polymerase NS5 and SLA in solution has not been performed. Here, we examine how solution conditions affect the size and shape of SLA and the formation of the NS5-SLA complex. We show that dengue virus NS5 binds SLA with a 1:1 stoichiometry and that the association reaction is primarily entropy driven. We also observe that the NS5-SLA interaction is influenced by the magnesium concentration in a complex manner. Binding is optimal with 1 mM MgCl 2 but decreases with both lower and higher magnesium concentrations. Additionally, data from a competition assay between SLA and single-stranded RNA (ssRNA) indicate that SLA competes with ssRNA for the same binding site on the NS5 polymerase. SLA 70 and SLA 80 , which contain the first 70 and 80 nucleotides (nt), respectively, bind NS5 with similar binding affinities. Dengue virus NS5 also binds SLAs from different serotypes, indicating that NS5 recognizes the overall shape of SLA as well as specific nucleotides. IMPORTANCE Dengue virus is an important human pathogen responsible for dengue hemorrhagic fever, whose global incidence has increased dramatically over the last several decades. Despite the clear medical importance of dengue virus infection, the mechanism of viral replication, a process commonly targeted by antiviral therapeutics, is not well understood. In particular, stem-loop A (SLA) and stem-loop B (SLB) located in the 5' untranslated region (UTR) are critical for binding the viral polymerase NS5 to initiate minus-strand RNA synthesis. However, little is known regarding the kinetic and thermodynamic parameters driving these interactions. Here, we quantitatively examine the

  20. Sequence and structure prediction of RNA-dependent RNA polymerase of lily symptomless virus isolated from L. × 'Casablanca'.

    Science.gov (United States)

    Xu, Pinsan; Li, Huangai; Liu, Jiwen; Luan, Yushi; Yin, Yalei; Bai, Jianfang

    2011-06-01

    The DNA sequence of the RNA-dependent RNA polymerase (RdRp) gene of lily symptomless virus (LSV), a lily-infecting member of the genus Carlavirus, was determined from nine overlapping cDNA fragments of different sizes. The complete sequence of this RdRp gene (HM070294) consisted of 5,847 nucleotides coding for a protein of 220 kDa. It had 97-98% sequence identity with RdRps of other known isolates at both the DNA and the amino acid level. Phylogenetic analysis indicated that this RdRp (designated as RdRp-DL) was closely related to the RdRp of the Korean isolate (AM516059), as well as to the RdRps from Passiflora latent virus (PLV) and Kalanchoe latent virus (KLV) of the genus Carlavirus. Hydrophobic analysis of RdRp-DL revealed a hydrophobic N-terminus and a hydrophilic C-terminus. Helices and Loops were the major secondary structures of RdRp-DL. In addition, RdRp-DL also had three coil structures. Four conserved domains were identified: typoviral methyltransferase, RNA-dependent RNA polymerase, P-loop-containing nucleoside triphosphate hydrolases and carlavirus endopeptidase. A model of the tertiary structure predicted by I-TASSER was obtained for each of these conserved domains. This is the first report of a detailed phylogenetic analysis of LSV RdRp with those of other members of the genus Carlavirus, and the first to predict the domain structures of LSV RdRp.

  1. An improved electrochemiluminescence polymerase chain reaction method for highly sensitive detection of plant viruses

    Energy Technology Data Exchange (ETDEWEB)

    Tang Yabing [MOE Key Laboratory of Laser Life Science and Institute of Laser Life Science, South China Normal University, Guangzhou 510631 (China); Xing Da [MOE Key Laboratory of Laser Life Science and Institute of Laser Life Science, South China Normal University, Guangzhou 510631 (China)]. E-mail: xingda@scnu.edu.cn; Zhu Debin [MOE Key Laboratory of Laser Life Science and Institute of Laser Life Science, South China Normal University, Guangzhou 510631 (China); Liu Jinfeng [MOE Key Laboratory of Laser Life Science and Institute of Laser Life Science, South China Normal University, Guangzhou 510631 (China)

    2007-01-23

    Recently, we have reported an electrochemiluminescence polymerase chain reaction (ECL-PCR) method for detection of genetically modified organisms. The ECL-PCR method was further improved in the current study by introducing a multi-purpose nucleic acid sequence that was specific to the tris(bipyridine) ruthenium (TBR) labeled probe, into the 5' terminal of the primers. The method was applied to detect plant viruses. Conserved sequence of the plant viruses was amplified by PCR. The product was hybridized with a biotin labeled probe and a TBR labeled probe. The hybridization product was separated by streptavidin-coated magnetic beads, and detected by measuring the ECL signals of the TBR labeled. Under the optimized conditions, the experiment results show that the detection limit is 50 fmol of PCR products, and the signal-to-noise ratio is in excess of 14.6. The method was used to detect banana streak virus, banana bunchy top virus, and papaya leaf curl virus. The experiment results show that this method could reliably identity viruses infected plant samples. The improved ECL-PCR approach has higher sensitivity and lower cost than previous approach. It can effectively detect the plant viruses with simplicity, stability, and high sensitivity.

  2. Computational Investigations on Polymerase Actions in Gene Transcription and Replication Combining Physical Modeling and Atomistic Simulations

    CERN Document Server

    Yu, Jin

    2015-01-01

    Polymerases are protein enzymes that move along nucleic acid chains and catalyze template-based polymerization reactions during gene transcription and replication. The polymerases also substantially improve transcription or replication fidelity through the non-equilibrium enzymatic cycles. We briefly review computational efforts that have been made toward understanding mechano-chemical coupling and fidelity control mechanisms of the polymerase elongation. The polymerases are regarded as molecular information motors during the elongation process. It requires a full spectrum of computational approaches from multiple time and length scales to understand the full polymerase functional cycle. We keep away from quantum mechanics based approaches to the polymerase catalysis due to abundant former surveys, while address only statistical physics modeling approach and all-atom molecular dynamics simulation approach. We organize this review around our own modeling and simulation practices on a single-subunit T7 RNA poly...

  3. Recruitment of RED-SMU1 complex by Influenza A Virus RNA polymerase to control Viral mRNA splicing.

    Directory of Open Access Journals (Sweden)

    Guillaume Fournier

    2014-06-01

    Full Text Available Influenza A viruses are major pathogens in humans and in animals, whose genome consists of eight single-stranded RNA segments of negative polarity. Viral mRNAs are synthesized by the viral RNA-dependent RNA polymerase in the nucleus of infected cells, in close association with the cellular transcriptional machinery. Two proteins essential for viral multiplication, the exportin NS2/NEP and the ion channel protein M2, are produced by splicing of the NS1 and M1 mRNAs, respectively. Here we identify two human spliceosomal factors, RED and SMU1, that control the expression of NS2/NEP and are required for efficient viral multiplication. We provide several lines of evidence that in infected cells, the hetero-trimeric viral polymerase recruits a complex formed by RED and SMU1 through interaction with its PB2 and PB1 subunits. We demonstrate that the splicing of the NS1 viral mRNA is specifically affected in cells depleted of RED or SMU1, leading to a decreased production of the spliced mRNA species NS2, and to a reduced NS2/NS1 protein ratio. In agreement with the exportin function of NS2, these defects impair the transport of newly synthesized viral ribonucleoproteins from the nucleus to the cytoplasm, and strongly reduce the production of infectious influenza virions. Overall, our results unravel a new mechanism of viral subversion of the cellular splicing machinery, by establishing that the human splicing factors RED and SMU1 act jointly as key regulators of influenza virus gene expression. In addition, our data point to a central role of the viral RNA polymerase in coupling transcription and alternative splicing of the viral mRNAs.

  4. Development of a reverse transcription polymerase chain reaction method for yellow fever virus detection.

    Science.gov (United States)

    Méndez, María C; Domingo, Cristina; Tenorio, Antonio; Pardo, Lissethe C; Rey, Gloria J; Méndez, Jairo A

    2013-09-01

    Yellow fever is considered a re-emerging disease and is endemic in tropical regions of Africa and South America. At present, there are no standardized or commercialized kits available for yellow fever virus detection. Therefore, diagnosis must be made by time-consuming routine techniques, and sometimes, the virus or its proteins are not detected. Furthermore, co-circulation with other flaviviruses, including dengue virus, increases the difficulty of diagnosis. To develop a specific reverse transcriptase-polymerase chain reaction (RT-PCR) and nested PCR-based assay to improve the detection and diagnosis of yellow fever virus using both serum and fresh tissue samples. RT-PCR primers were designed to amplify a short fragment of all yellow fever virus genotypes reported. A second set of primers was used in a nested PCR to increase sensitivity. Thirty-three clinical samples were tested with the standardized reaction. The expected amplicon was obtained in 25 out of 33 samples analyzed using this approach, and 2 more samples tested positive after a subsequent nested PCR approach. This improved technique not only ensures the specific detection of a wide range of yellow fever virus genotypes but also may increase the sensitivity of detection by introducing a second round of amplification, allowing a rapid differential diagnosis between dengue and yellow fever infection, which is required for effective surveillance and opportune epidemiologic measures.

  5. Ubiquitous cyanobacterial podoviruses in the global oceans unveiled through viral DNA polymerase gene sequences.

    Science.gov (United States)

    Huang, Sijun; Wilhelm, Steven W; Jiao, Nianzhi; Chen, Feng

    2010-10-01

    As a major cyanophage group, cyanobacterial podoviruses are important in regulating the biomass and population structure of picocyanobacteria in the ocean. However, little is known about their biogeography in the open ocean. This study represents the first survey of the biodiversity of cyanopodoviruses in the global oceans based on the viral encoded DNA polymerase (pol) gene. A total of 303 DNA pol sequences were amplified by PCR from 10 virus communities collected in the Atlantic and Pacific oceans and the South China Sea. At least five subclusters of cyanopodoviruses were identified in these samples, and one subcluster (subcluster VIII) was found in all sampling sites and comprised approximately 50% of total sequences. The diversity index based on the DNA pol gene sequences recovered through PCR suggests that cyanopodoviruses are less diverse in these oceanic samples than in a previously studied estuarine environment. Although diverse podoviruses were present in the global ocean, each sample was dominated by one major group of cyanopodoviruses. No clear biogeographic patterns were observed using statistical analysis. A metagenomic analysis based on the Global Ocean Sampling database indicates that other types of cyanopodovirus-like DNA pol sequences were present in the global ocean. Together, our study results suggest that cyanopodoviruses are widely distributed in the ocean but their community composition varies with local environments.

  6. Pengembangan Sejumlah Primer untuk Reverse Transcriptase Polymerase Chain Reaction Guna Melacak Virus Flu Burung di Indonesia (DEVELOPMENt OF PRIMERS FOR REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION TO DETECT AVIAN INFLUENZA VIRUS IN INDONESIA

    Directory of Open Access Journals (Sweden)

    Ni Luh Putu Indi Dharmayanti

    2016-07-01

    Full Text Available Until recently, two clades of of avian influenza viruses (AIVs designated as 2.3.2 and 2.2.3 havebeen circulating in Indonesia. Mutations of AIV genes have cretaed many more variants of the virus. It istherefore important to evaluate the appropriate methods used for the detection and diagnosis of AI virusin the field. Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR have been used as a standardmethod for detection of AIV in many laboratories in Indonesia. The success of RT-PCR for detection ofAIV virus is dependent on the nucleotide sequences of primer that match with the circulating of AIVs. Theaims of this study was to develop RT-PCR by designing primers for H5 subtype specific to the circulatingAIVs in the field. The primers were designed using Primer Design software, and optimization andvalidation of the primer were conducted using AIVs that have been characterized in the previous study.The primers were then used RT-PCR using AIV isolates from field samples and their sensitivity andspecificity were then determined. The results showed that the H5 primers designed in this study, H5-IDand H5-NLP, was able to detect the AIVs in field samples better than the H5-specific primers have beenused previously. In conclusion, H5 primers designed based on recent viruses in the field showed betterresults in the detection of AI virus as compared to the previous primers. As AIV-H5N1 subtype in the fieldwill continue to change and evolve, the use of primers designed in this study is recommended for diagnosisof H5 AIV.

  7. Whole Blood Polymerase Chain Reaction in a Neonate with Disseminated Herpes Simplex Virus Infection and Liver Failure

    Directory of Open Access Journals (Sweden)

    Jennifer A. Scoble

    2013-10-01

    Full Text Available A late preterm neonate born by cesarean section with intact membranes presented at 9 days of life with shock and liver failure. Surface cultures were negative but whole blood polymerase chain reaction was positive for herpes simplex virus type 2, underscoring the value of this test in early diagnosis of perinatally acquired disseminated herpes simplex virus infection without skin lesions.

  8. The cap-snatching endonuclease of influenza virus polymerase resides in the PA subunit.

    Science.gov (United States)

    Dias, Alexandre; Bouvier, Denis; Crépin, Thibaut; McCarthy, Andrew A; Hart, Darren J; Baudin, Florence; Cusack, Stephen; Ruigrok, Rob W H

    2009-04-16

    The influenza virus polymerase, a heterotrimer composed of three subunits, PA, PB1 and PB2, is responsible for replication and transcription of the eight separate segments of the viral RNA genome in the nuclei of infected cells. The polymerase synthesizes viral messenger RNAs using short capped primers derived from cellular transcripts by a unique 'cap-snatching' mechanism. The PB2 subunit binds the 5' cap of host pre-mRNAs, which are subsequently cleaved after 10-13 nucleotides by the viral endonuclease, hitherto thought to reside in the PB2 (ref. 5) or PB1 (ref. 2) subunits. Here we describe biochemical and structural studies showing that the amino-terminal 209 residues of the PA subunit contain the endonuclease active site. We show that this domain has intrinsic RNA and DNA endonuclease activity that is strongly activated by manganese ions, matching observations reported for the endonuclease activity of the intact trimeric polymerase. Furthermore, this activity is inhibited by 2,4-dioxo-4-phenylbutanoic acid, a known inhibitor of the influenza endonuclease. The crystal structure of the domain reveals a structural core closely resembling resolvases and type II restriction endonucleases. The active site comprises a histidine and a cluster of three acidic residues, conserved in all influenza viruses, which bind two manganese ions in a configuration similar to other two-metal-dependent endonucleases. Two active site residues have previously been shown to specifically eliminate the polymerase endonuclease activity when mutated. These results will facilitate the optimisation of endonuclease inhibitors as potential new anti-influenza drugs.

  9. Computational investigations on polymerase actions in gene transcription and replication: Combining physical modeling and atomistic simulations

    Science.gov (United States)

    Jin, Yu

    2016-01-01

    Polymerases are protein enzymes that move along nucleic acid chains and catalyze template-based polymerization reactions during gene transcription and replication. The polymerases also substantially improve transcription or replication fidelity through the non-equilibrium enzymatic cycles. We briefly review computational efforts that have been made toward understanding mechano-chemical coupling and fidelity control mechanisms of the polymerase elongation. The polymerases are regarded as molecular information motors during the elongation process. It requires a full spectrum of computational approaches from multiple time and length scales to understand the full polymerase functional cycle. We stay away from quantum mechanics based approaches to the polymerase catalysis due to abundant former surveys, while addressing statistical physics modeling approaches along with all-atom molecular dynamics simulation studies. We organize this review around our own modeling and simulation practices on a single subunit T7 RNA polymerase, and summarize commensurate studies on structurally similar DNA polymerases as well. For multi-subunit RNA polymerases that have been actively studied in recent years, we leave systematical reviews of the simulation achievements to latest computational chemistry surveys, while covering only representative studies published very recently, including our own work modeling structure-based elongation kinetic of yeast RNA polymerase II. In the end, we briefly go through physical modeling on elongation pauses and backtracking activities of the multi-subunit RNAPs. We emphasize on the fluctuation and control mechanisms of the polymerase actions, highlight the non-equilibrium nature of the operation system, and try to build some perspectives toward understanding the polymerase impacts from the single molecule level to a genome-wide scale. Project supported by the National Natural Science Foundation (Grant No. 11275022).

  10. Structure-Based Drug Design Targeting a Subunit Interaction of Influenza Virus RNA Polymerase

    Science.gov (United States)

    Sugiyama, Kanako; Obayashi, Eiji; Yoshida, Hisashi; Park, Sam-Yong

    Influenza A virus is a major human and animal pathogen with the potential to cause catastrophic loss of life. Influenza virus reproduces rapidly, mutates frequently, and occasionally crosses species barriers. The recent emergence of swine-origin influenza H1N1 and avian influenza related to highly pathogenic forms of the human virus has highlighted the urgent need for new effective treatments. Here, we describe two crystal structures of complexes made by fragments of PA and PB1, and PB1 and PB2. These novel interfaces are surprisingly small, yet they play a crucial role in regulating the 250 kDa polymerase complex, and are completely conserved among swine, avian and human influenza viruses. Given their importance to viral replication and strict conservation, the PA/PB1 and PB1/PB2 interfaces appear to be promising targets for novel anti-influenza drugs of use against all strains of influenza A virus. It is hoped that the structures presented here will assist the search for such compounds.

  11. Functional characterization of the triple gene block 1 (TGB1) gene of Pepino mosaic virus in tomato

    Science.gov (United States)

    Pepino mosaic virus (PepMV) has caused serious economic losses to many greenhouse tomato productions around the world. This potexvirus genome contains five major open reading frames (ORFs) encoding for a 164-kDa RNA-dependent RNA polymerase (RdRp), three triple gene block (TGB) proteins of 26, 14 an...

  12. Activity of polymerase proteins of vaccine and wild-type measles virus strains in a minigenome replication assay.

    Science.gov (United States)

    Bankamp, Bettina; Kearney, Sean P; Liu, Xin; Bellini, William J; Rota, Paul A

    2002-07-01

    The relative activities of five measles virus (MV) polymerase (L) proteins were compared in an intracellular, plasmid-based replication assay. When coexpressed with N and P proteins from an attenuated strain, L proteins from two attenuated viruses directed the production of up to eight times more reporter protein from an MV minigenome than the three wild-type L proteins. Northern blot analysis demonstrated that the differences in reporter protein production correlated with mRNA transcription levels. Increased activity of polymerases from attenuated viruses equally affected mRNA transcription and minigenome replication. The higher level of transcription may be a consequence of increased template availability or may be an independent effect of the elevated activity of the attenuated polymerases. Coexpression of wild-type L proteins with homologous N and P proteins did not affect the activity of the wild-type polymerases, indicating that the differential activity was a function of the L proteins alone. Use of a minigenome that incorporated two nucleotide changes found in the genomic leader of the three wild-type viruses did not raise the activity of the wild-type L proteins. These data demonstrate that increased polymerase activity differentiates attenuated from wild-type viruses and suggest that functions involved in RNA synthesis contribute to the attenuated phenotype of MV vaccine strains.

  13. Interplay Among Constitutes of Ebola Virus: Nucleoprotein, Polymerase L, Viral Proteins

    Science.gov (United States)

    Zhang, Minchuan; He, Peiming; Su, Jing; Singh, Dadabhai T.; Su, Hailei; Su, Haibin

    Ebola virus is a highly lethal filovirus, claimed thousands of people in its recent outbreak. Seven viral proteins constitute ebola viral structure, and four of them (nucleoprotein (NP), polymerase L, VP35 and VP30) participate majorly in viral replication and transcription. We have elucidated a conformation change of NP cleft by VP35 NP-binding protein domains through superimposing two experimental NP structure images and discussed the function of this conformation change in the replication and transcription with polymerase complex (L, VP35 and VP30). The important roles of VP30 in viral RNA synthesis have also been discussed. A “tapping” model has been proposed in this paper for a better understanding of the interplay among the four viral proteins (NP, polymerase L, VP35 and VP30). Moreover, we have pinpointed some key residue changes on NP (both NP N- and C-terminal) and L between Reston and Zaire by computational studies. Together, this paper provides a description of interactions among ebola viral proteins (NP, L, VP35, VP30 and VP40) in viral replication and transcription, and sheds light on the complex system of viral reproduction.

  14. Impact of genetic heterogeneity in polymerase of hepatitis B virus on dynamics of viral load and hepatitis B progression.

    Directory of Open Access Journals (Sweden)

    Chi-Jung Huang

    Full Text Available OBJECTIVE: The hepatitis B virus (HBV-polymerase region overlaps pre-S/S genes with high epitope density and plays an essential role in viral replication. We investigated whether genetic variation in the polymerase region determined long-term dynamics of viral load and the risk of hepatitis B progression in a population-based cohort study. METHODS: We sequenced the HBV-polymerase region using baseline plasma from treatment-naïve individuals with HBV-DNA levels≥1000 copies/mL in a longitudinal viral-load study of participants with chronic HBV infection followed-up for 17 years, and obtained sequences from 575 participants (80% with HBV genotype Ba and 17% with Ce. RESULTS: Patterns of viral sequence diversity across phases (i.e., immune-tolerant, immune-clearance, non/low replicative, and hepatitis B e antigen (HBeAg-negative hepatitis phases of HBV-infection, which were associated with viral and clinical features at baseline and during follow-up, were similar between HBV genotypes, despite greater diversity for genotype Ce vs. Ba. Irrespective of genotypes, however, HBeAg-negative participants had 1.5-to-2-fold higher levels of sequence diversity than HBeAg-positive participants (P<0.0001. Furthermore, levels of viral genetic divergence from the population consensus sequence, estimated by numbers of nucleotide substitutions, were inversely associated with long-term viral load even in HBeAg-negative participants. A mixed model developed through analysis of the entire HBV-polymerase region identified 153 viral load-associated single nucleotide polymorphisms in overall and 136 in HBeAg-negative participants, with distinct profiles between HBV genotypes. These polymorphisms were most evident at sites within or flanking T-cell epitopes. Seven polymorphisms revealed associations with both enhanced viral load and a more than 4-fold increased risk of hepatocellular carcinoma and/or liver cirrhosis. CONCLUSIONS: The data highlight a role of viral

  15. 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.

  16. Simultaneous detection of hemagglutinin and neuraminidase genes of novel influenza A (H7N9) by duplex real-time reverse transcription polymerase chain reaction.

    Science.gov (United States)

    Li, Yan; Wu, Tao; Qi, Xian; Ge, Yiyue; Guo, Xiling; Wu, Bin; Yu, Huiyan; Zhu, Yefei; Shi, Zhiyang; Wang, Hua; Cui, Lunbiao; Zhou, Minghao

    2013-12-01

    A novel reassortant influenza A (H7N9) virus emerged recently in China. In this study, a duplex real-time reverse transcription polymerase chain reaction (rRT-PCR) assay was developed for the simultaneous detection of hemagglutinin (HA) and neuraminidase (NA) genes of H7N9 influenza viruses. The sensitivity of the assay was determined to be 10 RNA copies per reaction for both HA and NA genes. No cross-reactivity was observed with other influenza virus subtypes or respiratory tract viruses. One hundred and forty-six clinical and environmental specimens were tested and compared with reference methods and were found to be consistent. The assay is suitable for large-scale screening due to short turnaround times and high specificity, sensitivity, and reproducibility. Copyright © 2013 Elsevier B.V. All rights reserved.

  17. Differentiation of sheeppox and goatpox viruses by polymerase Chain reaction-restriction fragment length polymorphism.

    Science.gov (United States)

    Venkatesan, Gnanavel; Balamurugan, Vinayagamurthy; Yogisharadhya, Revaniah; Kumar, Amit; Bhanuprakash, Veerakyathappa

    2012-12-01

    In the present study, the partial gene sequences of P32 protein, an immunogenic envelope protein of Capripoxviruses (CaPV), were analyzed to assess the genetic relationship among sheeppox and goatpox virus isolates, and restriction enzyme specific PCR-RFLP was developed to differentiate CaPV strains. A total of six goatpox virus (GTPV) and nine sheeppox virus (SPPV) isolates of Indian origin were included in the sequence analysis of the attachment gene. The sequence analysis revealed a high degree of sequence identity among all the Indian SPPV and GTPV isolates at both nucleotide and amino acid levels. Phylogenetic analysis showed three distinct clusters of SPPV, GTPV and Lumpy skin disease virus (LSDV) isolates. Further, multiple sequence alignment revealed a unique change at G120A in all GTPV isolates resulting in the formation of Dra I restriction site in lieu of EcoR I, which is present in SPPV isolates studied. This change was unique and exploited to develop restriction enzyme specific PCR-RFLP for detection and differentiation of SPPV and GTPV strains. The optimized PCR-RFLP was validated using a total of fourteen (n=14) cell culture isolates and twenty two (n=22) known clinical samples of CaPV. The Restriction Enzyme specific PCR-RFLP to differentiate both species will allow a rapid differential diagnosis during CaPV outbreaks particularly in mixed flocks of sheep and goats and could be an adjunct/supportive tool for complete gene or virus genome sequencing methods.

  18. Initiation of RNA Polymerization and Polymerase Encapsidation by a Small dsRNA Virus.

    Directory of Open Access Journals (Sweden)

    Aaron M Collier

    2016-04-01

    Full Text Available During the replication cycle of double-stranded (ds RNA viruses, the viral RNA-dependent RNA polymerase (RdRP replicates and transcribes the viral genome from within the viral capsid. How the RdRP molecules are packaged within the virion and how they function within the confines of an intact capsid are intriguing questions with answers that most likely vary across the different dsRNA virus families. In this study, we have determined a 2.4 Å resolution structure of an RdRP from the human picobirnavirus (hPBV. In addition to the conserved polymerase fold, the hPBV RdRP possesses a highly flexible 24 amino acid loop structure located near the C-terminus of the protein that is inserted into its active site. In vitro RNA polymerization assays and site-directed mutagenesis showed that: (1 the hPBV RdRP is fully active using both ssRNA and dsRNA templates; (2 the insertion loop likely functions as an assembly platform for the priming nucleotide to allow de novo initiation; (3 RNA transcription by the hPBV RdRP proceeds in a semi-conservative manner; and (4 the preference of virus-specific RNA during transcription is dictated by the lower melting temperature associated with the terminal sequences. Co-expression of the hPBV RdRP and the capsid protein (CP indicated that, under the conditions used, the RdRP could not be incorporated into the recombinant capsids in the absence of the viral genome. Additionally, the hPBV RdRP exhibited higher affinity towards the conserved 5'-terminal sequence of the viral RNA, suggesting that the RdRP molecules may be encapsidated through their specific binding to the viral RNAs during assembly.

  19. Analysis of Ribonucleotide 5'-Triphosphate Analogs as Potential Inhibitors of Zika Virus RNA-Dependent RNA Polymerase by Using Nonradioactive Polymerase Assays.

    Science.gov (United States)

    Lu, Gaofei; Bluemling, Gregory R; Collop, Paul; Hager, Michael; Kuiper, Damien; Gurale, Bharat P; Painter, George R; De La Rosa, Abel; Kolykhalov, Alexander A

    2017-03-01

    Zika virus (ZIKV) is an emerging human pathogen that is spreading rapidly through the Americas and has been linked to the development of microcephaly and to a dramatically increased number of Guillain-Barré syndrome cases. Currently, no vaccine or therapeutic options for the prevention or treatment of ZIKV infections exist. In the study described in this report, we expressed, purified, and characterized full-length nonstructural protein 5 (NS5) and the NS5 polymerase domain (NS5pol) of ZIKV RNA-dependent RNA polymerase. Using purified NS5, we developed an in vitro nonradioactive primer extension assay employing a fluorescently labeled primer-template pair. Both purified NS5 and NS5pol can carry out in vitro RNA-dependent RNA synthesis in this assay. Our results show that Mn2+ is required for enzymatic activity, while Mg2+ is not. We found that ZIKV NS5 can utilize single-stranded DNA but not double-stranded DNA as a template or a primer to synthesize RNA. The assay was used to compare the efficiency of incorporation of analog 5'-triphosphates by the ZIKV polymerase and to calculate their discrimination versus that of natural ribonucleotide triphosphates (rNTPs). The 50% inhibitory concentrations for analog rNTPs were determined in an alternative nonradioactive coupled-enzyme assay. We determined that, in general, 2'-C-methyl- and 2'-C-ethynyl-substituted analog 5'-triphosphates were efficiently incorporated by the ZIKV polymerase and were also efficient chain terminators. Derivatives of these molecules may serve as potential antiviral compounds to be developed to combat ZIKV infection. This report provides the first characterization of ZIKV polymerase and demonstrates the utility of in vitro polymerase assays in the identification of potential ZIKV inhibitors. Copyright © 2017 American Society for Microbiology.

  20. Human parainfluenza virus type 2 polymerase complex recognizes leader promoters of other species belonging to the genus Rubulavirus.

    Science.gov (United States)

    Matsumoto, Yusuke; Ohta, Keisuke; Nishio, Machiko

    2017-12-01

    Leader sequence, located at the 3' terminus of paramyxovirus genomes, determines the degree of viral transcription and replication. The essential nucleotides in the leader sequence that influence viral propagation, however, have not been investigated in detail. In the present study, we show that polymerase complex of human parainfluenza virus type 2 (hPIV2) uses a luciferase-encoding hPIV2 mini-genome possessing the leader sequence from other closely related viruses as a template. Furthermore, we demonstrate that although hPIV2 polymerase complex can recognize the leader sequence of hPIV4B, mumps virus (MuV) and PIV5 as well as Newcastle disease virus (NDV), it cannot recognize measles virus, hPIV1, Sendai virus (SeV) or hPIV3. We could obtain the chimeric hPIV2 possessing the leader sequence from hPIV4B, MuV and PIV5, but not from other species, including NDV and SeV. These results reveal that although hPIV2 polymerase complex can recognize the leader sequence from rubulaviruses to achieve efficient viral infection, this does not apply to viruses belonging to other genus. A comparison of leader sequence nucleotides among paramyxoviruses highlights the importance of the conservation in the first 13 nucleotides for infectious hPIV2 growth.

  1. An emerging avian influenza A virus H5N7 is a genetic reassortant of highly pathogenic genes

    DEFF Research Database (Denmark)

    Bragstad, K.; Jørgensen, Poul Henrik; Handberg, Kurt

    2006-01-01

    We full genome characterised the newly discovered avian influenza virus H5N7 subtype combination isolated from a stock of Danish game ducks to investigate the composition of the genome and possible features of high pathogenicity. It was found that the haemagglutinin and the acidic polymerase genes...... low pathogenic avian influenza A viruses. (c) 2006 Elsevier Ltd. All rights reserved....

  2. (FMO3) gene by polymerase chain reaction-restriction fragment ...

    African Journals Online (AJOL)

    Yomi

    2012-01-24

    Jan 24, 2012 ... 1National Engineering Laboratory for Animal Breeding, Ministry of Agriculture Key Laboratory of Animal Genetics and. Breeding, College of ... is a gene that is clustered on human chromosome 1. (HSA1) (Shephard et ... The procedure for extraction was modified from that of “Molecular Cloning” (Sambrook.

  3. Isolation, expression and functional analysis of a putative RNA-dependent RNA polymerase gene from maize (Zea mays L.).

    Science.gov (United States)

    He, Junguang; Dong, Zhigang; Jia, Zhiwei; Wang, Jianhua; Wang, Guoying

    2010-02-01

    RNA-dependent RNA polymerases (RdRPs) in plants have been reported to be involved in post-transcriptional gene silencing (PTGS) and antiviral defense. In this report, an RdRP gene from maize (ZmRdRP1) was obtained by rapid amplification of cDNA ends (RACE) and RT-PCR. The mRNA of ZmRdRP1 was composed of 3785 nucleotides, including a 167 nt 5' untranslated region (UTR), a 291 nt 3'UTR and a 3327 nt open reading frame (ORF), which encodes a putative protein of 1108 amino acids with an estimated molecular mass of 126.9 kDa and a predicated isoelectric point (pI) of 8.37. Real-time quantitative RT-PCR analysis showed that ZmRdRP1 was elicited by salicylic acid (SA) treatment, methyl jasmonate (MeJA) treatment and sugarcane mosaic virus (SCMV) infection. We silenced ZmRdRP1 by constitutively expressing an inverted-repeat fragment of ZmRdRP1 (ir-RdRP1) in transgenic maize plants. Further studies revealed that the ir-RdRP1 transgenic plants were more susceptible to SCMV infection than wild type plants. Virus-infected transgenic maize plants developed more serious disease symptoms and accumulated more virus than wild type plants. These findings suggested that ZmRdRP1 was involved in antiviral defense in maize.

  4. RNA polymerase II stalling promotes nucleosome occlusion and pTEFb recruitment to drive immortalization by Epstein-Barr virus.

    Directory of Open Access Journals (Sweden)

    Richard D Palermo

    2011-10-01

    Full Text Available Epstein-Barr virus (EBV immortalizes resting B-cells and is a key etiologic agent in the development of numerous cancers. The essential EBV-encoded protein EBNA 2 activates the viral C promoter (Cp producing a message of ~120 kb that is differentially spliced to encode all EBNAs required for immortalization. We have previously shown that EBNA 2-activated transcription is dependent on the activity of the RNA polymerase II (pol II C-terminal domain (CTD kinase pTEFb (CDK9/cyclin T1. We now demonstrate that Cp, in contrast to two shorter EBNA 2-activated viral genes (LMP 1 and 2A, displays high levels of promoter-proximally stalled pol II despite being constitutively active. Consistent with pol II stalling, we detect considerable pausing complex (NELF/DSIF association with Cp. Significantly, we observe substantial Cp-specific pTEFb recruitment that stimulates high-level pol II CTD serine 2 phosphorylation at distal regions (up to +75 kb, promoting elongation. We reveal that Cp-specific pol II accumulation is directed by DNA sequences unfavourable for nucleosome assembly that increase TBP access and pol II recruitment. Stalled pol II then maintains Cp nucleosome depletion. Our data indicate that pTEFb is recruited to Cp by the bromodomain protein Brd4, with polymerase stalling facilitating stable association of pTEFb. The Brd4 inhibitor JQ1 and the pTEFb inhibitors DRB and Flavopiridol significantly reduce Cp, but not LMP1 transcript production indicating that Brd4 and pTEFb are required for Cp transcription. Taken together our data indicate that pol II stalling at Cp promotes transcription of essential immortalizing genes during EBV infection by (i preventing promoter-proximal nucleosome assembly and ii necessitating the recruitment of pTEFb thereby maintaining serine 2 CTD phosphorylation at distal regions.

  5. Detection Of Caprine Arthritis Encephalitis Virus Gag-Gene By Rt ...

    African Journals Online (AJOL)

    Predominantly, cells of the monocyte-macrophage lineage were specifically infected by the virus as proviral DNA was detected in infected cultures by amplification of a 414 base-pair (bp) fragment of the viral gag-gene by Reverse Transcription- Polymerase Chain Reaction (RT-PCR) technique. The present study revealed ...

  6. Hepatitis B virus DNA polymerase gene polymorphism based ...

    African Journals Online (AJOL)

    2017-09-03

    Sep 3, 2017 ... genotypes in chronic HBV patients from Western India. Yashwant G Chavan1,2, Sharad R Pawar2, Minal Wani1, Amol D Raut2, Rabindra N Misra1,3. 1. Dr. D Y Patil Biotechnology and Bioinformatics Institute, Tathwade, Pune 411033. Dr. D Y Patil Vidyapeeth,. Sant Tukaram Nagar, Pimpri, Pune 411018.

  7. A new family of polymerases related to superfamily A DNA polymerases and T7-like DNA-dependent RNA polymerases

    Directory of Open Access Journals (Sweden)

    Aravind L

    2008-10-01

    Full Text Available Abstract Using sequence profile methods and structural comparisons we characterize a previously unknown family of nucleic acid polymerases in a group of mobile elements from genomes of diverse bacteria, an algal plastid and certain DNA viruses, including the recently reported Sputnik virus. Using contextual information from domain architectures and gene-neighborhoods we present evidence that they are likely to possess both primase and DNA polymerase activity, comparable to the previously reported prim-pol proteins. These newly identified polymerases help in defining the minimal functional core of superfamily A DNA polymerases and related RNA polymerases. Thus, they provide a framework to understand the emergence of both DNA and RNA polymerization activity in this class of enzymes. They also provide evidence that enigmatic DNA viruses, such as Sputnik, might have emerged from mobile elements coding these polymerases. Reviewers This article was reviewed by Eugene Koonin and Mark Ragan.

  8. Development and deployment of a rapid recombinase polymerase amplification Ebola virus detection assay in Guinea in 2015

    OpenAIRE

    Faye, Oumar; Faye, Ousmane; Soropogui, B.; Patel, Pranav; Abd El Wahed, Ahmed; Loucoubar, C.; Fall, G.; Kiory, D.; Magassouba, N.; Keita, S.; Kondé, M. K.; Diallo, A.; Koivogui, L.; Karlberg, H.; Mirazimi, Ali

    2015-01-01

    In the absence of a vaccine or specific treatments for Ebola virus disease (EVD), early identification of cases is crucial for the control of EVD epidemics. We evaluated a new extraction kit (SpeedXtract (SE), Qiagen) on sera and swabs in combination with an improved diagnostic reverse transcription recombinase polymerase amplification assay for the detection of Ebola virus (EBOV-RT-RPA). The performance of combined extraction and detection was best for swabs. Sensitivity and specificity of t...

  9. In Silico Analysis of Epitope-Based Vaccine Candidates against Hepatitis B Virus Polymerase Protein.

    Science.gov (United States)

    Zheng, Juzeng; Lin, Xianfan; Wang, Xiuyan; Zheng, Liyu; Lan, Songsong; Jin, Sisi; Ou, Zhanfan; Wu, Jinming

    2017-05-16

    Hepatitis B virus (HBV) infection has persisted as a major public health problem due to the lack of an effective treatment for those chronically infected. Therapeutic vaccination holds promise, and targeting HBV polymerase is pivotal for viral eradication. In this research, a computational approach was employed to predict suitable HBV polymerase targeting multi-peptides for vaccine candidate selection. We then performed in-depth computational analysis to evaluate the predicted epitopes' immunogenicity, conservation, population coverage, and toxicity. Lastly, molecular docking and MHC-peptide complex stabilization assay were utilized to determine the binding energy and affinity of epitopes to the HLA-A0201 molecule. Criteria-based analysis provided four predicted epitopes, RVTGGVFLV, VSIPWTHKV, YMDDVVLGA and HLYSHPIIL. Assay results indicated the lowest binding energy and high affinity to the HLA-A0201 molecule for epitopes VSIPWTHKV and YMDDVVLGA and epitopes RVTGGVFLV and VSIPWTHKV, respectively. Regions 307 to 320 and 377 to 387 were considered to have the highest probability to be involved in B cell epitopes. The T cell and B cell epitopes identified in this study are promising targets for an epitope-focused, peptide-based HBV vaccine, and provide insight into HBV-induced immune response.

  10. Prdm5 Regulates Collagen Gene Transcription by Association with RNA Polymerase II in Developing Bone

    DEFF Research Database (Denmark)

    Galli, Giorgio Giacomo; Honnens de Lichtenberg, Kristian; Carrara, Matteo

    2012-01-01

    and fibrillogenesis by binding inside the Col1a1 gene body and maintaining RNA polymerase II occupancy. In vivo, Prdm5 loss results in delayed ossification involving a pronounced impairment in the assembly of fibrillar collagens. Collectively, our results define a novel role for Prdm5 in sustaining...

  11. Mutations in genes encoding subunits of RNA polymerases I and III cause Treacher Collins syndrome.

    NARCIS (Netherlands)

    Dauwerse, J.G.; Dixon, J.; Seland, S.; Ruivenkamp, C.A.; Haeringen, A. van; Hoefsloot, L.H.; Peters, D.J.; Boers, A.C.; Daumer-Haas, C.; Maiwald, R.; Zweier, C.; Kerr, B.; Cobo, A.M.; Toral, J.F.; Hoogeboom, A.J.M.; Lohmann, D.R.; Hehr, U.; Dixon, M.J.; Breuning, M.H.; Wieczorek, D.

    2011-01-01

    We identified a deletion of a gene encoding a subunit of RNA polymerases I and III, POLR1D, in an individual with Treacher Collins syndrome (TCS). Subsequently, we detected 20 additional heterozygous mutations of POLR1D in 252 individuals with TCS. Furthermore, we discovered mutations in both

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

    Science.gov (United States)

    Ferron, François; Bussetta, Cécile; Dutartre, Hélène; Canard, Bruno

    2005-10-14

    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. 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. 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 polymerases.

  13. 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

  14. Comparative Profiling of Ubiquitin Proteasome System Interplay with Influenza A Virus PB2 Polymerase Protein Recapitulating Virus Evolution in Humans.

    Science.gov (United States)

    Biquand, Elise; Poirson, Juline; Karim, Marwah; Declercq, Marion; Malausse, Nicolas; Cassonnet, Patricia; Barbezange, Cyril; Straub, Marie-Laure; Jones, Louis; Munier, Sandie; Naffakh, Nadia; van der Werf, Sylvie; Jacob, Yves; Masson, Murielle; Demeret, Caroline

    2017-01-01

    The optimized exploitation of cell resources is one cornerstone of a successful infection. Differential mapping of host-pathogen protein-protein interactions (PPIs) on the basis of comparative interactomics of multiple strains is an effective strategy to highlight correlations between host proteome hijacking and biological or pathogenic traits. Here, we developed an interactomic pipeline to deliver high-confidence comparative maps of PPIs between a given pathogen and the human ubiquitin proteasome system (UPS). This subarray of the human proteome represents a range of essential cellular functions and promiscuous targets for many viruses. The screening pipeline was applied to the influenza A virus (IAV) PB2 polymerase proteins of five strains representing different levels of virulence in humans. An extensive PB2-UPS interplay has been detected that recapitulates the evolution of IAVs in humans. Functional validation with several IAV strains, including the seasonal H1N1 pdm09 and H3N2 viruses, confirmed the biological relevance of most identified UPS factors and revealed strain-independent and strain-specific effects of UPS factor invalidation on IAV infection. This strategy is applicable to proteins from any other virus or pathogen, providing a valuable resource with which to explore the UPS-pathogen interplay and its relationship with pathogenicity. IMPORTANCE Influenza A viruses (IAVs) are responsible for mild-to-severe seasonal respiratory illness of public health concern worldwide, and the risk of avian strain outbreaks in humans is a constant threat. Elucidating the requisites of IAV adaptation to humans is thus of prime importance. In this study, we explored how PB2 replication proteins of IAV strains with different levels of virulence in humans hijack a major protein modification pathway of the human host cell, the ubiquitin proteasome system (UPS). We found that the PB2 protein engages in an extended interplay with the UPS that evolved along with the virus

  15. Fluorescent antibody test, quantitative polymerase chain reaction pattern and clinical aspects of rabies virus strains isolated from main reservoirs in Brazil

    Directory of Open Access Journals (Sweden)

    Camila Appolinário

    Full Text Available ABSTRACTRabies virus (RABV isolated from different mammals seems to have unique characteristics that influence the outcome of infection. RABV circulates in nature and is maintained by reservoirs that are responsible for the persistence of the disease for almost 4000 years. Considering the different pattern of pathogenicity of RABV strains in naturally and experimentally infected animals, the aim of this study was to analyze the characteristics of RABV variants isolated from the main Brazilian reservoirs, being related to a dog (variant 2,Desmodus rotundus (variant 3, crab eating fox, marmoset, and Myotis spp. Viral replication in brain tissue of experimentally infected mouse was evaluated by two laboratory techniques and the results were compared to clinical evolution from five RABV variants. The presence of the RABV was investigated in brain samples by fluorescent antibody test (FAT and real time polymerase chain reaction (qRT-PCR for quantification of rabies virus nucleoprotein gene (N gene. Virus replication is not correlated with clinical signs and evolution. The pattern of FAT is associated with RABV replication levels. Virus isolates from crab eating fox and marmoset had a longer evolution period and higher survival rate suggesting that the evolution period may contribute to the outcome. RABV virus variants had independent characteristics that determine the clinical evolution and survival of the infected mice.

  16. Characterization of a nuclear localization signal in the foot-and-mouth disease virus polymerase

    Energy Technology Data Exchange (ETDEWEB)

    Sanchez-Aparicio, Maria Teresa; Rosas, Maria Flora [Centro de Biología Molecular, “Severo Ochoa” (CSIC-UAM), Cantoblanco 28049, Madrid (Spain); Sobrino, Francisco, E-mail: fsobrino@cbm.uam.es [Centro de Biología Molecular, “Severo Ochoa” (CSIC-UAM), Cantoblanco 28049, Madrid (Spain); Centro de Investigación en Sanidad Animal, INIA, Valdeolmos, 28130 Madrid (Spain)

    2013-09-15

    We have experimentally tested whether the MRKTKLAPT sequence in FMDV 3D protein (residues 16 to 24) can act as a nuclear localization signal (NLS). Mutants with substitutions in two basic residues within this sequence, K18E and K20E, were generated. A decreased nuclear localization was observed in transiently expressed 3D and its precursor 3CD, suggesting a role of K18 and K20 in nuclear targeting. Fusion of MRKTKLAPT to the green fluorescence protein (GFP) increased the nuclear localization of GFP, which was not observed when GFP was fused to the 3D mutated sequences. These results indicate that the sequence MRKTKLAPT can be functionally considered as a NLS. When introduced in a FMDV full length RNA replacements K18E and K20E led to production of revertant viruses that replaced the acidic residues introduced (E) by K, suggesting that the presence of lysins at positions 18 and 20 of 3D is essential for virus multiplication. - Highlights: • The FMDV 3D polymerase contains a nuclear localization signal. • Replacements K18E and K20E decrease nuclear localization of 3D and its precursor 3CD. • Fusion of the MRKTKLAPT 3D motif to GFP increases the nuclear localization of GFP. • Replacements K18E and K20E abolish the ability of MRKTKLAPT to relocate GFP. • RNAs harboring replacements K18E and K20E lead to recovery of revertant FMDVs.

  17. A recombinase polymerase amplification assay for rapid detection of Crimean-Congo Haemorrhagic fever Virus infection.

    Directory of Open Access Journals (Sweden)

    Laura C Bonney

    2017-10-01

    Full Text Available Crimean-Congo Haemorrhagic fever Virus (CCHFV is a rapidly emerging vector-borne pathogen and the cause of a virulent haemorrhagic fever affecting large parts of Europe, Africa, the Middle East and Asia.An isothermal recombinase polymerase amplification (RPA assay was successfully developed for molecular detection of CCHFV. The assay showed rapid (under 10 minutes detection of viral extracts/synthetic virus RNA of all 7 S-segment clades of CCHFV, with high target specificity. The assay was shown to tolerate the presence of inhibitors in crude preparations of mock field samples, indicating that this assay may be suitable for use in the field with minimal sample preparation. The CCHFV RPA was successfully used to screen and detect CCHFV positives from a panel of clinical samples from Tajikistan.The assay is a rapid, isothermal, simple-to-perform molecular diagnostic, which can be performed on a light, portable real-time detection device. It is ideally placed therefore for use as a field-diagnostic or in-low resource laboratories, for monitoring of CCHF outbreaks at the point-of-need, such as in remote rural regions in affected countries.

  18. A recombinase polymerase amplification assay for rapid detection of Crimean-Congo Haemorrhagic fever Virus infection.

    Science.gov (United States)

    Bonney, Laura C; Watson, Robert J; Afrough, Babak; Mullojonova, Manija; Dzhuraeva, Viktoriya; Tishkova, Farida; Hewson, Roger

    2017-10-01

    Crimean-Congo Haemorrhagic fever Virus (CCHFV) is a rapidly emerging vector-borne pathogen and the cause of a virulent haemorrhagic fever affecting large parts of Europe, Africa, the Middle East and Asia. An isothermal recombinase polymerase amplification (RPA) assay was successfully developed for molecular detection of CCHFV. The assay showed rapid (under 10 minutes) detection of viral extracts/synthetic virus RNA of all 7 S-segment clades of CCHFV, with high target specificity. The assay was shown to tolerate the presence of inhibitors in crude preparations of mock field samples, indicating that this assay may be suitable for use in the field with minimal sample preparation. The CCHFV RPA was successfully used to screen and detect CCHFV positives from a panel of clinical samples from Tajikistan. The assay is a rapid, isothermal, simple-to-perform molecular diagnostic, which can be performed on a light, portable real-time detection device. It is ideally placed therefore for use as a field-diagnostic or in-low resource laboratories, for monitoring of CCHF outbreaks at the point-of-need, such as in remote rural regions in affected countries.

  19. The RNA-dependent RNA polymerase of Citrus tristeza virus forms oligomers.

    Science.gov (United States)

    Cevik, Bayram

    2013-12-01

    The RNA-dependent RNA polymerases (RdRp) from Citrus tristeza virus (CTV) were tagged with HA and FLAG epitopes. Differentially tagged proteins were expressed either individually or concomitantly in Escherichia coli. Immunoprecipitation of the expressed proteins with anti-FLAG antibody followed by Western blot with anti-HA antibody demonstrated that molecules of RdRp from CTV interact to form oligomers. Yeast two-hybrid assays showed that molecules of RdRp interact in eukaryotic cells. Co-immunoprecipitation with anti-FLAG antibody of truncated HA-tagged RdRps (RdRpΔ1-166-HA, RdRpΔ1-390-HA, RdRp1-169-HA) co-expressed with full-length RdRp-FLAG showed that only RdRp1-169-HA interacted with the full-length FLAG-RdRp. Yeast two-hybrid assays with truncated RdRp constructs confirmed that the oligomerization site resides in the N-terminal region and that the first 169 aa of CTV RdRp are necessary and sufficient for oligomerization both in bacterial and yeast cells. Development of control strategies targeting viral RdRp oligomer formation may inhibit virus replication and prove useful in control of CTV. © 2013 Elsevier Inc. All rights reserved.

  20. Immunohistochemistry and Polymerase Chain Reaction for Detection Human Papilloma Virus in Warts: A Comparative Study

    Science.gov (United States)

    Lee, Hong Sun; Lee, Ji Hyun; Choo, Ji Yoon; Byun, Hee Jin; Jun, Jin Hyun

    2016-01-01

    Background Immunohistochemistry and polymerase chain reaction (PCR) are the most widely used methods for the detection of viruses. PCR is known to be a more sensitive and specific method than the immunohistochemical method at this time, but PCR has the disadvantages of high cost and skilled work to use widely. With the progress of technology, the immunohistochemical methods used in these days has come to be highly sensitive and actively used in the diagnostic fields. Objective To evaluate and compare the usefulness of immunohistochemistry and PCR for detection human papilloma virus (HPV) in wart lesions. Methods Nine biopsy samples of verruca vulgaris and 10 of condyloma accuminatum were examined. Immunohistochemical staining using monoclonal antibody to HPV L1 capsid protein and PCR were done for the samples. DNA sequencing of the PCR products and HPV genotyping were also done. Results HPV detection rate was 78.9% (88.9% in verruca vulgaris, 70.0% in condyloma accuminatum) on immunohistochemistry and 100.0% for PCR. HPV-6 genotype showed a lower positivity rate on immunohistochemistry (50.0%) as compared to that of the other HPV genotypes. Conclusion Immunohistochemistry for HPV L1 capsid protein showed comparable sensitivity for detection HPV. Considering the high cost and great effort needed for the PCR methods, we can use immunohistochemistry for HPV L1 capsid protein with the advantage of lower cost and simple methods for HPV detection. PMID:27489431

  1. Aire unleashes stalled RNA polymerase to induce ectopic gene expression in thymic epithelial cells.

    Science.gov (United States)

    Giraud, Matthieu; Yoshida, Hideyuki; Abramson, Jakub; Rahl, Peter B; Young, Richard A; Mathis, Diane; Benoist, Christophe

    2012-01-10

    Aire is a transcriptional regulator that induces expression of peripheral tissue antigens (PTA) in thymic medullary epithelial cells (MECs), driving immunological self-tolerance in differentiating T cells. To elucidate its mechanistic pathways, we examined its transcriptional impact in MECs in vivo by microarray analysis with mRNA-spanning probes. This analysis revealed initiation of Aire-activated genes to be comparable in Aire-deficient and wild-type MECs, but with a block to elongation after 50-100 bp in the absence of Aire, suggesting activation by release of stalled polymerases by Aire. In contrast, patterns of activation by transcription factors such as Klf4 were consistent with regulation of initiation. Mapping of Aire and RNA polymerase-II (Pol-II) by ChIP and high-throughput sequencing (ChIP-seq) revealed that Aire bound all Pol-II-rich transcriptional start sites (TSS), irrespective of its eventual effect. However, the genes it preferentially activated were characterized by a relative surfeit of stalled polymerases at the TSS, which resolved once Aire was introduced into cells. Thus, transcript mapping and ChIP-seq data indicate that Aire activates ectopic transcription not through specific recognition of PTA gene promoters but by releasing stalled polymerases.

  2. De novo polymerase activity and oligomerization of hepatitis C virus RNA-dependent RNA-polymerases from genotypes 1 to 5.

    Directory of Open Access Journals (Sweden)

    Pilar Clemente-Casares

    Full Text Available Hepatitis C virus (HCV shows a great geographical diversity reflected in the high number of circulating genotypes and subtypes. The response to HCV treatment is genotype specific, with the predominant genotype 1 showing the lowest rate of sustained virological response. Virally encoded enzymes are candidate targets for intervention. In particular, promising antiviral molecules are being developed to target the viral NS3/4A protease and NS5B polymerase. Most of the studies with the NS5B polymerase have been done with genotypes 1b and 2a, whilst information about other genotypes is scarce. Here, we have characterized the de novo activity of NS5B from genotypes 1 to 5, with emphasis on conditions for optimum activity and kinetic constants. Polymerase cooperativity was determined by calculating the Hill coefficient and oligomerization through a new FRET-based method. The V(max/K(m ratios were statistically different between genotype 1 and the other genotypes (p<0.001, mainly due to differences in V(max values, but differences in the Hill coefficient and NS5B oligomerization were noted. Analysis of sequence changes among the studied polymerases and crystal structures show the αF helix as a structural component probably involved in NS5B-NS5B interactions. The viability of the interaction of αF and αT helixes was confirmed by docking studies and calculation of electrostatic surface potentials for genotype 1 and point mutants corresponding to mutations from different genotypes. Results presented in this study reveal the existence of genotypic differences in NS5B de novo activity and oligomerization. Furthermore, these results allow us to define two regions, one consisting of residues Glu128, Asp129, and Glu248, and the other consisting of residues of αT helix possibly involved in NS5B-NS5B interactions.

  3. In vivo and in vitro expression analysis of the RNA-dependent RNA polymerase of Citrus tristeza virus.

    Science.gov (United States)

    Cevik, B; Lee, R F; Niblett, C L

    2008-01-01

    Expression of the RNA-dependent RNA polymerase (RdRp) of Citrus tristeza virus (CTV) was studied in vivo and in vitro using a polyclonal antiserum raised against the recombinant CTV-RdRp protein. Although a 57-kDa CTV-RdRp was expected to be expressed by a +1 translational frameshift at the carboxyl terminus of a 400-kDa polyprotein, a 50-kDa protein was detected in CTV-infected but not in healthy citrus tissue by Western blot. This suggests that the RdRp was cleaved from the CTV polyprotein. The 50-kDa protein was present in both the cytoplasmic and membrane fractions, but it accumulated mainly in the membrane fraction, where most of the replication-associated proteins of RNA viruses are found. When the expression of a cloned CTV-RdRp gene encoding a 60-kDa fusion protein was studied in vitro in a rabbit reticulocyte lysate system, two smaller proteins of about 50 kDa and 10 kDa were detected in addition to the expected 60-kDa protein. All three proteins were immunoprecipitated with the anti-CTV-RdRp serum, suggesting that the 50-kDa and 10-kDa proteins were fragments of the 60-kDa CTV-RdRp fusion protein. When the expression of the RdRp was analyzed at different times during in vitro translation, the 60-kDa and 50-kDa proteins were detected at all time points, and a small amount of the 10-kDa protein was detected after 30 min of translation. These results suggest that the CTV-RdRp may also be cleaved in vitro in the rabbit reticulocyte lysate.

  4. Detection of stress resistance genes in transgenic maize by multiplex and touchdown polymerase chain reaction

    Directory of Open Access Journals (Sweden)

    Bannikova M. A.

    2015-10-01

    Full Text Available Aim. To develop a methodology for detection of the genes of resistance to the stress factors in transgenic maize by multiplex (mPCR and touchdown polymerase chain reactions. Methods. isolation of total DNA by CTAB method, purification of DNA from RNA and proteins, electrophoresis of total DNA and amplification products in agarose gel, polymerase chain reaction. Results. The protocol of multiplex and touchdown polymerase chain reactions has been developed for simultaneous verification of the quality of total DNA extracted from the studied maize plant samples and detection of the following genes that determine resistance to the stress factors in the transgenic maize and maize transformation events: BT176, MON810, MON88017, DAS1507, DAS59122, MIR604, GA21, NK603 (mPCR, Bt11, MON863, MON89034, T25 (touchdown PCR. The multiplex PCR and touchdown PCR were developed using the reference samples. Conclusions. The proposed protocol of mPCR and touchdown PCR reactions can be used for mass analysis of maize samples to detect the genes of tolerance/resistance to herbicides and genes of resistance to insects reliably, authentically, quickly and cheaply.

  5. Mitochondrial Genes of Dinoflagellates Are Transcribed by a Nuclear-Encoded Single-Subunit RNA Polymerase.

    Directory of Open Access Journals (Sweden)

    Chang Ying Teng

    Full Text Available Dinoflagellates are a large group of algae that contribute significantly to marine productivity and are essential photosynthetic symbionts of corals. Although these algae have fully-functioning mitochondria and chloroplasts, both their organelle genomes have been highly reduced and the genes fragmented and rearranged, with many aberrant transcripts. However, nothing is known about their RNA polymerases. We cloned and sequenced the gene for the nuclear-encoded mitochondrial polymerase (RpoTm of the dinoflagellate Heterocapsa triquetra and showed that the protein presequence targeted a GFP construct into yeast mitochondria. The gene belongs to a small gene family, which includes a variety of 3'-truncated copies that may have originated by retroposition. The catalytic C-terminal domain of the protein shares nine conserved sequence blocks with other single-subunit polymerases and is predicted to have the same fold as the human enzyme. However, the N-terminal (promoter binding/transcription initiation domain is not well-conserved. In conjunction with the degenerate nature of the mitochondrial genome, this suggests a requirement for novel accessory factors to ensure the accurate production of functional mRNAs.

  6. Diagnosis of enzootic pneumonia in Danish cattle: reverse transcription-polymerase chain reaction assay for detection of bovine respiratory syncytial virus in naturally and experimentally infected cattle

    DEFF Research Database (Denmark)

    Larsen, Lars Erik; Tjørnehøj, Kirsten; Viuff, B.

    1999-01-01

    A reverse transcription-polymerase chain reaction (RT-PCR) assay was developed for detection of bovine respiratory syncytial virus (BRSV) in lung tissue of naturally and experimentally infected cattle. Primers were selected from the gene coding the F fusion protein, which is relatively conserved...... among BRSV isolates. The RT-PCR assay was highly specific, it yielded positive reactions only when performed on BRSV-infected cell cultures or tissues. The detection limit of the RT-PCR assay was assessed as 5 TCID50. BRSV was detected in tissues of the respiratory tract and in the tracheobroncheal...... lymph node of calves euthanized 2-8 days after experimental infection with BRSV, whereas samples of other tissues and samples from mock-infected animals were negative at all time points. Examination of lung samples from 8 different regions of the lungs revealed that although the virus was most often...

  7. RNA Polymerase Structure, Function, Regulation, Dynamics, Fidelity, and Roles in GENE EXPRESSION | Center for Cancer Research

    Science.gov (United States)

    Multi-subunit RNA polymerases (RNAP) are ornate molecular machines that translocate on a DNA template as they generate a complementary RNA chain. RNAPs are highly conserved in evolution among eukarya, eubacteria, archaea, and some viruses. As such, multi-subunit RNAPs appear to be an irreplaceable advance in the evolution of complex life on earth. Because of their stepwise movement on DNA, RNAPs are considered to be molecular motors, and because RNAPs catalyze a templated polymerization reaction, they are central to biological information flow.

  8. Common changes in global gene expression induced by RNA polymerase inhibitors in Shigella flexneri.

    Directory of Open Access Journals (Sweden)

    Hua Fu

    Full Text Available Characterization of expression profile of organisms in response to antimicrobials provides important information on the potential mechanism of action of the drugs. The special expression signature can be used to predict whether other drugs act on the same target. Here, the common response of Shigella flexneri to two inhibitors of RNA polymerase was examined using gene expression profiling. Consistent with similar effects of the two drugs, the gene expression profiles indicated that responses of the bacteria to these drugs were roughly the same, with 225 genes affected commonly. Of them, 88 were induced and 137 were repressed. Real-time PCR was performed for selected genes to verify the microarray results. Analysis of the expression data revealed that more than 30% of the plasmid-encoded genes on the array were up-regulated by the antibiotics including virF regulon, other virulence-related genes, and genes responsible for plasmid replication, maintenance, and transfer. In addition, some chromosome-encoded genes involved in virulence and genes acquired from horizontal transfer were also significantly up-regulated. However, the expression of genes encoding the beta-subunit of RNA polymerase was increased moderately. The repressed genes include those that code for products associated with the ribosome, citrate cycle, glycolysis, thiamine biosynthesis, purine metabolism, fructose metabolism, mannose metabolism, and cold shock proteins. This study demonstrates that the two antibiotics induce rapid cessation of RNA synthesis resulting in inhibition of translation components. It also indicates that the production of virulence factors involved in intercellular dissemination, tissue invasion and inflammatory destruction may be enhanced through derepressing horizontal transfer genes by the drugs.

  9. Detection of respiratory viruses by real-time polymerase chain reaction in outpatients with acute respiratory infection

    Directory of Open Access Journals (Sweden)

    Ronaldo Bragança Martins Júnior

    2014-09-01

    Full Text Available Viruses are the major contributors to the morbidity and mortality of upper and lower acute respiratory infections (ARIs for all age groups. The aim of this study was to determine the frequencies for a large range of respiratory viruses using a sensitive molecular detection technique in specimens from outpatients of all ages with ARIs. Nasopharyngeal aspirates were obtained from 162 individuals between August 2007-August 2009. Twenty-three pathogenic respiratory agents, 18 respiratory viruses and five bacteria were investigated using multiplex real-time reverse transcriptase polymerase chain reaction (RT-PCR and indirect immunofluorescence assay (IIF. Through IIF, 33 (20.4% specimens with respiratory virus were recognised, with influenza virus representing over half of the positive samples. Through a multiplex real-time RT-PCR assay, 88 (54.3% positive samples were detected; the most prevalent respiratory viral pathogens were influenza, human rhinovirus and respiratory syncytial virus (RSV. Six cases of viral co-detection were observed, mainly involving RSV. The use of multiplex real-time RT-PCR increased the viral detection by 33.9% and revealed a larger number of respiratory viruses implicated in ARI cases, including the most recently described respiratory viruses [human bocavirus, human metapneumovirus, influenza A (H1N1 pdm09 virus, human coronavirus (HCoV NL63 and HCoV HKU1].

  10. Mutations to A/Puerto Rico/8/34 PB1 gene improves seasonal reassortant influenza A virus growth kinetics.

    Science.gov (United States)

    Plant, Ewan P; Liu, Teresa M; Xie, Hang; Ye, Zhiping

    2012-12-17

    It is desirable for influenza vaccine virus strains to have phenotypes that include good growth and hemagglutinin (HA) protein yield. The quality of these characteristics varies among the vaccine viruses and is usually due to multigenic effects. Many influenza A virus vaccine viruses are made as reassortants of the high yield virus A/Puerto Rico/8/34 (PR/8) and a circulating seasonal virus. Co-infection of eggs with the two viruses, and selection of reassortants with the HA and neuraminidase (NA) segments from the seasonal virus, can result in viruses that contain a mixture of internal genes derived from both the high yield virus and the circulating virus. Segment 2 (PB1), which encodes the RNA-dependent RNA polymerase, frequently cosegregates with the seasonal HA and NA segments. We asked whether mutations based on the seasonal PB1 genes could improve vaccine virus strains. Here we report that mutations to the PR/8 PB1 gene, based on differences observed between seasonal and PR/8 PB1 genes, accelerate egg and cell culture based replication for a reassortant virus containing HA and NA segments from the low yield A/Wyoming/03/2003 (H3N2) vaccine virus. Published by Elsevier Ltd.

  11. Ethanolic Extract of Melia Fructus Has Anti-influenza A Virus Activity by Affecting Viral Entry and Viral RNA Polymerase.

    Science.gov (United States)

    Jin, Young-Hee; Choi, Jang-Gi; Cho, Won-Kyung; Ma, Jin Yeul

    2017-01-01

    Meliae Fructus (MF) is the dried ripe fruit of Melia toosendan Siebold et Zuccarini, Meliaceae family. MF is widely used in traditional medicine to treat inflammation and helminthic infection and has anti-bacterial, anti-oxidant, anti-cancer, anti-inflammatory, and analgesic activities. However, potential anti-influenza properties of MF have yet to be investigated. We determined whether an ethanolic extract of MF (EMF) has anti-viral activity via an EMF pre-, co-, and post-treatment assay, using the Influenza A/PR/8/34 and H3N2 virus on Madin-Darby canine kidney (MDCK) cells. The EMF had anti-influenza virus activity in pre- and co-treated cells in a dose-dependent manner, but not in post-treated cell. EMF inhibited the activity of hemagglutinin (HA) and neuraminidase (NA) of influenza virus. EMF inhibited viral HA, nucleoprotein (NP), matrix protein 2 (M2), non-structural protein 1 (NS1), polymerase acidic protein (PA), polymerase basic protein 1 (PB1), and polymerase basic protein 2 (PB2) mRNA synthesis at 5 h post infection (hpi), however, the levels of PA, PB1, and PB2 mRNA were increased in pre- and co-EMF treated cells compared with control virus-infected and EMF post-treated cells at 18 hpi. The level of M2 protein expression was also decreased upon pre- and co-treatment with EMF. The PA protein was accumulated and localized in not only the nucleus but also the cytoplasm of virus-infected MDCK cells at 18 hpi. Pre-EMF treatment inhibited the expression of pAKT, which is induced by influenza virus infection, at the stage of virus entry. We also found that treatment of EMF up-regulated the antiviral protein Mx1, which may play a partial role in inhibiting influenza virus infection in pre- and co-EMF treated MDCK cells. In summary, these results strongly suggested that an ethanolic extract of Meliae Fructus inhibited influenza A virus infection by affecting viral entry, PA proteins of the RNA polymerase complex, and Mx1 induction and may be a potential and

  12. Ethanolic Extract of Melia Fructus Has Anti-influenza A Virus Activity by Affecting Viral Entry and Viral RNA Polymerase

    Science.gov (United States)

    Jin, Young-Hee; Choi, Jang-Gi; Cho, Won-Kyung; Ma, Jin Yeul

    2017-01-01

    Meliae Fructus (MF) is the dried ripe fruit of Melia toosendan Siebold et Zuccarini, Meliaceae family. MF is widely used in traditional medicine to treat inflammation and helminthic infection and has anti-bacterial, anti-oxidant, anti-cancer, anti-inflammatory, and analgesic activities. However, potential anti-influenza properties of MF have yet to be investigated. We determined whether an ethanolic extract of MF (EMF) has anti-viral activity via an EMF pre-, co-, and post-treatment assay, using the Influenza A/PR/8/34 and H3N2 virus on Madin-Darby canine kidney (MDCK) cells. The EMF had anti-influenza virus activity in pre- and co-treated cells in a dose-dependent manner, but not in post-treated cell. EMF inhibited the activity of hemagglutinin (HA) and neuraminidase (NA) of influenza virus. EMF inhibited viral HA, nucleoprotein (NP), matrix protein 2 (M2), non-structural protein 1 (NS1), polymerase acidic protein (PA), polymerase basic protein 1 (PB1), and polymerase basic protein 2 (PB2) mRNA synthesis at 5 h post infection (hpi), however, the levels of PA, PB1, and PB2 mRNA were increased in pre- and co-EMF treated cells compared with control virus-infected and EMF post-treated cells at 18 hpi. The level of M2 protein expression was also decreased upon pre- and co-treatment with EMF. The PA protein was accumulated and localized in not only the nucleus but also the cytoplasm of virus-infected MDCK cells at 18 hpi. Pre-EMF treatment inhibited the expression of pAKT, which is induced by influenza virus infection, at the stage of virus entry. We also found that treatment of EMF up-regulated the antiviral protein Mx1, which may play a partial role in inhibiting influenza virus infection in pre- and co-EMF treated MDCK cells. In summary, these results strongly suggested that an ethanolic extract of Meliae Fructus inhibited influenza A virus infection by affecting viral entry, PA proteins of the RNA polymerase complex, and Mx1 induction and may be a potential and

  13. The RNA-dependent-RNA polymerase, an emerging antiviral drug target for the Hendra virus.

    Science.gov (United States)

    Velkov, Tony; Carbone, Vincenzo; Akter, Jesmin; Sivanesan, Sivashangarie; Li, Jian; Beddoe, Travis; Marsh, Glenn A

    2014-01-01

    Australia is facing a major national medical challenge with the emergence of the Hendra virus (HeV) as a medically and economically important pathogen of humans and animals. Clinical symptoms of human HeV infection can include fever, hypotension, dizziness, encephalitis, respiratory haemorrhage and edema. The window of opportunity for successful patient treatment remains unknown, but is likely to be very narrow. Currently, very few effective therapeutic options are available for the case management of severe HeV infections or the rapid silencing of local outbreaks. This underscores the need for more activity in the drug discovery arena to develop much needed therapeutics that specifically targets this deadly disease. The structural analysis of HeV is very much in its infancy, which leaves many gaps in our understanding of the biology of HeV and makes structure-guided drug design difficult. Structural studies of the viral RNAdependent- RNA polymerase (RdRp), which is the heart of the viral replication machinery, will set the stage for rational drug design and fill a major gap in our understanding of the HeV replication machinery. This review examines the current knowledge based on the multi-domain architecture of the Hendra RdRp and highlights which essential domain functions represent tangible targets for drug development against this deadly disease.

  14. Analysis ulcerative colitis for presence Epstein-Barr virus DNA sequences by polymerase chain reaction technique

    Directory of Open Access Journals (Sweden)

    Sahar Mehrabani khasraghi

    2016-02-01

    Full Text Available Introduction: Ulcerative colitis (UC is one type of inflammatory bowel disease (IBD. The purpose of this study is to explore the prevalence of Epstein–Barr virus (EBV in UC patients in comparison with healthy subjects using the polymerase chain reaction (PCR method. Methods: In this case-control study, five biopsies of patients with UC and 30 healthy people as controls were selected. Sampling was performed by endoscopic biopsy operation. After DNA extraction, PCR was used to determine EBV genome by specific primers. Statistical analysis was performed using the chi-square test. Results: The results of PCR indicated that EBV genome was detected in 60.0% of samples in the case group, and 36.7% of samples in the control group were positive for EBV. Thus, no significant association was observed between the prevalence of EBV and incidence of UC in comparison with the control group (P = 0.36. Conclusion: The findings presented herein demonstrate no direct molecular evidence to support an association of EBV with UC. These results, do not exclude the possibility oncogenic role of EBV to infect the different colon cell.

  15. Detection of Citrus leprosis virus C using specific primers and TaqMan probe in one-step real-time reverse-transcription polymerase chain reaction assays.

    Science.gov (United States)

    Choudhary, Nandlal; Wei, G; Govindarajulu, A; Roy, Avijit; Li, Wenbin; Picton, Deric D; Nakhla, M K; Levy, L; Brlansky, R H

    2015-11-01

    Citrus leprosis virus C (CiLV-C), a causal agent of the leprosis disease in citrus, is mostly present in the South and Central America and spreading toward the North America. To enable better diagnosis and inhibit the further spread of this re-emerging virus a quantitative (q) real-time reverse transcription polymerase chain reaction (qRT-PCR) assay is needed for early detection of CiLV-C when the virus is present in low titer in citrus leprosis samples. Using the genomic sequence of CiLV-C, specific primers and probe were designed and synthesized to amplify a 73 nt amplicon from the movement protein (MP) gene. A standard curve of the 73 nt amplicon MP gene was developed using known 10(10)-10(1) copies of in vitro synthesized RNA transcript to estimate the copy number of RNA transcript in the citrus leprosis samples. The one-step qRT-PCR detection assays for CiLV-C were determined to be 1000 times more sensitive when compared to the one-step conventional reverse transcription polymerase chain reaction (RT-PCR) CiLV-C detection method. To evaluate the quality of the total RNA extracts, NADH dehydrogenase gene specific primers (nad5) and probe were included in reactions as an internal control. The one-step qRT-PCR specificity was successfully validated by testing for the presence of CiLV-C in the total RNA extracts of the citrus leprosis samples collected from Belize, Costa Rica, Mexico and Panama. Implementation of the one-step qRT-PCR assays for CiLV-C diagnosis should assist regulatory agencies in surveillance activities to monitor the distribution pattern of CiLV-C in countries where it is present and to prevent further dissemination into citrus growing countries where there is no report of CiLV-C presence. Published by Elsevier B.V.

  16. Transcriptional interference by RNA polymerase III affects expression of the Polr3e gene.

    Science.gov (United States)

    Yeganeh, Meghdad; Praz, Viviane; Cousin, Pascal; Hernandez, Nouria

    2017-02-15

    Overlapping gene arrangements can potentially contribute to gene expression regulation. A mammalian interspersed repeat (MIR) nested in antisense orientation within the first intron of the Polr3e gene, encoding an RNA polymerase III (Pol III) subunit, is conserved in mammals and highly occupied by Pol III. Using a fluorescence assay, CRISPR/Cas9-mediated deletion of the MIR in mouse embryonic stem cells, and chromatin immunoprecipitation assays, we show that the MIR affects Polr3e expression through transcriptional interference. Our study reveals a mechanism by which a Pol II gene can be regulated at the transcription elongation level by transcription of an embedded antisense Pol III gene. © 2017 Yeganeh et al.; Published by Cold Spring Harbor Laboratory Press.

  17. 3D-QSAR and molecular docking studies on designing inhibitors of the hepatitis C virus NS5B polymerase

    Science.gov (United States)

    Li, Wenlian; Si, Hongzong; Li, Yang; Ge, Cuizhu; Song, Fucheng; Ma, Xiuting; Duan, Yunbo; Zhai, Honglin

    2016-08-01

    Viral hepatitis C infection is one of the main causes of the hepatitis after blood transfusion and hepatitis C virus (HCV) infection is a global health threat. The HCV NS5B polymerase, an RNA dependent RNA polymerase (RdRp) and an essential role in the replication of the virus, has no functional equivalent in mammalian cells. So the research and development of efficient NS5B polymerase inhibitors provides a great strategy for antiviral therapy against HCV. A combined three-dimensional quantitative structure-activity relationship (QSAR) modeling was accomplished to profoundly understand the structure-activity correlation of a train of indole-based inhibitors of the HCV NS5B polymerase to against HCV. A comparative molecular similarity indices analysis (COMSIA) model as the foundation of the maximum common substructure alignment was developed. The optimum model exhibited statistically significant results: the cross-validated correlation coefficient q2 was 0.627 and non-cross-validated r2 value was 0.943. In addition, the results of internal validations of bootstrapping and Y-randomization confirmed the rationality and good predictive ability of the model, as well as external validation (the external predictive correlation coefficient rext2 = 0.629). The information obtained from the COMSIA contour maps enables the interpretation of their structure-activity relationship. Furthermore, the molecular docking study of the compounds for 3TYV as the protein target revealed important interactions between active compounds and amino acids, and several new potential inhibitors with higher activity predicted were designed basis on our analyses and supported by the simulation of molecular docking. Meanwhile, the OSIRIS Property Explorer was introduced to help select more satisfactory compounds. The satisfactory results from this study may lay a reliable theoretical base for drug development of hepatitis C virus NS5B polymerase inhibitors.

  18. Analysis of Respiratory Syncytial Virus in Clinical Samples by Reverse Transcriptase-Polymerase Chain Reaction Restriction Mapping

    OpenAIRE

    Valdivia, Angel; Savón, Clara; Chacón, Danay; Sarmiento, Luis; Morier, Luis; Otero, Anselmo; Soto, Yudira; Oropesa, Suset; Goyenechea, Angel

    1997-01-01

    The aim of this study was to develop a polymerase chain reaction (PCR) for the detection of respiratory syncytial virus (RSV) genomes. The primers were designed from published sequences and selected from conserved regions of the genome encoding for the N protein of subgroups A and B of RSV. PCR was applied to 20 specimens from children admitted to the respiratory ward of "William Soler" Pediatric Hospital in Havana City with a clinical diagnosis of bronchiolitis. The PCR was compared with vir...

  19. 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

  20. Endosymbiont gene functions impaired and rescued by polymerase infidelity at poly(A) tracts

    Science.gov (United States)

    Tamas, Ivica; Wernegreen, Jennifer J.; Nystedt, Björn; Kauppinen, Seth N.; Darby, Alistair C.; Gomez-Valero, Laura; Lundin, Daniel; Poole, Anthony M.; Andersson, Siv G. E.

    2008-01-01

    Among host-dependent bacteria that have evolved by extreme reductive genome evolution, long-term bacterial endosymbionts of insects have the smallest (160–790 kb) and most A + T-rich (>70%) bacterial genomes known to date. These genomes are riddled with poly(A) tracts, and 5–50% of genes contain tracts of 10 As or more. Here, we demonstrate transcriptional slippage at poly(A) tracts within genes of Buchnera aphidicola associated with aphids and Blochmannia pennsylvanicus associated with ants. Several tracts contain single frameshift deletions; these apparent pseudogenes showed patterns of constraint consistent with purifying selection on the encoded proteins. Transcriptional slippage yielded a heterogeneous population of transcripts with variable numbers of As in the tract. Across several frameshifted genes, including B. aphidicola cell wall biosynthesis genes and a B. pennsylvanicus histidine biosynthesis gene, 12–50% of transcripts contained corrected reading frames that could potentially yield full-length proteins. In situ immunostaining confirmed the production of the cell wall biosynthetic enzyme UDP-N-acetylmuramyl pentapeptide synthase encoded by the frameshifted murF gene. Simulation studies indicated an overrepresentation of poly(A) tracts in endosymbiont genomes relative to other A + T-rich bacterial genomes. Polymerase infidelity at poly(A) tracts rescues the functionality of genes with frameshift mutations and, conversely, reduces the efficiency of expression for in-frame genes carrying poly(A) regions. These features of homopolymeric tracts could be exploited to manipulate gene expression in small synthetic genomes. PMID:18815381

  1. Development of Plant Expression Vector with Taq DNA Polymerase Gene to Yield Heat-tolerant Maize Lines

    National Research Council Canada - National Science Library

    Ya-Jun Zhang; Ling-Yan Zhou; Yu-Xian Bai; Wen-Bin Yi; Kun Cu; Xiaoxiong Nie; Xue-Lian Liang

    2017-01-01

    In this study, we have focused on using Taq DNA polymerase gene to develop heat-tolerant maize transgenic lines, which would aid in controlling crop loss due to heat stress and potentially increase the economic value...

  2. The Escherichia coli gapA gene is transcribed by the vegetative RNA polymerase holoenzyme E sigma 70 and by the heat shock RNA polymerase E sigma 32.

    Science.gov (United States)

    Charpentier, B; Branlant, C

    1994-02-01

    Escherichia coli D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is produced by the gapA gene and is structurally related to eukaryotic GAPDHs. These facts led to the proposal that the gapA gene originated by a horizontal transfer of genetic information. The yields and start sites of gapA mRNAs produced in various fermentation conditions and genetic contexts were analyzed by primer extension. The transcriptional regulatory region of the gapA gene was found to contain four promoter sequences, three recognized by the vegetative RNA polymerase E sigma 70 and one recognized by the heat shock RNA polymerase E sigma 32. Transcription of gapA by E sigma 32 is activated in the logarithmic phase under conditions of starvation and of heat shock. Using a GAPDH- strain, we found that GAPDH production has a positive effect on cell growth at 43 degrees C. Thus, E. coli GAPDH displays some features of heat shock proteins. One of the gapA promoter sequences transcribed by E sigma 70 is subject to catabolic repression. Another one has growth phase-dependent efficiency. This complex area of differentially regulated promoters allows the production of large amounts of gapA transcripts in a wide variety of environmental conditions. On the basis of these data, the present view of E sigma 32 RNA polymerase function has to be enlarged, and the various hypotheses on E. coli gapA gene origin have to be reexamined.

  3. The Translesion Polymerase Pol η Is Required for Efficient Epstein-Barr Virus Infectivity and Is Regulated by the Viral Deubiquitinating Enzyme BPLF1.

    Science.gov (United States)

    Dyson, Ossie F; Pagano, Joseph S; Whitehurst, Christopher B

    2017-10-01

    Epstein-Barr virus (EBV) infection and lytic replication are known to induce a cellular DNA damage response. We previously showed that the virally encoded BPLF1 protein interacts with and regulates several members of the translesion synthesis (TLS) pathway, a DNA damage tolerance pathway, and that these cellular factors enhance viral infectivity. BPLF1 is a late lytic cycle gene, but the protein is also packaged in the viral tegument, indicating that BPLF1 may function both early and late during infection. The BPLF1 protein expresses deubiquitinating activity that is strictly conserved across the Herpesviridae ; mutation of the active site cysteine results in a loss of enzymatic activity. Infection with an EBV BPLF1 knockout virus results in decreased EBV infectivity. Polymerase eta (Pol η), a specialized DNA repair polymerase, functions in TLS and allows for DNA replication complexes to bypass lesions in DNA. Here we report that BPLF1 interacts with Pol η and that Pol η protein levels are increased in the presence of functional BPLF1. BPLF1 promotes a nuclear relocalization of Pol η molecules which are focus-like in appearance, consistent with the localization observed when Pol η is recruited to sites of DNA damage. Knockdown of Pol η resulted in decreased production of infectious virus, and further, Pol η was found to bind to EBV DNA, suggesting that it may allow for bypass of damaged viral DNA during its replication. The results suggest a mechanism by which EBV recruits cellular repair factors, such as Pol η, to sites of viral DNA damage via BPLF1, thereby allowing for efficient viral DNA replication. IMPORTANCE Epstein-Barr virus is the causative agent of infectious mononucleosis and infects approximately 90% of the world's population. It causes lymphomas in individuals with acquired and innate immune disorders and is strongly associated with Hodgkin's lymphoma, Burkitt's lymphoma, diffuse large B-cell lymphomas, nasopharyngeal carcinoma (NPC), and

  4. Inhibition of viral RNA polymerases by nucleoside and nucleotide analogs: therapeutic applications against positive-strand RNA viruses beyond hepatitis C virus.

    Science.gov (United States)

    Deval, Jerome; Symons, Julian A; Beigelman, Leo

    2014-12-01

    A number of important human infections are caused by positive-strand RNA viruses, yet almost none can be treated with small molecule antiviral therapeutics. One exception is the chronic infection caused by hepatitis C virus (HCV), against which new generations of potent inhibitors are being developed. One of the main molecular targets for anti-HCV drugs is the viral RNA-dependent RNA polymerase, NS5B. This review summarizes the search for nucleoside and nucleotide analogs that inhibit HCV NS5B, which led to the FDA approval of sofosbuvir in 2013. Advances in anti-HCV therapeutics have also stimulated efforts to develop nucleoside analogs against other positive-strand RNA viruses. Although it remains to be validated in the clinic, the prospect of using nucleoside analogs to treat acute infections caused by RNA viruses represents an important paradigm shift and a new frontier for future antiviral therapies. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. Validation of reference genes for quantifying changes in gene expression in virus-infected tobacco.

    Science.gov (United States)

    Baek, Eseul; Yoon, Ju-Yeon; Palukaitis, Peter

    2017-10-01

    To facilitate quantification of gene expression changes in virus-infected tobacco plants, eight housekeeping genes were evaluated for their stability of expression during infection by one of three systemically-infecting viruses (cucumber mosaic virus, potato virus X, potato virus Y) or a hypersensitive-response-inducing virus (tobacco mosaic virus; TMV) limited to the inoculated leaf. Five reference-gene validation programs were used to establish the order of the most stable genes for the systemically-infecting viruses as ribosomal protein L25 > β-Tubulin > Actin, and the least stable genes Ubiquitin-conjugating enzyme (UCE) genes were EF1α > Cysteine protease > Actin, and the least stable genes were GAPDH genes, three defense responsive genes were examined to compare their relative changes in gene expression caused by each virus. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Identification of Leptospira serovars by RFLP of the RNA polymerase beta subunit gene (rpoB).

    Science.gov (United States)

    Jung, Lenice Roteia Cardoso; Bomfim, Maria Rosa Quaresma; Kroon, Erna Geessien; Nunes, Álvaro Cantini

    2015-06-01

    Leptospires are usually classified by methods based on DNA-DNA hybridization and the conventional cross-agglutination absorption test, which uses polyclonal antibodies against lipopolysaccharides. In this study, the amplification of the rpoB gene, which encodes the beta-subunit of RNA polymerase, was used as an alternative tool to identify Leptospira. DNA extracts from sixty-eight serovars were obtained, and the hypervariable region located between 1990 and 2500-bp in the rpoB gene was amplified by polymerase chain reaction (PCR). The 600-bp amplicons of the rpoB gene were digested with the restriction endonucleases TaqI, Tru1I, Sau3AI and MslI, and the restriction fragments were separated by 6% polyacrylamide gel electrophoresis. Thirty-five fragment patters were obtained from the combined data of restriction fragment length polymorphism (PCR-RFLP) analysis and used to infer the phylogenetic relationships among the Leptospira species and serovars. The species assignments obtained were in full agreement with the established taxonomic classifications. Twenty-two serovars were effectively identified based on differences in their molecular profiles. However, the other 46 serovars remained clustered in groups that included more than one serovar of different species. This study demonstrates the value of RFLP analysis of PCR-amplified rpoB as an initial method for identifying Leptospira species and serovars.

  7. Identification, molecular cloning and expression analysis of five RNA-dependent RNA polymerase genes in Salvia miltiorrhiza.

    Directory of Open Access Journals (Sweden)

    Fenjuan Shao

    Full Text Available RNA-dependent RNA polymerases (RDRs act as key components of the small RNA biogenesis pathways and play significant roles in post-transcriptional gene silencing (PTGS and antiviral defense. However, there is no information about the RDR gene family in Salvia miltiorrhiza, an emerging model medicinal plant with great economic value. Through genome-wide predication and subsequent molecular cloning, five full-length S. miltiorrhiza RDR genes, termed SmRDR1-SmRDR5, were identified. The length of SmRDR cDNAs varies between 3,262 (SmRDR5 and 4,130 bp (SmRDR3. The intron number of SmRDR genes varies from 3 (SmRDR1, SmRDR3 and SmRDR4 to 17 (SmRDR5. All of the deduced SmRDR protein sequences contain the conserved RdRp domain. Moreover, SmRDR2 and SmRDR4 have an additional RRM domain. Based on the phylogenetic tree constructed with sixteen RDRs from Arabidopsis, rice and S. miltiorrhiza, plant RDRs may be divided into four groups (RDR1-RDR4. The RDR1 group contains an AtRDR and an OsRDR, while includes two SmRDRs. On the contrary, the RDR3 group contains three AtRDRs and two OsRDRs, but has only one SmRDR. SmRDRs were differentially expressed in flowers, leaves, stems and roots of S. miltiorrhiza and responsive to methyl jasmonate treatment and cucumber mosaic virus infection. The results suggest the involvement of RDRs in S. miltiorrhiza development and response to abiotic and biotic stresses. It provides a foundation for further studying the regulation and biological functions of SmRDRs and the biogenesis pathways of small RNAs in S. miltiorrhiza.

  8. Report on waterborne diseases: The polymerase chain reaction for the identification of enteric viruses in water; Rapporto sulle malattie infettive di origine idricamerizzazione a catena per l`identificazione dei virus enterici nell`acqua

    Energy Technology Data Exchange (ETDEWEB)

    Muscillo, M.; La Rosa, G. [Istituto Superiore di Sanita, Rome (Italy). Lab. di Igiene Ambientale

    1995-12-01

    A variety of human infectious diseases are associated with the pollution of water by enteric viruses. The epidemiological data on cases associated with drinking and recreational water show Norwalk, hepatitis A and E viruses, rotavirus and enteroviruses as the etiological agents. The polymerase chain reaction (PCR) is certainly the most reliable technique available for the rapid identification of these viruses in water samples.

  9. Analysis of Colorectal Cancer and Polyps for the Presence of Herpes Simplex Virus and Epstein - Barr virus DNA Sequences by Polymerase Chain Reaction

    Directory of Open Access Journals (Sweden)

    Sahar Mehrabani-Khasraghi

    2015-10-01

    Full Text Available Background: Colorectal cancer is one of the most common malignancies worldwide with more than one million new cases diagnosed each year. The aim of this study is to investigate the prevalence of herpes simplex virus and Epstein-Barr virus in patients with colorectal carcinomas and polyps in comparison with healthy subjects by using the polymerase chain reaction technique. Methods: In this analytical case-control study, we selected 15 patients with colorectal cancer, 20 patients with colorectal polyps and 35 patients without malignancy as controls. Biopsy specimens were frozen under sterile conditions at -20ºC. After DNA extraction, analysis of polymerase chain reaction to detect herpes simplex virus and Epstein-Barr virus DNA in tissue samples was performed. Statistical analysis was performed with the χ2 test. Results: We observed herpes simplex DNA in 33.3% of tumor samples (5 of 15 and 20% from the non-malignant control group (7 of 35. There was no herpes simplex DNA in the polyp tissues (0 of 20. Epstein-Barr DNA was found in 60% of tumor samples (9 of 15, 35% of polyp samples (7 of 20, and 40% of the non-malignant control group (14 of 35. Statistical analysis showed no significant association between the prevalence of herpes simplex and Epstein- Barr viruses and the incidence of colorectal cancer and polyps compared with the control group. Conclusion: The results demonstrate a lack of direct molecular evidence to support an association between herpes simplex and Epstein-Barr viruses with human colorectal malignancies. These results do not exclude a possible oncogenic role of these viruses to infect different colon cells.

  10. Bacterial Suppression of RNA Polymerase II-Dependent Host Gene Expression

    Directory of Open Access Journals (Sweden)

    Ines Ambite

    2016-07-01

    Full Text Available Asymptomatic bacteriuria (ABU is a bacterial carrier state in the urinary tract that resembles commensalism at other mucosal sites. ABU strains often lack the virulence factors that characterize uropathogenic Escherichia coli (E. coli strains and therefore elicit weak innate immune responses in the urinary tract. In addition, ABU strains are active modifiers of the host environment, which they influence by suppressing RNA polymerase II (Pol II-dependent host gene expression. In patients inoculated with the ABU strain E. coli 83972, gene expression was markedly reduced after 24 h (>60% of all regulated genes. Specific repressors and activators of Pol II-dependent transcription were modified, and Pol II Serine 2 phosphorylation was significantly inhibited, indicating reduced activity of the polymerase. This active inhibition included disease–associated innate immune response pathways, defined by TLR4, IRF-3 and IRF-7, suggesting that ABU strains persist in human hosts by active suppression of the antibacterial defense. In a search for the mechanism of inhibition, we compared the whole genome sequences of E. coli 83972 and the uropathogenic strain E. coli CFT073. In addition to the known loss of virulence genes, we observed that the ABU strain has acquired several phages and identified the lytic Prophage 3 as a candidate Pol II inhibitor. Intact phage particles were released by ABU during in vitro growth in human urine. To address if Prophage 3 affects Pol II activity, we constructed a Prophage 3 negative deletion mutant in E. coli 83972 and compared the effect on Pol II phosphorylation between the mutant and the E. coli 83972 wild type (WT strains. No difference was detected, suggesting that the Pol II inhibitor is not encoded by the phage. The review summarizes the evidence that the ABU strain E. coli 83972 modifies host gene expression by inhibition of Pol II phosphorylation, and discusses the ability of ABU strains to actively create an

  11. A novel multiplex polymerase chain reaction assay for profile analyses of gene expression in peripheral blood

    Directory of Open Access Journals (Sweden)

    Jia Xingwang

    2012-07-01

    Full Text Available Abstract Background Studies have demonstrated that inflammation has a key role in the pathogenesis of atherosclerosis due to the abnormal gene expressions of multiple cytokines. We established an accurate and precise method to observe gene expression in whole blood that might provide specific diagnostic information for coronary artery disease (CAD and other related diseases. Methods The fifteen selected CAD-related genes (IL1B, IL6, IL8, IFNG, MCP-1, VWF, MTHFR, SELL, TNFalpha, ubiquitin, MCSF, ICAM1, ID2, HMOX1 and LDLR and two housekeeping genes (ACTB and GK as internal references have been measured simultaneously with a newly developed multiplex polymerase chain reaction (multi-PCR method. Moreover, the precision was evaluated, and a procedure for distinguishing patients from the normal population has been developed based upon analyses of peripheral blood. A total of 148 subjects were divided into group A (control group without plaques, group B (calcified plaques and group C (non-calcified plaques, and combination group according dual-source CT criteria. Gene expression in blood was analyzed by multi-PCR, and levels of glucose and lipids measured in 50 subjects to explore the relationship among them. Results The precision results of the multi-PCR system revealed within-run and between-run CV values of 3.695–12.537% and 4.405–13.405%, respectively. The profiles of cytokine gene expression in peripheral blood were set: a positive correlation between glucose and MCSF, HMOX1 or TNFalpha were found. We also found that triglyceride levels were negatively correlated with SELL gene expression in 50 subjects. Compared with controls, gene expression levels of IL1B, IL6, IL8 and MCP-1 increased significantly in group C. Conclusions A new multiple gene expression analysis system has been developed. The primary data suggested that gene expression was related to CAD. This system might be used for risk assessment of CVDs and other related diseases.

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

    Directory of Open Access Journals (Sweden)

    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

  13. Evolution of eukaryotic single-stranded DNA viruses of the Bidnaviridae family from genes of four other groups of widely different viruses

    Science.gov (United States)

    Krupovic, Mart; Koonin, Eugene V.

    2014-06-01

    Single-stranded (ss)DNA viruses are extremely widespread, infect diverse hosts from all three domains of life and include important pathogens. Most ssDNA viruses possess small genomes that replicate by the rolling-circle-like mechanism initiated by a distinct virus-encoded endonuclease. However, viruses of the family Bidnaviridae, instead of the endonuclease, encode a protein-primed type B DNA polymerase (PolB) and hence break this pattern. We investigated the provenance of all bidnavirus genes and uncover an unexpected turbulent evolutionary history of these unique viruses. Our analysis strongly suggests that bidnaviruses evolved from a parvovirus ancestor from which they inherit a jelly-roll capsid protein and a superfamily 3 helicase. The radiation of bidnaviruses from parvoviruses was probably triggered by integration of the ancestral parvovirus genome into a large virus-derived DNA transposon of the Polinton (polintovirus) family resulting in the acquisition of the polintovirus PolB gene along with terminal inverted repeats. Bidnavirus genes for a receptor-binding protein and a potential novel antiviral defense modulator are derived from dsRNA viruses (Reoviridae) and dsDNA viruses (Baculoviridae), respectively. The unusual evolutionary history of bidnaviruses emphasizes the key role of horizontal gene transfer, sometimes between viruses with completely different genomes but occupying the same niche, in the emergence of new viral types.

  14. CDK9 inhibitors define elongation checkpoints at both ends of RNA polymerase II-transcribed genes.

    Science.gov (United States)

    Laitem, Clélia; Zaborowska, Justyna; Isa, Nur F; Kufs, Johann; Dienstbier, Martin; Murphy, Shona

    2015-05-01

    Transcription through early-elongation checkpoints requires phosphorylation of negative transcription elongation factors (NTEFs) by the cyclin-dependent kinase (CDK) 9. Using CDK9 inhibitors and global run-on sequencing (GRO-seq), we have mapped CDK9 inhibitor-sensitive checkpoints genome wide in human cells. Our data indicate that early-elongation checkpoints are a general feature of RNA polymerase (pol) II-transcribed human genes and occur independently of polymerase stalling. Pol II that has negotiated the early-elongation checkpoint can elongate in the presence of inhibitors but, remarkably, terminates transcription prematurely close to the terminal polyadenylation (poly(A)) site. Our analysis has revealed an unexpected poly(A)-associated elongation checkpoint, which has major implications for the regulation of gene expression. Interestingly, the pattern of modification of the C-terminal domain of pol II terminated at this new checkpoint largely mirrors the pattern normally found downstream of the poly(A) site, thus suggesting common mechanisms of termination.

  15. RNA polymerase II interacts with the promoter region of the noninduced hsp70 gene in Drosophila melanogaster cells.

    Science.gov (United States)

    Gilmour, D S; Lis, J T

    1986-11-01

    By using a protein-DNA cross-linking method (D. S. Gilmour and J. T. Lis, Mol. Cell. Biol. 5:2009-2018, 1985), we examined the in vivo distribution of RNA polymerase II on the hsp70 heat shock gene in Drosophila melanogaster Schneider line 2 cells. In heat shock-induced cells, a high level of RNA polymerase II was detected on the entire gene, while in noninduced cells, the RNA polymerase II was confined to the 5' end of the hsp70 gene, predominantly between nucleotides -12 and +65 relative to the start of transcription. This association of RNA polymerase II was apparent whether the cross-linking was performed by a 10-min UV irradiation of chilled cells with mercury vapor lamps or by a 40-microsecond irradiation of cells with a high-energy xenon flash lamp. We hypothesize that RNA polymerase II has access to, and a high affinity for, the promoter region of this gene before induction, and this poised RNA polymerase II may be critical in the mechanism of transcription activation.

  16. Diverse Gene-Silencing Mechanisms with Distinct Requirements for RNA Polymerase Subunits in Zea mays

    Science.gov (United States)

    Sloan, Amy E.; Sidorenko, Lyudmila; McGinnis, Karen M.

    2014-01-01

    In Zea mays, transcriptional regulation of the b1 (booster1) gene requires a distal enhancer and MEDIATOR OF PARAMUTATION1 (MOP1), MOP2, and MOP3 proteins orthologous to Arabidopsis components of the RNA-dependent DNA methylation pathway. We compared the genetic requirements for MOP1, MOP2, and MOP3 for endogenous gene silencing by two hairpin transgenes with inverted repeats of the a1 (anthocyaninless1) gene promoter (a1pIR) and the b1 gene enhancer (b1IR), respectively. The a1pIR transgene induced silencing of endogenous A1 in mop1-1 and mop3-1, but not in Mop2-1 homozygous plants. This finding suggests that transgene-derived small interfering RNAs (siRNAs) circumvented the requirement for MOP1, a predicted RNA-dependent RNA polymerase, and MOP3, the predicted largest subunit of RNA polymerase IV (Pol IV). Because the Arabidopsis protein orthologous to MOP2 is the second largest subunit of Pol IV and V, our results may indicate that hairpin-induced siRNAs cannot bypass the requirement for the predicted scaffolding activity of Pol V. In contrast to a1pIR, the b1IR transgene silenced endogenous B1 in all three homozygous mutant genotypes—mop1-1, Mop2-1, and mop3-1—suggesting that transgene mediated b1 silencing did not involve MOP2-containing Pol V complexes. Based on the combined results for a1, b1, and three previously described loci, we propose a speculative hypothesis of locus-specific deployment of Pol II, MOP2-containing Pol V, or alternative versions of Pol V with second largest subunits other than MOP2 to explain the mechanistic differences in silencing at specific loci, including one example associated with paramutation. PMID:25164883

  17. [DETECTION OF VENEZUELAN EQUINE ENCEPHALOMYELITIS VIRUS RNA IN BIOLOGICAL SAMPLES BY REVERSE-TRANSCRIPTION POLYMERASE CHAIN REACTION].

    Science.gov (United States)

    Petrov, A A; Pyshnaya, N S; Lebedev, V N; Kulish, V S; Stovba, L F; Kazantsev, A V; Borisevich, S V

    2015-01-01

    Detection-and identification of Venezuelan equine encephalomyelitis (VEE) virus RNA in biological samples by reverse-transcription polymerase chain reaction (RT-PCR) and RT-PCR in real time (rRT-PCR). VEE, Sindbis, West Nile, Japanese and tick-borne encephalitis viruses were studied. Cell culture of chicken fibroblasts, outbred mice and rats, Javanese macaques were used in the experiments. Biological activity determination of the running culture of causative agents used in the experiments was carried out by negative colony method in monolayer cell culture under agar coating. and using intra-cerebral infection of mice. Reagent kits developed in the 48th Central Research Institute and Institute of Analytical Instrument Engineering were used during execution of experiments of VEE virus RNA detection by RT-PCR and rRT-PCR. VEE virus was detected in biological samples by various methods. Data from RT-PCR and rRT-PCR are in accordance with the results of virus detection in samples using sensitive animals. Use of molecular-diagnostics methods for detection in biological samples of a causative agent of a dangerous infectious disease is important for procuring biological safety of Russian Federation.

  18. Molecular Detection of Dirofilaria immitis Specific Gene from Infected Dog Blood Sample Using Polymerase Chain Reaction

    Science.gov (United States)

    OH, In Young; KIM, Kyung Tae; SUNG, Ho Joong

    2017-01-01

    Background: Dirofilaria immitis, a filarial nematode, is the most important parasite-affecting dogs, causing cardiopulmonary dirofilariasis. Current diagnostic tools for detecting D. immitis include morphological assays, antigen detection, and X-ray. Herein, we developed a method for the molecular detection of D. immitis in blood using polymerase chain reaction (PCR). Methods: The study was conducted at Eulji University, Republic of Korea in 2016. To detect D. immitis-specific gene regions, we aligned the cytochrome c oxidase subunit I (COI) genes of seven filarial nematodes and designed primers targeting the unique region. We used dog glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-targeted primers as the internal control. We conducted PCR-amplified genomic DNA from canine blood samples. The products were confirmed by sequencing. Results: Gene alignment revealed a D. immitis COI-specific gene region, and the activity of designed primers was confirmed by PCR and sequencing. Plasmid DNA made from the PCR products was a positive control. The limit of detection for our method was 50 copies. The D. immitis COI and dog GAPDH genes could be discriminated from blood samples simultaneously. Conclusion: This study provides a method for highly specific and sensitive molecular diagnosis of D. immitis used as a diagnostic and therapeutic tool from the early stage of infection. PMID:28979354

  19. Molecular Detection of Dirofilaria immitis Specific Gene from Infected Dog Blood Sample Using Polymerase Chain Reaction

    Directory of Open Access Journals (Sweden)

    In Young OH

    2017-09-01

    Full Text Available Background: Dirofilaria immitis, a filarial nematode, is the most important parasite-affecting dogs, causing cardiopulmonary dirofilariasis. Current diagnostic tools for detecting D. immitis include morphological assays, antigen detection, and X-ray. Herein, we developed a method for the molecular detection of D. immitis in blood using polymerase chain reaction (PCR.Methods: The study was conducted at Eulji University, Republic of Korea in 2016. To detect D. immitis-specific gene regions, we aligned the cytochrome c oxidase subunit I (COI genes of seven filarial nematodes and designed primers targeting the unique region. We used dog glyceraldehyde-3-phosphate dehydrogenase (GAPDH-targeted primers as the internal control. We conducted PCR-amplified genomic DNA from canine blood samples. The products were confirmed by sequencing.Results: Gene alignment revealed a D. immitis COI-specific gene region, and the activity of designed primers was confirmed by PCR and sequencing. Plasmid DNA made from the PCR products was a positive control. The limit of detection for our method was 50 copies. The D. immitis COI and dog GAPDH genes could be discriminated from blood samples simultaneously.Conclusion: This study provides a method for highly specific and sensitive molecular diagnosis of D. immitis used as a diagnostic and therapeutic tool from the early stage of infection.

  20. Molecular Detection of Dirofilaria immitis Specific Gene from Infected Dog Blood Sample Using Polymerase Chain Reaction.

    Science.gov (United States)

    Oh, In Young; Kim, Kyung Tae; Sung, Ho Joong

    2017-01-01

    Dirofilaria immitis, a filarial nematode, is the most important parasite-affecting dogs, causing cardiopulmonary dirofilariasis. Current diagnostic tools for detecting D. immitis include morphological assays, antigen detection, and X-ray. Herein, we developed a method for the molecular detection of D. immitis in blood using polymerase chain reaction (PCR). The study was conducted at Eulji University, Republic of Korea in 2016. To detect D. immitis-specific gene regions, we aligned the cytochrome c oxidase subunit I (COI) genes of seven filarial nematodes and designed primers targeting the unique region. We used dog glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-targeted primers as the internal control. We conducted PCR-amplified genomic DNA from canine blood samples. The products were confirmed by sequencing. Gene alignment revealed a D. immitis COI-specific gene region, and the activity of designed primers was confirmed by PCR and sequencing. Plasmid DNA made from the PCR products was a positive control. The limit of detection for our method was 50 copies. The D. immitis COI and dog GAPDH genes could be discriminated from blood samples simultaneously. This study provides a method for highly specific and sensitive molecular diagnosis of D. immitis used as a diagnostic and therapeutic tool from the early stage of infection.

  1. Frameshift mutations of poly(adenosine diphosphate-ribose) polymerase genes in gastric and colorectal cancers with microsatellite instability.

    Science.gov (United States)

    Kim, Min Sung; An, Chang Hyeok; Kim, Sung Soo; Yoo, Nam Jin; Lee, Sug Hyung

    2011-09-01

    Poly(adenosine diphosphate-ribose) polymerases consist of 16 members that modify nuclear proteins by building adenosine diphosphate-ribose polymers. Poly(adenosine diphosphate-ribose) polymerase 1, the prototype poly(adenosine diphosphate-ribose) polymerase, and some poly(adenosine diphosphate-ribose) polymerases are involved in many cellular processes including DNA damage response/repair, cell death, and inflammation. Inactivation of poly(adenosine diphosphate-ribose) polymerase proteins frequently enhances genomic instability and apoptosis inactivation, suggesting their roles in cancer development. However, genetic alterations of poly(adenosine diphosphate-ribose) polymerase genes have not been reported in cancers. In a public database, we found that poly(adenosine diphosphate-ribose) polymerase 1, poly(adenosine diphosphate-ribose) polymerase 11, poly(adenosine diphosphate-ribose) polymerase 14, poly(adenosine diphosphate-ribose) polymerase 15, tankyrase-1 (TNKS1), and tankyrase-2 (TNKS2) genes have mononucleotide repeats in coding DNA sequences. To see whether these genes are mutated in cancers with microsatellite instability, we analyzed the mononucleotide repeats in 30 gastric cancers with high microsatellite instability, 13 gastric cancers with low microsatellite instability, 45 gastric cancers with stable microsatellite instability, 40 colorectal cancers with high microsatellite instability, 14 colorectal cancers with low microsatellite instability, and 45 colorectal cancers with stable microsatellite instability by single-strand conformation polymorphism. We found poly(adenosine diphosphate-ribose) polymerase 14, TNKS1, and TNKS2 mutations in 8, 4, and 18 cancers, respectively. They were detected in cancers with high microsatellite instability but not in cancers with low microsatellite instability or stable microsatellite instability. The gastric cancers and colorectal cancers with high microsatellite instability harbored one or more mutations of the poly

  2. Detection of coat protein gene of nervous necrosis virus using loop-mediated isothermal amplification.

    Science.gov (United States)

    Hwang, Jinik; Suh, Sung-Suk; Park, Mirye; Oh, Myung-Joo; Kim, Jong-Oh; Lee, Sukchan; Lee, Taek-Kyun

    2016-03-01

    To establish a novel and highly specific loop-mediated isothermal amplification (LAMP) assay for the identification of nervous necrosis virus (NNV) infection. A set of synthesized primers was used to match the sequences of a specific region of the nnv gene from the National Center for Biotechnology Information database, not originating from NNV-infected fish, the efficiency and specificity of LAMP were measured dependent on the concentration of DNA polymerase and the reaction temperature and time. In addition, to determine species-specific LAMP primers, cross reactivity testing was applied to the reaction between NVV and other virus families including viral hemorrhagic septicemia virus and marine birnavirus. The optimized LAMP reaction carried out at 64 °C for 60 min, and above 4 U Bst DNA polymerase. The sensitivity of LAMP for the detection of nnv was thus about 10 times greater than the sensitivity of polymerase chain reaction. The LAMP assay primers were specific for the detection NNV infection in Epinephelus septemfasciatus. The development of LAMP primers based on genetic information from a public database, not virus-infected samples, may provide a very simple and convenient method to identify viral infection in aquatic organisms. Copyright © 2016 Hainan Medical College. Production and hosting by Elsevier B.V. All rights reserved.

  3. The emerging GII.P16-GII.4 Sydney 2012 norovirus lineage is circulating worldwide, arose by late-2014 and contains polymerase changes that may increase virus transmission.

    Directory of Open Access Journals (Sweden)

    Christopher Ruis

    Full Text Available Noroviruses are a leading cause of human gastroenteritis worldwide. The norovirus genotype GII.4 is the most prevalent genotype in the human population and has caused six pandemics since 1995. A novel norovirus lineage containing the GII.P16 polymerase and pandemic GII.4 Sydney 2012 capsid was recently detected in Asia and Germany. We demonstrate that this lineage is also circulating within the UK and USA and has been circulating since October 2014 or earlier. While the lineage does not contain unique substitutions in the capsid, it does contain polymerase substitutions close to positions known to influence polymerase function and virus transmission. These polymerase substitutions are shared with a GII.P16-GII.2 virus that dominated outbreaks in Germany in Winter 2016. We suggest that the substitutions in the polymerase may have resulted in a more transmissible virus and the combination of this polymerase and the pandemic GII.4 capsid may result in a highly transmissible virus. Further surveillance efforts will be required to determine whether the GII.P16-GII.4 Sydney 2012 lineage increases in frequency over the coming months.

  4. Experimental therapies: gene therapies and oncolytic viruses.

    Science.gov (United States)

    Hulou, M Maher; Cho, Choi-Fong; Chiocca, E Antonio; Bjerkvig, Rolf

    2016-01-01

    Glioblastoma is the most common and aggressive primary brain tumor in adults. Over the past three decades, the overall survival time has only improved by a few months, therefore novel alternative treatment modalities are needed to improve clinical management strategies. Such strategies should ultimately extend patient survival. At present, the extensive insight into the molecular biology of gliomas, as well as into genetic engineering techniques, has led to better decision processes when it comes to modifying the genome to accommodate suicide genes, cytokine genes, and tumor suppressor genes that may kill cancer cells, and boost the host defensive immune system against neoantigenic cytoplasmic and nuclear targets. Both nonreplicative viral vectors and replicating oncolytic viruses have been developed for brain cancer treatment. Stem cells, microRNAs, nanoparticles, and viruses have also been designed. These have been armed with transgenes or peptides, and have been used both in laboratory-based experiments as well as in clinical trials, with the aim of improving selective killing of malignant glioma cells while sparing normal brain tissue. This chapter reviews the current status of gene therapies for malignant gliomas and highlights the most promising viral and cell-based strategies under development. © 2016 Elsevier B.V. All rights reserved.

  5. Detection of Zaire Ebola virus by real-time reverse transcription-polymerase chain reaction, Sierra Leone, 2014.

    Science.gov (United States)

    Liu, Licheng; Sun, Yang; Kargbo, Brima; Zhang, Chuntao; Feng, Huahua; Lu, Huijun; Liu, Wenseng; Wang, Chengyu; Hu, Yi; Deng, Yongqiang; Jiang, Jiafu; Kang, Xiaoping; Yang, Honglei; Jiang, Yongqiang; Yang, Yinhui; Kargbo, David; Qian, Jun; Chen, Weijun

    2015-09-15

    During the 2014 Ebola virus disease (EVD) outbreak, a real-time quantitative polymerase chain reaction was established to detect and identify the Zaire Ebola virus. We describe the use of this assay to screen 315 clinical samples from EVD suspected person in Sierra Leone. The detection rate in blood samples was 77.81% (207/266), and there were relatively higher detection rate (79.32% and 81.42%, respectively) during the first two weeks after onset of symptoms. In the two weeks that followed, the detection rate declined to 66.67% and 25.00%, respectively. There was the highest virus load at the first week and then decreased. The detection rate in swab samples was 89.79% (44/49). This may be benefit from the included patients. 46 of 49 swab samples were collected from died patients. Taken together, the results presented here indicate that the assay specifically and sensitively detects Zaire Ebola virus. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. Rapid detection of respiratory syncytial virus in nasopharyngeal aspirates by reverse transcription and polymerase chain reaction amplification.

    Science.gov (United States)

    Paton, A W; Paton, J C; Lawrence, A J; Goldwater, P N; Harris, R J

    1992-01-01

    A rapid method for detection of respiratory syncytial virus (RSV) in nasopharyngeal aspirates, involving a combination of reverse transcription and polymerase chain reaction amplification (RT-PCR), has been developed. The RT-PCR assay employs oligonucleotide primers specific for the region of the RSV genome which encodes the F1 subunit of the fusion (F) glycoprotein. Other respiratory viruses do not give a positive reaction. The RT-PCR assay was tested on 202 nasopharyngeal aspirates collected from children with clinical signs of respiratory infection, and the results from RT-PCR were compared with those obtained from virus culture and direct detection by enzyme immunoassay (EIA). RT-PCR results were positive in 118 of 125 samples from which RSV was cultured, as well as in 4 of 7 samples which were culture negative but EIA positive. RT-PCR results were negative in 68 of 70 culture-negative, EIA-negative samples, which included 11 samples from which other respiratory viruses were isolated. The speed, sensitivity (94.6%), and specificity (greater than 97%) of the RT-PCR assay suggest that this technique could be useful for rapid detection of RSV in clinical samples. Images PMID:1374080

  7. Epstein–Barr virus latent genes

    Science.gov (United States)

    Kang, Myung-Soo; Kieff, Elliott

    2015-01-01

    Latent Epstein–Barr virus (EBV) infection has a substantial role in causing many human disorders. The persistence of these viral genomes in all malignant cells, yet with the expression of limited latent genes, is consistent with the notion that EBV latent genes are important for malignant cell growth. While the EBV-encoded nuclear antigen-1 (EBNA-1) and latent membrane protein-2A (LMP-2A) are critical, the EBNA-leader proteins, EBNA-2, EBNA-3A, EBNA-3C and LMP-1, are individually essential for in vitro transformation of primary B cells to lymphoblastoid cell lines. EBV-encoded RNAs and EBNA-3Bs are dispensable. In this review, the roles of EBV latent genes are summarized. PMID:25613728

  8. Promoter Structure of the RNA Polymerase II Large Subunit Gene in Caenorhabditis elegans and C. briggsae.

    Science.gov (United States)

    Bird, D M; Kaloshian, I; Molinari, S

    1997-06-01

    The 5'-end of the Caenorhabditis elegans ama-1 gene transcript, which encodes the largest subunit of RNA polymerase II, was cloned. Sequencing revealed that the message is trans-spliced. To characterize the Ce-ama-1 promoter, DNA sequence spanning 3 kb upstream from the initiation codon was determined. Typical elements, such as TATA and Spl sites, were absent. The homologue of ama-1 in C. briggsae, Cb-ama-1, was isolated and its 5' flanking sequence compared with that of Ce-ama-1, revealing only limited similarity, although both sequences included a potential initiator-class transcriptional regulator and phased repeats of an ATC motif. The latter elements are postulated to facilitate DNA bending and may play a role in transcription regulation.

  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 Impact factor: 4.271, year: 2016

  10. Mutations Conferring Resistance to Viral DNA Polymerase Inhibitors in Camelpox Virus Give Different Drug-Susceptibility Profiles in Vaccinia Virus

    Czech Academy of Sciences Publication Activity Database

    Duraffour, S.; Andrei, G.; Topalis, D.; Krečmerová, Marcela; Crance, J. M.; Garin, D.; Snoeck, R.

    2012-01-01

    Roč. 86, č. 13 (2012), s. 7310-7325 ISSN 0022-538X Institutional support: RVO:61388963 Keywords : camelpox virus * CMLV * vaccinia virus VACV * acyclic nucleoside phosphonates * HPMPDAP * cidofovir * drug resistance Subject RIV: CC - Organic Chemistry Impact factor: 5.076, year: 2012

  11. Detection of varicella-zoster virus and herpes simplex virus by the polymerase chain reaction with degenerate primers

    NARCIS (Netherlands)

    Jacobs, J.J.L.; Folkers, E.; Vreeswijk, J.

    1999-01-01

    Varicella-zoster virus (VZV) and herpes simplex virus (HSV) are human pathogens of significance involved in multiple diseases with either typical or atypical clinical features. In neonates and immunocompromised patients these alphaherpesviruses may cause life-threatening diseases such as

  12. Epstein-Barr virus BPLF1 deubiquitinates PCNA and attenuates polymerase η recruitment to DNA damage sites.

    Science.gov (United States)

    Whitehurst, Christopher B; Vaziri, Cyrus; Shackelford, Julia; Pagano, Joseph S

    2012-08-01

    PCNA is monoubiquitinated in response to DNA damage and fork stalling and then initiates recruitment of specialized polymerases in the DNA damage tolerance pathway, translesion synthesis (TLS). Since PCNA is reported to associate with Epstein-Barr virus (EBV) DNA during its replication, we investigated whether the EBV deubiquitinating (DUB) enzyme encoded by BPLF1 targets ubiquitinated PCNA and disrupts TLS. An N-terminal BPLF1 fragment (a BPLF1 construct containing the first 246 amino acids [BPLF1 1-246]) associated with PCNA and attenuated its ubiquitination in response to fork-stalling agents UV and hydroxyurea in cultured cells. Moreover, monoubiquitinated PCNA was deubiquitinated after incubation with purified BPLF1 1-246 in vitro. BPLF1 1-246 dysregulated TLS by reducing recruitment of the specialized repair polymerase polymerase η (Polη) to the detergent-resistant chromatin compartment and virtually abolished localization of Polη to nuclear repair foci, both hallmarks of TLS. Expression of BPLF1 1-246 decreased viability of UV-treated cells and led to cell death, presumably through deubiquitination of PCNA and the inability to repair damaged DNA. Importantly, deubiquitination of PCNA could be detected endogenously in EBV-infected cells in comparison with samples expressing short hairpin RNA (shRNA) against BPLF1. Further, the specificity of the interaction between BPLF1 and PCNA was dependent upon a PCNA-interacting peptide (PIP) domain within the N-terminal region of BPLF1. Both DUB activity and PIP sequence are conserved in the members of the family Herpesviridae. Thus, deubiquitination of PCNA, normally deubiquitinated by cellular USP1, by the viral DUB can disrupt repair of DNA damage by compromising recruitment of TLS polymerase to stalled replication forks. PCNA is the first cellular target identified for BPLF1 and its deubiquitinating activity.

  13. Genetic polymorphism of thymidine kinase (TK) and DNA polymerase (pol) of clinical varicella-zoster virus (VZV) isolates collected over three decades.

    Science.gov (United States)

    Hoffmann, Anja; Döring, Kristin; Seeger, Natalja Tatjana; Bühler, Martina; Schacke, Michael; Krumbholz, Andi; Sauerbrei, Andreas

    2017-10-01

    Genotypic resistance testing of varicella-zoster virus (VZV) strains to antivirals is of high relevance in immunocompromised patients with VZV reactivations unresponsive to therapy. However, the knowledge on mutations associated with natural gene polymorphism or resistance is limited. To examine the genotype of the thymidine kinase (TK) and DNA polymerase (pol) of unselected clinical VZV isolates collected between 1984 and 2014 and to verify the phenotype related to novel amino acid (aa) substitutions. The TK and DNA pol genes of 169 VZV isolates were analyzed by amplification and sequencing. Sequences were compared to that of the reference strain Dumas. The phenotype to acyclovir and other antivirals was examined in isolates with novel aa substitutions using modified plaque reduction assay. In the TK of four strains, four different aa substitutions were detected, apart from the known change S288L that was present in all strains compared to Dumas. All four substitutions have hitherto not been described in the literature and were phenotypically classified as natural gene polymorphisms although two out of them (S51L, K186R) were localized in conserved gene centers. The DNA pol of 34 isolates exhibited 19 different substitutions, 14 out of them were novel, and two (R753K, V777I) were within conserved gene regions. Again, these changes were characterized as natural gene polymorphisms. Non-synonymous mutations in VZV TK or DNA pol conferring natural gene polymorphism are rare events. Nevertheless, the phenotypic characterization of 18 novel polymorphisms can help to provide a better identification of resistance mutations. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. A Novel, Essential Control for Clonality Analysis with Human Androgen Receptor Gene Polymerase Chain Reaction

    Science.gov (United States)

    van Dijk, Jeroen P.; Heuver, Leonie H.; van der Reijden, Bert A.; Raymakers, Reinier A.; de Witte, Theo; Jansen, Joop H.

    2002-01-01

    The most widely used technique for determining clonality based on X-chromosome inactivation is the human androgen receptor gene polymerase chain reaction (PCR). The reliability of this assay depends critically on the digestion of DNA before PCR with the methylation-sensitive restriction enzyme HpaII. We have developed a novel method for quantitatively monitoring the HpaII digestion in individual samples. Using real-time quantitative PCR we measured the efficiency of HpaII digestion by measuring the amplification of a gene that escapes X-chromosome inactivation (XE169) before and after digestion. This method was tested in blood samples from 30 individuals: 2 healthy donors and 28 patients with myelodysplastic syndrome. We found a lack of XE169 DNA reduction after digestion in the granulocytes of two myelodysplastic syndrome patients leading to a false polyclonal X-chromosome inactivation pattern. In all other samples a significant reduction of XE169 DNA was observed after HpaII digestion. The median reduction was 220-fold, ranging from a 9.0-fold to a 57,000-fold reduction. Also paraffin-embedded malignant tissue was investigated from two samples of patients with mantle cell lymphoma and two samples of patients with colon carcinoma. In three of these cases inefficient HpaII digestion led to inaccurate X-chromosome inactivation pattern ratios. We conclude that monitoring the efficiency of the HpaII digestion in a human androgen receptor gene PCR setting is both necessary and feasible. PMID:12213708

  15. Variation in the Genetic Repertoire of Viruses Infecting Micromonas pusilla Reflects Horizontal Gene Transfer and Links to Their Environmental Distribution.

    Science.gov (United States)

    Finke, Jan F; Winget, Danielle M; Chan, Amy M; Suttle, Curtis A

    2017-05-19

    Prasinophytes, a group of eukaryotic phytoplankton, has a global distribution and is infected by large double-stranded DNA viruses (prasinoviruses) in the family Phycodnaviridae . This study examines the genetic repertoire, phylogeny, and environmental distribution of phycodnaviruses infecting Micromonas pusilla , other prasinophytes and chlorophytes. Based on comparisons among the genomes of viruses infecting M. pusilla and other phycodnaviruses, as well as the genome from a host isolate of M. pusilla , viruses infecting M. pusilla (MpVs) share a limited set of core genes, but vary strongly in their flexible pan-genome that includes numerous metabolic genes, such as those associated with amino acid synthesis and sugar manipulation. Surprisingly, few of these presumably host-derived genes are shared with M. pusilla , but rather have their closest non-viral homologue in bacteria and other eukaryotes, indicating horizontal gene transfer. A comparative analysis of full-length DNA polymerase (DNApol) genes from prasinoviruses with their overall gene content, demonstrated that the phylogeny of DNApol gene fragments reflects the gene content of the viruses; hence, environmental DNApol gene sequences from prasinoviruses can be used to infer their overall genetic repertoire. Thus, the distribution of virus ecotypes across environmental samples based on DNApol sequences implies substantial underlying differences in gene content that reflect local environmental conditions. Moreover, the high diversity observed in the genetic repertoire of prasinoviruses has been driven by horizontal gene transfer throughout their evolutionary history, resulting in a broad suite of functional capabilities and a high diversity of prasinovirus ecotypes.

  16. Dynamic bookmarking of primary response genes by p300 and RNA polymerase II complexes.

    Science.gov (United States)

    Byun, Jung S; Wong, Madeline M; Cui, Wenwu; Idelman, Gila; Li, Quentin; De Siervi, Adriana; Bilke, Sven; Haggerty, Cynthia M; Player, Audrey; Wang, Yong Hong; Thirman, Michael J; Kaberlein, Joseph J; Petrovas, Constantinos; Koup, Richard A; Longo, Dan; Ozato, Keiko; Gardner, Kevin

    2009-11-17

    Profiling the dynamic interaction of p300 with proximal promoters of human T cells identified a class of genes that rapidly coassemble p300 and RNA polymerase II (pol II) following mitogen stimulation. Several of these p300 targets are immediate early genes, including FOS, implicating a prominent role for p300 in the control of primary genetic responses. The recruitment of p300 and pol II rapidly transitions to the assembly of several elongation factors, including the positive transcriptional elongation factor (P-TEFb), the bromodomain-containing protein (BRD4), and the elongin-like eleven nineteen lysine-rich leukemia protein (ELL). However, transcription at many of these rapidly induced genes is transient, wherein swift departure of P-TEFb, BRD4, and ELL coincides with termination of transcriptional elongation. Unexpectedly, both p300 and pol II remain accumulated or "bookmarked" at the proximal promoter long after transcription has terminated, demarking a clear mechanistic separation between the recruitment and elongation phases of transcription in vivo. The bookmarked pol II is depleted of both serine-2 and serine-5 phosphorylation of its C-terminal domain and remains proximally positioned at the promoter for hours. Surprisingly, these p300/pol II bookmarked genes can be readily reactivated, and elongation factors can be reassembled by subsequent addition of nonmitogenic agents that, alone, have minimal effects on transcription in the absence of prior preconditioning by mitogen stimulation. These findings suggest that p300 is likely to play an important role in biological processes in which transcriptional bookmarking or preconditioning influences cellular growth and development through the dynamic priming of genes for response to rechallenge by secondary stimuli.

  17. Stability of Barley stripe mosaic virus-induced gene silencing in barley

    DEFF Research Database (Denmark)

    Bruun-Rasmussen, Marianne; Madsen, Christian Toft; Jessing, Stine

    2007-01-01

    Virus-induced gene silencing (VIGS) can be used as a powerful tool for functional genomics studies in plants. With this approach, it is possible to target most genes and downregulate the messenger (m)RNA in a sequence-specific manner. Barley stripe mosaic virus (BSMV) is an established VIGS vector...... for barley and wheat; however, silencing using this vector is generally transient, with efficient silencing often being confined to the first two or three systemically infected leaves. To investigate this further, part of the barley Phytoene desaturase (PDS) gene was inserted into BSMV and the resulting...... photobleaching in infected barley plants was used as a reporter for silencing. In addition, downregulation of PDS mRNA was measured by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). Using fragments of PDS ranging from 128 to 584 nucleotides in BSMV, we observed that insert length...

  18. VARIATION OF NON-CODING REGION AND CODING REGION OF 5’-TERMINAL CRNA OF POLYMERASE BASIC 1 OF AVIAN INFLUENZA VIRUS SUBTYPE H5N1

    Directory of Open Access Journals (Sweden)

    Gusti Ayu Yuniati Kencana

    2009-03-01

    Full Text Available The sequence of the Non-Coding Region (NCR and Coding Region (CR of 5’-terminal cRNA of thepolymerase basic 1 (PB1 gene as a major factor for the species adaptation of avian influenza virussubtype H5N1 (AIV H5N1 has been analysed. The information could be a virological signal for theemergence of a new strain with pandemic potential. Total RNA from twenty six (26 avian influenzasubtype H5N1 isolates were amplified using reverse-transcriptase-polymerase chain reaction (RT-PCRwith a universal forward primer for influenza virus and specifically designed backward primers. Fifteen(15 PB1 gene fragments could be amplified. RT-PCR products were sequenced and analyzed using Mega4software. The length of NCR of PB1 gene was found to be 24 bases and mostly shows conserved sequence,with an exception of Dk/Badung/2006 isolate which has C-7T substitution. A/T composition of PB1 NCRwas 54,2%, while the Dk/Badung/2006 isolate was 58,3%. Species and geographical specificity could not befound in the genetic distance, the amino acid polymorphism, as well as the phylogenetic analysis of t

  19. Detection and differentiation of genotype I and III Japanese encephalitis virus in mosquitoes by multiplex reverse transcriptase-polymerase chain reaction.

    Science.gov (United States)

    Chen, Y Y; Lin, J W; Fan, Y C; Chiou, S S

    2014-02-01

    Japanese encephalitis (JE) is a disease that threatens both human and animal populations in Asian countries, and the causative agent of JE, Japanese encephalitis virus (JEV), has recently changed from genotype III (GIII) to genotype I (GI). However, a test for the rapid differentiation of GI and GIII JEV is still unavailable, especially one that can be used for mosquito-based surveillance. We have designed GI- and GIII-specific primer sets for the rapid detection and differentiation of GI and GIII JEV by multiplex reverse transcriptase-polymerase chain reaction (multiplex RT-PCR). The GI-specific and GIII-specific primer sets were able to specifically amplify the target gene from GI and GIII JEV, respectively. The limitations of detection were 0.00225 and 0.225 pfu for the GI-specific and GIII-specific primers, respectively. Using a mixture of GI-specific and GIII-specific primers, the multiplex RT-PCR was able to specifically detect and differentiate GI and GIII JEV. The multiplex RT-PCR was able to successfully differentiate GI and GIII virus in JEV-infected mosquitoes. Thus, a sensitive and specific multiplex RT-PCR system for the rapid detection and differentiation of GI and GIII JEV has been developed, and this test is likely to be valuable when carrying out mosquito-based JEV surveillance. © 2012 Blackwell Verlag GmbH.

  20. Ring test evaluation of the detection of influenza A virus in swine oral fluids by real-time, reverse transcription polymerase chain reaction (rRT-PCR) and virus isolation

    Science.gov (United States)

    The probability of detecting influenza A virus (IAV) in oral fluid (OF) specimens was calculated for each of 13 real-time, reverse transcription polymerase chain reaction (rRT-PCR) and 7 virus isolation (VI) assays. To conduct the study, OF was inoculated with H1N1 or H3N2 IAV and serially 10-fold d...

  1. Detection of Viral Hemorrhagic Septicemia Virus by Quantitative Reverse Transcription Polymerase Chain Reaction from Two Fish Species at Two Sites in Lake Superior

    Science.gov (United States)

    Cornwell, Emily R.; Eckerlin, Geofrey E.; Getchell, Rodman G.; Groocock, Geoffrey H.; Thompson, Tarin M.; Batts, William N.; Casey, Rufina N.; Kurath, Gael; Winton, James R.; Bowser, Paul R.; Bain, Mark B.; Casey, James W.

    2011-01-01

    Viral hemorrhagic septicemia virus (VHSV) was first detected in the Laurentian Great Lakes in 2005 during a mortality event in the Bay of Quinte, Lake Ontario. Subsequent analysis of archived samples determined that the first known isolation of VHSV in the Laurentian Great Lakes was from a muskellunge Esox masquinongy collected in Lake St. Clair in 2003. By the end of 2008, mortality events and viral isolations had occurred in all of the Laurentian Great Lakes except Lake Superior. In 2009, a focused disease surveillance program was designed to determine whether VHSV was also present in Lake Superior. In this survey, 874 fish from 7 sites along the U.S. shoreline of Lake Superior were collected during June 2009. Collections were focused on nearshore species known to be susceptible to VHSV. All fish were dissected individually by using aseptic techniques and were tested for the presence of VHSV genetic material by use of a quantitative reverse transcription (qRT) polymerase chain reaction (PCR) targeting the viral nucleoprotein gene. Seventeen fish from two host species at two different sites tested positive at low levels for VHSV. All attempts to isolate virus in cell culture were unsuccessful. However, the presence of viral RNA was confirmed independently in five fish by using a nested PCR that targeted the glycoprotein (G) gene. Partial G gene sequences obtained from three fish were identical to the corresponding sequence from the original 2003 VHSV isolate (MI03) from muskellunge. These detections represent the earliest evidence for the presence of VHSV in Lake Superior and illustrate the utility of the highly sensitive qRT-PCR assay for disease surveillance in aquatic animals.

  2. Detection of viral hemorrhagic septicemia virus by quantitative reverse transcription polymerase chain reaction from two fish species at two sites in Lake Superior.

    Science.gov (United States)

    Cornwell, Emily R; Eckerlin, Geofrey E; Getchell, Rodman G; Groocock, Geoffrey H; Thompson, Tarin M; Batts, William N; Casey, Rufina N; Kurath, Gael; Winton, James R; Bowser, Paul R; Bain, Mark B; Casey, James W

    2011-12-01

    Viral hemorrhagic septicemia virus (VHSV) was first detected in the Laurentian Great Lakes in 2005 during a mortality event in the Bay of Quinte, Lake Ontario. Subsequent analysis of archived samples determined that the first known isolation of VHSV in the Laurentian Great Lakes was from a muskellunge Esox masquinongy collected in Lake St. Clair in 2003. By the end of 2008, mortality events and viral isolations had occurred in all of the Laurentian Great Lakes except Lake Superior. In 2009, a focused disease surveillance program was designed to determine whether VHSV was also present in Lake Superior. In this survey, 874 fish from 7 sites along the U.S. shoreline of Lake Superior were collected during June 2009. Collections were focused on nearshore species known to be susceptible to VHSV. All fish were dissected individually by using aseptic techniques and were tested for the presence of VHSV genetic material by use of a quantitative reverse transcription (qRT) polymerase chain reaction (PCR) targeting the viral nucleoprotein gene. Seventeen fish from two host species at two different sites tested positive at low levels for VHSV. All attempts to isolate virus in cell culture were unsuccessful. However, the presence of viral RNA was confirmed independently in five fish by using a nested PCR that targeted the glycoprotein (G) gene. Partial G gene sequences obtained from three fish were identical to the corresponding sequence from the original 2003 VHSV isolate (MI03) from muskellunge. These detections represent the earliest evidence for the presence of VHSV in Lake Superior and illustrate the utility of the highly sensitive qRT-PCR assay for disease surveillance in aquatic animals.

  3. RNA-dependent RNA polymerase 6 of rice (Oryza sativa) plays role in host defense against negative-strand RNA virus, Rice stripe virus.

    Science.gov (United States)

    Jiang, Lin; Qian, Dan; Zheng, Hong; Meng, Lin-Yan; Chen, Jie; Le, Wen-Jing; Zhou, Tong; Zhou, Yi-Jun; Wei, Chun-Hong; Li, Yi

    2012-02-01

    RNA-dependent RNA polymerases (RDRs) from fungi, plants and some invertebrate animals play fundamental roles in antiviral defense. Here, we investigated the role of RDR6 in the defense of economically important rice plants against a negative-strand RNA virus (Rice stripe virus, RSV) that causes enormous crop damage. In three independent transgenic lines (OsRDR6AS line A, B and C) in which OsRDR6 transcription levels were reduced by 70-80% through antisense silencing, the infection and disease symptoms of RSV were shown to be significantly enhanced. The hypersusceptibilities of the OsRDR6AS plants were attributed not to enhanced insect infestation but to enhanced virus infection. The rise in symptoms was associated with the increased accumulation of RSV genomic RNA in the OsRDR6AS plants. The deep sequencing data showed reduced RSV-derived siRNA accumulation in the OsRDR6AS plants compared with the wild type plants. This is the first report of the antiviral role of a RDR in a monocot crop plant in the defense against a negative-strand RNA virus and significantly expands upon the current knowledge of the antiviral roles of RDRs in the defense against different types of viral genomes in numerous groups of plants. Copyright © 2011 Elsevier B.V. All rights reserved.

  4. 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

  5. Enrichment of deleterious variants of mitochondrial DNA polymerase gene (POLG1) in bipolar disorder.

    Science.gov (United States)

    Kasahara, Takaoki; Ishiwata, Mizuho; Kakiuchi, Chihiro; Fuke, Satoshi; Iwata, Nakao; Ozaki, Norio; Kunugi, Hiroshi; Minabe, Yoshio; Nakamura, Kazuhiko; Iwata, Yasuhide; Fujii, Kumiko; Kanba, Shigenobu; Ujike, Hiroshi; Kusumi, Ichiro; Kataoka, Muneko; Matoba, Nana; Takata, Atsushi; Iwamoto, Kazuya; Yoshikawa, Takeo; Kato, Tadafumi

    2017-08-01

    Rare missense variants, which likely account for a substantial portion of the genetic 'dark matter' for a common complex disease, are challenging because the impacts of variants on disease development are difficult to substantiate. This study aimed to examine the impacts of amino acid substitution variants in the POLG1 found in bipolar disorder, as an example and proof of concept, in three different modalities of assessment: in silico predictions, in vitro biochemical assays, and clinical evaluation. We then tested whether deleterious variants in POLG1 contributed to the genetics of bipolar disorder. We searched for variants in the POLG1 gene in 796 Japanese patients with bipolar disorder and 767 controls and comprehensively investigated all 23 identified variants in the three modalities of assessment. POLG1 encodes mitochondrial DNA polymerase and is one of the causative genes for a Mendelian-inheritance mitochondrial disease, which is occasionally accompanied by mood disorders. The healthy control data from the Tohoku Medical Megabank Organization were also employed. Although the frequency of carriers of deleterious variants varied from one method to another, every assessment achieved the same conclusion that deleterious POLG1 variants were significantly enriched in the variants identified in patients with bipolar disorder compared to those in controls. Together with mitochondrial dysfunction in bipolar disorder, the present results suggested deleterious POLG1 variants as a credible risk for the multifactorial disease. © 2016 The Authors. Psychiatry and Clinical Neurosciences published by John Wiley & Sons Australia, Ltd on behalf of Japanese Society of Psychiatry and Neurology.

  6. Halogenated imidazole derivatives block RNA polymerase II elongation along mitogen inducible genes

    Directory of Open Access Journals (Sweden)

    Rubel Tymon

    2010-01-01

    Full Text Available Abstract Background Aberrant activation of protein kinases is one of the essential oncogenic driving forces inherent to the process of tumorigenesis. The protein kinase CK2 plays an important role in diverse biological processes, including cell growth and proliferation as well as in the governing and transduction of prosurvival signals. Increased expression of CK2 is a hallmark of some cancers, hence its antiapoptotic properties may be relevant to cancer onset. Thus, the designing and synthesis of the CK2 inhibitors has become an important pursuit in the search for cancer therapies. Results Using a high-throughput microarray approach, we demonstrate that two potent inhibitors of CK2, 4,5,6,7-tetrabromo-benzimidazole (TBBz and 2-Dimethyloamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT, blocked mitogen induced mRNA expression of immediate early genes. Given the impact of these inhibitors on the process of transcription, we investigated their effects on RNA Polymerase II (RNAPII elongation along the mitogen inducible gene, EGR1 (early growth response 1, using chromatin immunoprecipitation (ChIP assay. ChIP analysis demonstrated that both drugs arrest RNAPII elongation. Finally, we show that CDK9 kinase activity, essential for the triggering of RNAPII elongation, was blocked by TBBz and to lesser degree by DMAT. Conclusions Our approach revealed that small molecules derived from halogenated imidazole compounds may decrease cell proliferation, in part, by inhibiting pathways that regulate transcription elongation.

  7. A Double Polymerase Chain Reaction Method for Detecting African ...

    African Journals Online (AJOL)

    Purpose: To establish a double polymerase chain reaction (PCR) method for the simultaneous detection of African swine fever virus (ASFV) and swine vesicular disease virus (SVDV). Methods: By using reference sequences of ASFV and SVDV, this study synthesized parts of the genes connected to the 19-T vector which ...

  8. Analysis of colorectal cancer and polyp for presence herpes simplex virus and cytomegalovirus DNA sequences by polymerase chain reaction

    Directory of Open Access Journals (Sweden)

    Sahar Mehrabani khasraghi

    2016-05-01

    Full Text Available Introduction: In recent years, it was demonstrated that there is a clear association between the complicated course of colorectal cancer (CRC and the presence of herpes viruses. Despite a great number of published reports, the exact pathogenic role of herpes viruses remains unclear in these patients. The purpose of this study is to explore the prevalence of herpes simplex virus (HSV and cytomegalovirus (CMV in patients with CRC and polyp in comparison with healthy subjects using the polymerase chain reaction (PCR method. Methods: In this case-control study, 15 biopsies of patients with CRC and 20 colorectal polyp sample were selected. From each patient, two tissue samples were obtained: one sample from malignant tissue, and the other from normal colorectal tissue in an area located 15 cm away from the malignant tissue. Furthermore, 35 samples from healthy people as controls were selected. After DNA extraction, PCR was used to determine HSV and CMV genomes by specific primers. A statistical analysis was performed using the chi-square test. Results: Five CRC patients (33.3% had HSV DNA detected in both the malignant and the matched normal tissue. Five CRC patients (33.3% and seven polyp patients (35.0% had CMV DNA detected in both the malignant and the matched normal tissue. HSV DNA was found in 20% and CMV DNA in 37.1% of samples from healthy people as a control group. Thus, no significant association was observed between the prevalence of HSV and CMV, and an incidence of CRC and polyps according to the location of the samples as compared with the control group. Conclusion: The findings demonstrated that there is no direct molecular evidence to support the association between HSV and CMV and human colorectal malignancies. However, the results from this study do not exclude a possible oncogenic role of these viruses in the neoplastic development of colon cells.

  9. Identification of a novel multiple kinase inhibitor with potent antiviral activity against influenza virus by reducing viral polymerase activity

    Energy Technology Data Exchange (ETDEWEB)

    Sasaki, Yutaka; Kakisaka, Michinori; Chutiwitoonchai, Nopporn [Viral Infectious Diseases Unit, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198 (Japan); Tajima, Shigeru [Department of Virology I, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku, Tokyo 162-8640 (Japan); Hikono, Hirokazu; Saito, Takehiko [Influenza and Prion Disease Research Center, National Institute of Animal Health, National Agriculture and Food Research Organization (NARO), 3-1-5 Kannondai, Tsukuba, Ibaraki 305-0856 (Japan); Aida, Yoko, E-mail: aida@riken.jp [Viral Infectious Diseases Unit, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198 (Japan)

    2014-07-18

    Highlights: • Screening of 50,000 compounds and subsequent lead optimization identified WV970. • WV970 has antiviral effects against influenza A, B and highly pathogenic viral strains. • WV970 inhibits viral genome replication and transcription. • A target database search suggests that WV970 may bind to a number of kinases. • KINOMEscan screening revealed that WV970 has inhibitory effects on 15 kinases. - Abstract: Neuraminidase inhibitors are the only currently available influenza treatment, although resistant viruses to these drugs have already been reported. Thus, new antiviral drugs with novel mechanisms of action are urgently required. In this study, we identified a novel antiviral compound, WV970, through cell-based screening of a 50,000 compound library and subsequent lead optimization. This compound exhibited potent antiviral activity with nanomolar IC{sub 50} values against both influenza A and B viruses but not non-influenza RNA viruses. Time-of-addition and indirect immunofluorescence assays indicated that WV970 acted at an early stage of the influenza life cycle, but likely after nuclear entry of viral ribonucleoprotein (vRNP). Further analyses of viral RNA expression and viral polymerase activity indicated that WV970 inhibited vRNP-mediated viral genome replication and transcription. Finally, structure-based virtual screening and comprehensive human kinome screening were used to demonstrate that WV970 acts as a multiple kinase inhibitor, many of which are associated with influenza virus replication. Collectively, these results strongly suggest that WV970 is a promising anti-influenza drug candidate and that several kinases associated with viral replication are promising drug targets.

  10. Variations of Human DNA Polymerase Genes as Biomarkers of Prostate Cancer Progression

    Science.gov (United States)

    2013-07-01

    Athens, Greece, 07/10 Makridakis N. Error-prone polymerase mutations and prostate cancer progression, COBRE /Cancer Genetics group seminar, Tulane...New Orleans, LA, 03/11 12 Makridakis N. Error-prone polymerases, genomic instability and prostate cancer progression, COBRE / External...Orleans, LA, 09/11 Makridakis N. Error-prone polymerases, genomic instability and prostate cancer progression, COBRE / External Advisory Board

  11. Genetic diversity of avian infectious bronchitis viruses in Japan based on analysis of s2 glycoprotein gene.

    Science.gov (United States)

    Mase, Masaji; Inoue, Toshikazu; Yamaguchi, Shigeo; Imada, Tadao

    2009-03-01

    To understand the genetic diversity of the S2 gene of infectious bronchitis viruses (IBV) isolated in Japan, we determined the nucleotide sequences of these IBVs using the reverse transcriptase polymerase chain reaction method coupled with direct sequencing. IBV isolated in Japan were classified into six different groups by phylogenetic analysis based on the S2 gene. However, the classification based on the S2 gene of IBV isolated in Japan was different for some of the strains from those obtained with our previous analysis of the S1 gene. This suggested that genetic recombination between the virus strains classified into different genetic groups had occurred in poultry, and that recombinant viruses might be epidemic in Japan.

  12. Role of polymerase chain reaction in the diagnosis of Trichomonas vaginalis infection in human immunodeficiency virus-infected individuals from India (South

    Directory of Open Access Journals (Sweden)

    Hema Paul

    2012-01-01

    Full Text Available Background: Trichomonas vaginalis is a protozoan parasite and an etiological agent for trichomoniasis, a sexually transmitted infection (STI. Fifty to eighty percentage of women with trichomoniasis are asymptomatic and in the absence of treatment the infection persists longer. Aim: To evaluate the role of polymerase chain reaction (PCR in the diagnosis of trichomoniasis and also to look at the frequency of infection among human immunodeficiency virus (HIV infected women. Methods: A non-nested PCR was standardized to detect 102 bp size amplified product of the adhesin gene of T. vaginalis. The real time performance of this assay was performed with vaginal swab samples from 198 HIV-seropositive women who attended the infectious disease clinic and compared with wet mount and culture in Diamond′s modified media. Results: Among the prospectively studied 198 HIV-infected women, 1 (0.51% was positive by wet mount, 6 (3.03% were positive by culture and 10 (5.02% were positive by the PCR. There was a significant observed agreement between the PCR and culture (k=0.74, Z=10.7, P<0.0000. Conclusion: Our study showed that the PCR assay for the amplification of adhesion gene is a highly sensitive method to screen the high risk group individuals like HIV-positive women for Trichomonas vaginalis compared to the culture. Testing algorithm should be, wet mount and if negative, test by PCR as it is rapid compared to culture which takes 7 days.

  13. Selective inhibition of the reverse transcription of duck hepatitis B virus by binding of 2',3'-dideoxyguanosine 5'-triphosphate to the viral polymerase.

    Science.gov (United States)

    Howe, A Y; Robins, M J; Wilson, J S; Tyrrell, D L

    1996-01-01

    Hepatitis B virus (HBV) replication is mediated by the viral polymerase that possesses three functional domains: primer, DNA polymerase/reverse transcriptase, and RNase H. Using the Pekin duck as an animal model, we demonstrate a novel mechanism of inhibition of duck hepatitis B virus (DHBV) by 2,6-diaminopurine 2',3'-dideoxyriboside (ddDAPR), a prodrug of 2',3'-dideoxyguanosine (ddG). A selective and irreversible inhibition of DHBV DNA replication is found in ducklings treated with high doses of ddDAPR (20 to 50 mg/kg), but not with similar doses of 2',3'-dideoxycytidine (ddC). The inhibition mediated by ddDAPR occurs at a very early stage of the reverse transcription. Despite the inhibition of DHBV DNA replication by ddDAPR, the DNA polymerase and reverse transcriptase activities of the polymerase are found to remain active when tested on exogenous templates in activity gels. We have demonstrated direct binding of [alpha-32P]ddGTP to the DHBV polymerase expressed in an in vitro transcription and translation system. These results suggest that the binding of ddGTP to the polymerase blocks the initial DNA replication.

  14. Replication-competent infectious hepatitis B virus vectors carrying substantially sized transgenes by redesigned viral polymerase translation.

    Directory of Open Access Journals (Sweden)

    Zihua Wang

    Full Text Available Viral vectors are engineered virus variants able to deliver nonviral genetic information into cells, usually by the same routes as the parental viruses. For several virus families, replication-competent vectors carrying reporter genes have become invaluable tools for easy and quantitative monitoring of replication and infection, and thus also for identifying antivirals and virus susceptible cells. For hepatitis B virus (HBV, a small enveloped DNA virus causing B-type hepatitis, such vectors are not available because insertions into its tiny 3.2 kb genome almost inevitably affect essential replication elements. HBV replicates by reverse transcription of the pregenomic (pg RNA which is also required as bicistronic mRNA for the capsid (core protein and the reverse transcriptase (Pol; their open reading frames (ORFs overlap by some 150 basepairs. Translation of the downstream Pol ORF does not involve a conventional internal ribosome entry site (IRES. We reasoned that duplicating the overlap region and providing artificial IRES control for translation of both Pol and an in-between inserted transgene might yield a functional tricistronic pgRNA, without interfering with envelope protein expression. As IRESs we used a 22 nucleotide element termed Rbm3 IRES to minimize genome size increase. Model plasmids confirmed its activity even in tricistronic arrangements. Analogous plasmids for complete HBV genomes carrying 399 bp and 720 bp transgenes for blasticidin resistance (BsdR and humanized Renilla green fluorescent protein (hrGFP produced core and envelope proteins like wild-type HBV; while the hrGFP vector replicated poorly, the BsdR vector generated around 40% as much replicative DNA as wild-type HBV. Both vectors, however, formed enveloped virions which were infectious for HBV-susceptible HepaRG cells. Because numerous reporter and effector genes with sizes of around 500 bp or less are available, the new HBV vectors should become highly useful tools to

  15. Replication-competent infectious hepatitis B virus vectors carrying substantially sized transgenes by redesigned viral polymerase translation.

    Science.gov (United States)

    Wang, Zihua; Wu, Li; Cheng, Xin; Liu, Shizhu; Li, Baosheng; Li, Haijun; Kang, Fubiao; Wang, Junping; Xia, Huan; Ping, Caiyan; Nassal, Michael; Sun, Dianxing

    2013-01-01

    Viral vectors are engineered virus variants able to deliver nonviral genetic information into cells, usually by the same routes as the parental viruses. For several virus families, replication-competent vectors carrying reporter genes have become invaluable tools for easy and quantitative monitoring of replication and infection, and thus also for identifying antivirals and virus susceptible cells. For hepatitis B virus (HBV), a small enveloped DNA virus causing B-type hepatitis, such vectors are not available because insertions into its tiny 3.2 kb genome almost inevitably affect essential replication elements. HBV replicates by reverse transcription of the pregenomic (pg) RNA which is also required as bicistronic mRNA for the capsid (core) protein and the reverse transcriptase (Pol); their open reading frames (ORFs) overlap by some 150 basepairs. Translation of the downstream Pol ORF does not involve a conventional internal ribosome entry site (IRES). We reasoned that duplicating the overlap region and providing artificial IRES control for translation of both Pol and an in-between inserted transgene might yield a functional tricistronic pgRNA, without interfering with envelope protein expression. As IRESs we used a 22 nucleotide element termed Rbm3 IRES to minimize genome size increase. Model plasmids confirmed its activity even in tricistronic arrangements. Analogous plasmids for complete HBV genomes carrying 399 bp and 720 bp transgenes for blasticidin resistance (BsdR) and humanized Renilla green fluorescent protein (hrGFP) produced core and envelope proteins like wild-type HBV; while the hrGFP vector replicated poorly, the BsdR vector generated around 40% as much replicative DNA as wild-type HBV. Both vectors, however, formed enveloped virions which were infectious for HBV-susceptible HepaRG cells. Because numerous reporter and effector genes with sizes of around 500 bp or less are available, the new HBV vectors should become highly useful tools to better

  16. Herpes simplex virus type 1 and human DNA polymerase interactions with 2'-deoxyguanosine 5'-triphosphate analogues. Kinetics of incorporation into DNA and induction of inhibition.

    Science.gov (United States)

    Reardon, J E

    1989-11-15

    The ability of herpes simplex virus type 1 (HSV-1) DNA polymerase, HeLa polymerase alpha, and HeLa polymerase beta to utilize several dGTP analogues has been investigated using a defined synthetic template primer. The relative efficiencies of the triphosphates of 9-[(2-hydroxyethoxy)methyl]guanine (acyclovir triphosphate, ACVTP), 9-[(1,3-dihydroxy-2-propoxy)methyl] guanine (ganciclovir triphosphate, DHPGTP), and 2',3'-dideoxyguanosine (ddGTP) as substrates for the three polymerases were: HSV-1 polymerase, dGTP greater than ACVTP approximately equal to DHPGTP greater than ddGTP; polymerase alpha, dGTP greater than ACVTP approximately equal to DHPGTP much greater than ddGTP; polymerase beta, ddGTP greater than dGTP much greater than ACVTP approximately equal to DHPGTP. The potent inhibition of HSV-1 polymerase by ACVTP has been shown previously to be due to the formation of a dead-end complex upon binding of the next 2'-deoxynucleoside 5'-triphosphate encoded by the template after incorporation of acyclovir monophosphate into the 3' end of the primer (Reardon, J. E., and Spector, T. (1989) J. Biol. Chem. 264, 7405-7411). This mechanism was shown here to be a general mechanism for inhibition of polymerases by the obligate chain terminators, ACVTP and ddGTP. The ACVTP-induced inhibition was 30-fold more potent with HSV-1 polymerase than with polymerase alpha. This difference may contribute to the antiviral selectivity of this nucleotide analogue. The effect of ganciclovir monophosphate incorporation (a nonobligate chain terminator) on subsequent primer extension was also evaluated. With HSV-1 polymerase and polymerase alpha, although there was a considerable reduction in the efficiency of utilization of the 3'-DHPGMP-terminal primer, contrasting kinetic behavior was observed. With HSV-1 polymerase, insertion of DHPGTP resulted in a significant reduction in Vmax for subsequent nucleotide incorporations. In contrast, with polymerase alpha, a relatively small decrease in

  17. A noncanonical poly(A) RNA polymerase gene affects morphology in Phoma medicaginis.

    Science.gov (United States)

    Choi, Kihyuck; Marek, Stephen M

    2017-11-15

    Phoma medicaginis (syn. Ascochyta medicaginicola Qchen & L. Cai) causes spring black stem and leaf spot, an important disease of alfalfa and annual medics. P. medicaginis forms uninucleate conidia in melanized pycnidia and is genetically tractable using Agrobacterium mediated transformation (ATMT), resulting in random integration of T-DNA that occasionally generates pycnidial mutants. The T-DNA tagged mutant, P265 displayed smaller pycnidia and more aerial hyphae than the wild type. A single T-DNA disrupted a putative noncanonical poly(A) RNA polymerase gene, Pmncpap1, which in yeast interacts with ribonucleotide reductase (RNR). As in yeast mutants, P265 showed sensitivity to hydroxyurea (HU), a RNR inhibitor. To characterize the role of Pmncpap1, targeted ΔPmncpap1 mutants were created using a hygromycin selectable marker flanked by 1 Kbp regions of Pmncpap1. ΔPmncpap1 mutants possessed similar morphological features to those of P265. The plasmid for rescue of PmncPAP1, pCAM-Nat1 (nourseothricin selection) was constructed and used to introduce full-length PmncPAP1 into mutants. Rescued P265 showed partial recovery of wild type and the original T-DNA was lost due to homologous integration. To our knowledge, this is the first ncPAP to be examined in a filamentous fungus. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. A field based detection method for Rose rosette virus using isothermal probe-based Reverse transcription-recombinase polymerase amplification assay.

    Science.gov (United States)

    Babu, Binoy; Washburn, Brian K; Ertek, Tülin Sarigül; Miller, Steven H; Riddle, Charles B; Knox, Gary W; Ochoa-Corona, Francisco M; Olson, Jennifer; Katırcıoğlu, Yakup Zekai; Paret, Mathews L

    2017-09-01

    Rose rosette disease, caused by Rose rosette virus (RRV; genus Emaravirus) is a major threat to the rose industry in the U.S. The only strategy currently available for disease management is early detection and eradication of the infected plants, thereby limiting its potential spread. Current RT-PCR based diagnostic methods for RRV are time consuming and are inconsistent in detecting the virus from symptomatic plants. Real-time RT-qPCR assay is highly sensitive for detection of RRV, but it is expensive and requires well-equipped laboratories. Both the RT-PCR and RT-qPCR cannot be used in a field-based testing for RRV. Hence a novel probe based, isothermal reverse transcription-recombinase polymerase amplification (RT-exoRPA) assay, using primer/probe designed based on the nucleocapsid gene of the RRV has been developed. The assay is highly specific and did not give a positive reaction to other viruses infecting roses belonging to both inclusive and exclusive genus. Dilution assays using the in vitro transcript showed that the primer/probe set is highly sensitive, with a detection limit of 1 fg/μl. In addition, a rapid technique for the extraction of viral RNA (<5min) has been standardized from RRV infected tissue sources, using PBS-T buffer (pH 7.4), which facilitates the virus adsorption onto the PCR tubes at 4°C for 2min, followed by denaturation to release the RNA. RT-exoRPA analysis of the infected plants using the primer/probe indicated that the virus could be detected from leaves, stems, petals, pollen, primary roots and secondary roots. In addition, the assay was efficiently used in the diagnosis of RRV from different rose varieties, collected from different states in the U.S. The entire process, including the extraction can be completed in 25min, with less sophisticated equipments. The developed assay can be used with high efficiency in large scale field testing for rapid detection of RRV in commercial nurseries and landscapes. Copyright © 2017 Elsevier B

  19. Kaposi's sarcoma-associated herpesvirus LANA recruits the DNA polymerase clamp loader to mediate efficient replication and virus persistence.

    Science.gov (United States)

    Sun, Qiming; Tsurimoto, Toshiki; Juillard, Franceline; Li, Lin; Li, Shijun; De León Vázquez, Erika; Chen, She; Kaye, Kenneth

    2014-08-12

    Kaposi's sarcoma-associated herpesvirus (KSHV) latently infects tumor cells and persists as a multiple-copy, extrachromosomal, circular episome. To persist, the viral genome must replicate with each cell cycle. The KSHV latency-associated nuclear antigen (LANA) mediates viral DNA replication and persistence, but little is known regarding the underlying mechanisms. We find that LANA recruits replication factor C (RFC), the DNA polymerase clamp [proliferating cell nuclear antigen (PCNA)] loader, to drive DNA replication efficiently. Mutated LANA lacking RFC interaction was deficient for LANA-mediated DNA replication and episome persistence. RFC depletion had a negative impact on LANA's ability to replicate and maintain viral DNA in cells containing artificial KSHV episomes or in infected cells, leading to loss of virus. LANA substantially increased PCNA loading onto DNA in vitro and recruited RFC and PCNA to KSHV DNA in cells. These findings suggest that PCNA loading is a rate-limiting step in DNA replication that is incompatible with viral survival. LANA enhancement of PCNA loading permits efficient virus replication and persistence, revealing a previously unidentified mechanism for KSHV latency.

  20. Hepatitis B virus polymerase blocks pattern recognition receptor signaling via interaction with DDX3: implications for immune evasion.

    Directory of Open Access Journals (Sweden)

    Haifeng Wang

    Full Text Available Viral infection leads to induction of pattern-recognition receptor signaling, which leads to interferon regulatory factor (IRF activation and ultimately interferon (IFN production. To establish infection, many viruses have strategies to evade the innate immunity. For the hepatitis B virus (HBV, which causes chronic infection in the liver, the evasion strategy remains uncertain. We now show that HBV polymerase (Pol blocks IRF signaling, indicating that HBV Pol is the viral molecule that effectively counteracts host innate immune response. In particular, HBV Pol inhibits TANK-binding kinase 1 (TBK1/IkappaB kinase-epsilon (IKKepsilon, the effector kinases of IRF signaling. Intriguingly, HBV Pol inhibits TBK1/IKKepsilon activity by disrupting the interaction between IKKepsilon and DDX3 DEAD box RNA helicase, which was recently shown to augment TBK1/IKKepsilon activity. This unexpected role of HBV Pol may explain how HBV evades innate immune response in the early phase of the infection. A therapeutic implication of this work is that a strategy to interfere with the HBV Pol-DDX3 interaction might lead to the resolution of life-long persistent infection.

  1. Hepatitis C Virus RNA-Dependent RNA Polymerase Interacts with the Akt/PKB Kinase and Induces Its Subcellular Relocalization

    Science.gov (United States)

    Valero, María Llanos; Sabariegos, Rosario; Cimas, Francisco J.; Perales, Celia; Domingo, Esteban; Sánchez-Prieto, Ricardo

    2016-01-01

    Hepatitis C virus (HCV) interacts with cellular components and modulates their activities for its own benefit. These interactions have been postulated as a target for antiviral treatment, and some candidate molecules are currently in clinical trials. The multifunctional cellular kinase Akt/protein kinase B (PKB) must be activated to increase the efficacy of HCV entry but is rapidly inactivated as the viral replication cycle progresses. Viral components have been postulated to be responsible for Akt/PKB inactivation, but the underlying mechanism remained elusive. In this study, we show that HCV polymerase NS5B interacts with Akt/PKB. In the presence of transiently expressed NS5B or in replicon- or virus-infected cells, NS5B changes the cellular localization of Akt/PKB from the cytoplasm to the perinuclear region. Sequestration of Akt/PKB by NS5B could explain its exclusion from its participation in early Akt/PKB inactivation. The NS5B-Akt/PKB interaction represents a new regulatory step in the HCV infection cycle, opening possibilities for new therapeutic options. PMID:27021315

  2. Development and deployment of a rapid recombinase polymerase amplification Ebola virus detection assay in Guinea in 2015.

    Science.gov (United States)

    Faye, Oumar; Faye, Ousmane; Soropogui, Barré; Patel, Pranav; El Wahed, Ahmed Abd; Loucoubar, Cheikh; Fall, Gamou; Kiory, Davy; Magassouba, N'Faly; Keita, Sakoba; Kondé, Mandy Kader; Diallo, Alpha Amadou; Koivogui, Lamine; Karlberg, Helen; Mirazimi, Ali; Nentwich, Oliver; Piepenburg, Olaf; Niedrig, Matthias; Weidmann, Manfred; Sall, Amadou Alpha

    2015-01-01

    In the absence of a vaccine or specific treatments for Ebola virus disease (EVD), early identification of cases is crucial for the control of EVD epidemics. We evaluated a new extraction kit (SpeedXtract (SE), Qiagen) on sera and swabs in combination with an improved diagnostic reverse transcription recombinase polymerase amplification assay for the detection of Ebola virus (EBOV-RT-RPA). The performance of combined extraction and detection was best for swabs. Sensitivity and specificity of the combined SE and EBOV-RT-RPA were tested in a mobile laboratory consisting of a mobile glovebox and a Diagnostics-in-a-Suitcase powered by a battery and solar panel, deployed to Matoto Conakry, Guinea as part of the reinforced surveillance strategy in April 2015 to reach the goal of zero cases. The EBOV-RT-RPA was evaluated in comparison to two real-time PCR assays. Of 928 post-mortem swabs, 120 tested positive, and the combined SE and EBOV-RT-RPA yielded a sensitivity and specificity of 100% in reference to one real-time RT-PCR assay. Another widely used real-time RT-PCR was much less sensitive than expected. Results were provided very fast within 30 to 60 min, and the field deployment of the mobile laboratory helped improve burial management and community engagement.

  3. Nucleoproteins of Negative Strand RNA Viruses; RNA Binding, Oligomerisation and Binding to Polymerase Co-Factor

    Directory of Open Access Journals (Sweden)

    Thibaut Crépin

    2010-01-01

    Full Text Available Commentary on Tawar, R.G.; Duquerroy, S.; Vonrhein, C.; Varela, P.F.; Damier-Piolle, L.; Castagné, N.; MacLellan, K.; Bedouelle, H.; Bricogne, G.; Bhella, D.; Eléouët, J.-F.; Rey, F.A. Crystal structure of a nucleocapsid-like nucleoprotein-RNA complex of respiratory syncytial virus. Science 2009, 326, 1279-1283.

  4. The avian-origin PB1 gene segment facilitated replication and transmissibility of the H3N2/1968 pandemic influenza virus.

    Science.gov (United States)

    Wendel, Isabel; Rubbenstroth, Dennis; Doedt, Jennifer; Kochs, Georg; Wilhelm, Jochen; Staeheli, Peter; Klenk, Hans-Dieter; Matrosovich, Mikhail

    2015-04-01

    The H2N2/1957 and H3N2/1968 pandemic influenza viruses emerged via the exchange of genomic RNA segments between human and avian viruses. The avian hemagglutinin (HA) allowed the hybrid viruses to escape preexisting immunity in the human population. Both pandemic viruses further received the PB1 gene segment from the avian parent (Y. Kawaoka, S. Krauss, and R. G. Webster, J Virol 63:4603-4608, 1989), but the biological significance of this observation was not understood. To assess whether the avian-origin PB1 segment provided pandemic viruses with some selective advantage, either on its own or via cooperation with the homologous HA segment, we modeled by reverse genetics the reassortment event that led to the emergence of the H3N2/1968 pandemic virus. Using seasonal H2N2 virus A/California/1/66 (Cal) as a surrogate precursor human virus and pandemic virus A/Hong Kong/1/68 (H3N2) (HK) as a source of avian-derived PB1 and HA gene segments, we generated four reassortant recombinant viruses and compared pairs of viruses which differed solely by the origin of PB1. Replacement of the PB1 segment of Cal by PB1 of HK facilitated viral polymerase activity, replication efficiency in human cells, and contact transmission in guinea pigs. A combination of PB1 and HA segments of HK did not enhance replicative fitness of the reassortant virus compared with the single-gene PB1 reassortant. Our data suggest that the avian PB1 segment of the 1968 pandemic virus served to enhance viral growth and transmissibility, likely by enhancing activity of the viral polymerase complex. Despite the high impact of influenza pandemics on human health, some mechanisms underlying the emergence of pandemic influenza viruses still are poorly understood. Thus, it was unclear why both H2N2/1957 and H3N2/1968 reassortant pandemic viruses contained, in addition to the avian HA, the PB1 gene segment of the avian parent. Here, we addressed this long-standing question by modeling the emergence of the H3N2

  5. Hairpin RNA Targeting Multiple Viral Genes Confers Strong Resistance to Rice Black-Streaked Dwarf Virus

    Directory of Open Access Journals (Sweden)

    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.

  6. Replicative homeostasis II: Influence of polymerase fidelity on RNA virus quasispecies biology: Implications for immune recognition, viral autoimmunity and other "virus receptor" diseases

    Directory of Open Access Journals (Sweden)

    Sallie Richard

    2005-08-01

    Full Text Available Abstract Much of the worlds' population is in active or imminent danger from established infectious pathogens, while sporadic and pandemic infections by these and emerging agents threaten everyone. RNA polymerases (RNApol generate enormous genetic and consequent antigenic heterogeneity permitting both viruses and cellular pathogens to evade host defences. Thus, RNApol causes more morbidity and premature mortality than any other molecule. The extraordinary genetic heterogeneity defining viral quasispecies results from RNApol infidelity causing rapid cumulative genomic RNA mutation a process that, if uncontrolled, would cause catastrophic loss of sequence integrity and inexorable quasispecies extinction. Selective replication and replicative homeostasis, an epicyclical regulatory mechanism dynamically linking RNApol fidelity and processivity with quasispecies phenotypic diversity, modulating polymerase fidelity and, hence, controlling quasispecies behaviour, prevents this happening and also mediates immune escape. Perhaps more importantly, ineluctable generation of broad phenotypic diversity after viral RNA is translated to protein quasispecies suggests a mechanism of disease that specifically targets, and functionally disrupts, the host cell surface molecules – including hormone, lipid, cell signalling or neurotransmitter receptors – that viruses co-opt for cell entry. This mechanism – "Viral Receptor Disease (VRD" – may explain so-called "viral autoimmunity", some classical autoimmune disorders and other diseases, including type II diabetes mellitus, and some forms of obesity. Viral receptor disease is a unifying hypothesis that may also explain some diseases with well-established, but multi-factorial and apparently unrelated aetiologies – like coronary artery and other vascular diseases – in addition to diseases like schizophrenia that are poorly understood and lack plausible, coherent, pathogenic explanations.

  7. Activation Pathway of a Nucleoside Analog Inhibiting Respiratory Syncytial Virus Polymerase.

    Science.gov (United States)

    Jordan, Paul C; Stevens, Sarah K; Tam, Yuen; Pemberton, Ryan P; Chaudhuri, Shuvam; Stoycheva, Antitsa D; Dyatkina, Natalia; Wang, Guangyi; Symons, Julian A; Deval, Jerome; Beigelman, Leo

    2017-01-20

    Human respiratory syncytial virus (RSV) is a negative-sense RNA virus and a significant cause of respiratory infection in infants and the elderly. No effective vaccines or antiviral therapies are available for the treatment of RSV. ALS-8176 is a first-in-class nucleoside prodrug inhibitor of RSV replication currently under clinical evaluation. ALS-8112, the parent molecule of ALS-8176, undergoes intracellular phosphorylation, yielding the active 5'-triphosphate metabolite. The host kinases responsible for this conversion are not known. Therefore, elucidation of the ALS-8112 activation pathway is key to further understanding its conversion mechanism, particularly given its potent antiviral effects. Here, we have identified the activation pathway of ALS-8112 and show it is unlike other antiviral cytidine analogs. The first step, driven by deoxycytidine kinase (dCK), is highly efficient, while the second step limits the formation of the active 5'-triphosphate species. ALS-8112 is a 2'- and 4'-modified nucleoside analog, prompting us to investigate dCK recognition of other 2'- and 4'-modified nucleosides. Our biochemical approach along with computational modeling contributes to an enhanced structure-activity profile for dCK. These results highlight an exciting potential to optimize nucleoside analogs based on the second activation step and increased attention toward nucleoside diphosphate and triphosphate prodrugs in drug discovery.

  8. Microwave or autoclave treatments destroy the infectivity of infectious bronchitis virus and avian pneumovirus but allow detection by reverse transcriptase-polymerase chain reaction.

    Science.gov (United States)

    Elhafi, G; Naylor, C J; Savage, C E; Jones, R C

    2004-06-01

    A method is described for enabling safe transit of denatured virus samples for polymerase chain reaction (PCR) identification without the risk of unwanted viable viruses. Cotton swabs dipped in avian infectious bronchitis virus (IBV) or avian pneumovirus (APV) were allowed to dry. Newcastle disease virus and avian influenza viruses were used as controls. Autoclaving and microwave treatment for as little as 20 sec destroyed the infectivity of all four viruses. However, both IBV and APV could be detected by reverse transcriptase (RT)-PCR after autoclaving and as long as 5 min microwave treatment (Newcastle disease virus and avian influenza viruses were not tested). Double microwave treatment of IBV and APV with an interval of 2 to 7 days between was tested. After the second treatment, RT-PCR products were readily detected in all samples. Swabs from the tracheas and cloacas of chicks infected with IBV shown to contain infectious virus were microwaved. Swabs from both sources were positive by RT-PCR. Microwave treatment appears to be a satisfactory method of inactivating virus while preserving nucleic acid for PCR identification.

  9. In Vitro Resistance Study of AG-021541, a Novel Nonnucleoside Inhibitor of the Hepatitis C Virus RNA-Dependent RNA Polymerase

    Energy Technology Data Exchange (ETDEWEB)

    Shi, S.T.; Herlihy, K.J.; Graham, J.P.; Fuhrman, S.A.; Doan, C.; Parge, H.; Hickey, M.; Gao, J.; Yu, X.; Chau, F.; Gonzalez, J.; Li, H.; Lewis, C.; Patrick, A.K.; Duggal, R.

    2009-05-27

    A novel class of nonnucleoside hepatitis C virus (HCV) polymerase inhibitors characterized by a dihydropyrone core was identified by high-throughput screening. Crystallographic studies of these compounds in complex with the polymerase identified an allosteric binding site close to the junction of the thumb and finger domains, approximately 30 A away from the catalytic center. AG-021541, a representative compound from this series, displayed measurable in vitro antiviral activity against the HCV genotype 1b subgenomic replicon with a mean 50% effective concentration of 2.9 muM. To identify mutations conferring in vitro resistance to AG-021541, resistance selection was carried out using HCV replicon cells either by serial passages in increasing concentrations of AG-021541 or by direct colony formation at fixed concentrations of the compound. We identified several amino acid substitutions in the AG-021541-binding region of the polymerase, including M423(T/V/I), M426T, I482(S/T), and V494A, with M423T as the predominant change observed. These mutants conferred various levels of resistance to AG-021541 and structurally related compounds but remained sensitive to interferon and HCV polymerase inhibitors known to interact with the active site or other allosteric sites of the protein. In addition, dihydropyrone polymerase inhibitors retained activity against replicons that contain signature resistance changes to other polymerase inhibitors, including S282T, C316N, M414T, and P495(S/L), indicating their potential to be used in combination therapies with these polymerase inhibitors. AG-021541-resistant replicon cell lines provide a valuable tool for mechanism-of-action studies of dihydropyrone polymerase inhibitors. The clinical relevance of in vitro resistance to HCV polymerase inhibitors remains to be investigated.

  10. Isolation and characterization of the genes for two small RNAs of herpesvirus papio and their comparison with Epstein-Barr virus-encoded EBER RNAs.

    OpenAIRE

    Howe, J G; Shu, M D

    1988-01-01

    Genes for the Epstein-Barr virus-encoded RNAs (EBERs), two low-molecular-weight RNAs encoded by the human gammaherpesvirus Epstein-Barr virus (EBV), hybridize to two small RNAs in a baboon cell line that contains a similar virus, herpesvirus papio (HVP). The genes for the HVP RNAs (HVP-1 and HVP-2) are located together in the small unique region at the left end of the viral genome and are transcribed by RNA polymerase III in a rightward direction, similar to the EBERs. There is significant si...

  11. Rapid differentiation and identification of potential severe strains of Citrus tristeza virus by real-time reverse transcription-polymerase chain reaction assays.

    Science.gov (United States)

    Yokomi, R K; Saponari, M; Sieburth, P J

    2010-04-01

    A multiplex Taqman-based real-time reverse transcription (RT) polymerase chain reaction (PCR) assay was developed to identify potential severe strains of Citrus tristeza virus (CTV) and separate genotypes that react with the monoclonal antibody MCA13. Three strain-specific probes were developed using intergene sequences between the major and minor coat protein genes (CPi) in a multiplex reaction. Probe CPi-VT3 was designed for VT and T3 genotypes; probe CPi-T36 for T36 genotypes; and probe CPi-T36-NS to identify isolates in an outgroup clade of T36-like genotypes mild in California. Total nucleic acids extracted by chromatography on silica particles, sodium dodecyl sulfate-potassium acetate, and CTV virion immunocapture all yielded high quality templates for real-time PCR detection of CTV. These assays successfully differentiated CTV isolates from California, Florida, and a large panel of CTV isolates from an international collection maintained in Beltsville, MD. The utility of the assay was validated using field isolates collected in California and Florida.

  12. Development of a reverse transcription quantitative polymerase chain reaction-based assay for broad coverage detection of African and Asian Zika virus lineages.

    Science.gov (United States)

    Yang, Yang; Wong, Gary; Ye, Baoguo; Li, Shihua; Li, Shanqin; Zheng, Haixia; Wang, Qiang; Liang, Mifang; Gao, George F; Liu, Lei; Liu, Yingxia; Bi, Yuhai

    2017-06-01

    The Zika virus (ZIKV) is an arbovirus that has spread rapidly worldwide within recent times. There is accumulating evidence that associates ZIKV infections with Guillain-Barré Syndrome (GBS) and microcephaly in humans. The ZIKV is genetically diverse and can be separated into Asian and African lineages. A rapid, sensitive, and specific assay is needed for the detection of ZIKV across various pandemic regions. So far, the available primers and probes do not cover the genetic diversity and geographic distribution of all ZIKV strains. To this end, we have developed a one-step quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay based on conserved sequences in the ZIKV envelope (E) gene. The detection limit of the assay was determined to be five RNA transcript copies and 2.94 × 10-3 50% tissue culture infectious doses (TCID50) of live ZIKV per reaction. The assay was highly specific and able to detect five different ZIKV strains covering the Asian and African lineages without nonspecific amplification, when tested against other flaviviruses. The assay was also successful in testing for ZIKV in clinical samples. Our assay represents an improvement over the current methods available for the detection ZIKV and would be valuable as a diagnostic tool in various pandemic regions.

  13. The proximal sequence element (PSE) plays a major role in establishing the RNA polymerase specificity of Drosophila U-snRNA genes.

    Science.gov (United States)

    Jensen, R C; Wang, Y; Hardin, S B; Stumph, W E

    1998-01-15

    Most small nuclear RNA (snRNA) genes are transcribed by RNA polymerase II, but some (e.g., U6) are transcribed by RNA polymerase III. In vertebrates a TATA box at a fixed distance downstream of the proximal sequence element (PSE) acts as a dominant determinant for recruiting RNA polymerase III to U6 gene promoters. In contrast, vertebrate snRNA genes that contain a PSE but lack a TATA box are transcribed by RNA polymerase II. In plants, transcription of both classes of snRNA genes requires a TATA box in addition to an upstream sequence element (USE), and polymerase specificity is determined by the spacing between these two core promoter elements. In these examples, the PSE (or USE) is interchangeable between the two classes of snRNA genes. Here we report the surprising finding that the Drosophila U1 and U6 PSEs cannot functionally substitute for each other; rather, determination of RNA polymerase specificity is an intrinsic property of the PSE sequence itself. The alteration of two or three base pairs near the 3'-end of the U1 and U6 PSEs was sufficient to switch the RNA polymerase specificity of Drosophila snRNA promoters in vitro. These findings reveal a novel mechanism for achieving RNA polymerase specificity at insect snRNA promoters.

  14. Cervical cancer isolate PT3, super-permissive for adeno-associated virus replication, over-expresses DNA polymerase δ, PCNA, RFC and RPA

    Directory of Open Access Journals (Sweden)

    Melchert Russell B

    2009-04-01

    Full Text Available Abstract Background Adeno-associated virus (AAV type 2 is an important virus due to its use as a safe and effective human gene therapy vector and its negative association with certain malignancies. AAV, a dependo-parvovirus, autonomously replicates in stratified squamous epithelium. Such tissue occurs in the nasopharynx and anogenitals, from which AAV has been clinically isolated. Related autonomous parvoviruses also demonstrate cell tropism and preferentially replicate in oncogenically transformed cells. Combining these two attributes of parvovirus tropism, squamous and malignant, we assayed if AAV might replicate in squamous cervical carcinoma cell isolates. Results Three primary isolates (PT1-3 and two established cervical cancer cell lines were compared to normal keratinocytes (NK for their ability to replicate AAV. One isolate, PT3, allowed for high levels of AAV DNA replication and virion production compared to others. In research by others, four cellular components are known required for in vitro AAV DNA replication: replication protein A (RPA, replication factor C (RFC, proliferating cell nuclear antigen (PCNA, and DNA polymerase delta (POLD1. Thus, we examined PT3 cells for expression of these components by DNA microarray and real-time quantitative PCR. All four components were over-expressed in PT3 over two representative low-permissive cell isolates (NK and PT1. However, this super-permissiveness did not result in PT3 cell death by AAV infection. Conclusion These data, for the first time, provide evidence that these four cellular components are likely important for AAV in vivo DNA replication as well as in vitro. These data also suggest that PT3 will be a useful reagent for investigating the AAV-permissive transcriptome and AAV anti-cancer effect.

  15. Cervical cancer isolate PT3, super-permissive for adeno-associated virus replication, over-expresses DNA polymerase delta, PCNA, RFC and RPA.

    Science.gov (United States)

    Kang, Bum Yong; You, Hong; Bandyopadhyay, Sarmistha; Agrawal, Nalini; Melchert, Russell B; Basnakian, Alexei G; Liu, Yong; Hermonat, Paul L

    2009-04-23

    Adeno-associated virus (AAV) type 2 is an important virus due to its use as a safe and effective human gene therapy vector and its negative association with certain malignancies. AAV, a dependo-parvovirus, autonomously replicates in stratified squamous epithelium. Such tissue occurs in the nasopharynx and anogenitals, from which AAV has been clinically isolated. Related autonomous parvoviruses also demonstrate cell tropism and preferentially replicate in oncogenically transformed cells. Combining these two attributes of parvovirus tropism, squamous and malignant, we assayed if AAV might replicate in squamous cervical carcinoma cell isolates. Three primary isolates (PT1-3) and two established cervical cancer cell lines were compared to normal keratinocytes (NK) for their ability to replicate AAV. One isolate, PT3, allowed for high levels of AAV DNA replication and virion production compared to others. In research by others, four cellular components are known required for in vitro AAV DNA replication: replication protein A (RPA), replication factor C (RFC), proliferating cell nuclear antigen (PCNA), and DNA polymerase delta (POLD1). Thus, we examined PT3 cells for expression of these components by DNA microarray and real-time quantitative PCR. All four components were over-expressed in PT3 over two representative low-permissive cell isolates (NK and PT1). However, this super-permissiveness did not result in PT3 cell death by AAV infection. These data, for the first time, provide evidence that these four cellular components are likely important for AAV in vivo DNA replication as well as in vitro. These data also suggest that PT3 will be a useful reagent for investigating the AAV-permissive transcriptome and AAV anti-cancer effect.

  16. Loss of human ribosomal gene CpG methylation enhances cryptic RNA polymerase II transcription and disrupts ribosomal RNA processing.

    Science.gov (United States)

    Gagnon-Kugler, Thérèse; Langlois, Frédéric; Stefanovsky, Victor; Lessard, Frédéric; Moss, Tom

    2009-08-28

    Epigenetic methyl-CpG silencing of the ribosomal RNA (rRNA) genes is thought to downregulate rRNA synthesis in mammals. In contrast, we now show that CpG methylation in fact positively influences rRNA synthesis and processing. Human HCT116 cells, inactivated for DNMT1 and DNMT3b or treated with aza-dC, lack CpG methylation and reactivate a large fraction of normally silent rRNA genes. Unexpectedly, these cells display reduced rRNA synthesis and processing and accumulate unprocessed 45S rRNA. Reactivation of the rRNA genes is associated with their cryptic transcription by RNA polymerase II. Ectopic expression of cryptic rRNA gene transcripts recapitulates the defects associated with loss of CpG methylation. The data demonstrate that rRNA gene silencing prevents cryptic RNA polymerase II transcription of these genes. Lack of silencing leads to the partial disruption of rRNA synthesis and rRNA processing, providing an explanation for the cytotoxic effects of loss of CpG methylation.

  17. A mutation in the DNA polymerase accessory factor of herpes simplex virus 1 restores viral DNA replication in the presence of raltegravir.

    Science.gov (United States)

    Zhou, Bin; Yang, Kui; Wills, Elizabeth; Tang, Liang; Baines, Joel D

    2014-10-01

    Previous reports showed that raltegravir, a recently approved antiviral compound that targets HIV integrase, can inhibit the nuclease function of human cytomegalovirus (HCMV terminase) in vitro. In this study, subtoxic levels of raltegravir were shown to inhibit the replication of four different herpesviruses, herpes simplex virus 1 (HSV-1), HSV-2, HCMV, and mouse cytomegalovirus, by 30- to 700-fold, depending on the dose and the virus tested. Southern blotting and quantitative PCR revealed that raltegravir inhibits DNA replication of HSV-1 rather than cleavage of viral DNA. A raltegravir-resistant HSV-1 mutant was generated by repeated passage in the presence of 200 μM raltegravir. The genomic sequence of the resistant virus, designated clone 7, contained mutations in 16 open reading frames. Of these, the mutations F198S in unique long region 15 (UL15; encoding the large terminase subunit), A374V in UL32 (required for DNA cleavage and packaging), V296I in UL42 (encoding the DNA polymerase accessory factor), and A224S in UL54 (encoding ICP27, an important transcriptional regulator) were introduced independently into the wild-type HSV-1(F) genome, and the recombinant viruses were tested for raltegravir resistance. Viruses bearing both the UL15 and UL32 mutations inserted within the genome of the UL42 mutant were also tested. While the UL15, UL32, and UL54 mutant viruses were fully susceptible to raltegravir, any virus bearing the UL42 mutation was as resistant to raltegravir as clone 7. Overall, these results suggest that raltegravir may be a valuable therapeutic agent against herpesviruses and the antiviral activity targets the DNA polymerase accessory factor rather than the nuclease activity of the terminase. This paper shows that raltegravir, the antiretrovirus drug targeting integrase, is effective against various herpesviruses. Drug resistance mapped to the herpesvirus DNA polymerase accessory factor, which was an unexpected finding. Copyright © 2014

  18. Epstein-Barr virus DNAemia in Iranian liver transplant recipients and assessment of its variation in posttransplant lymphproliferative disorder patients by quantitative polymerase chain reaction assay.

    Science.gov (United States)

    Jamalidoust, Marzieh; Geramizadeh, Bita; Pouladfar, Gholamreza; Namayandeh, Mandana; Asaie, Sadaf; Aliabadi, Nasrin; Nikeghbalian, Saman; Ziyaeyan, Mazyar

    2015-04-01

    Epstein-Barr virus primary infection and/or reactivation may play a major role in the incidence of posttransplant lymphoproliferative disorder in organ recipients. We assessed Epstein-Barr virus viral load in liver transplant patients suspected of having Epstein-Barr virus/ posttransplant lymphoproliferative disorder at specified times after transplant and evaluated the clinical findings and posttransplant complications. In the 696 patients who underwent liver transplant in this retrospective study, Epstein-Barr virus viral load was examined intermittently in 127 liver transplant recipients who were suspected to have Epstein-Barr virus infection/disease. Sampling was performed during 4 years from July 2009 to May 2013 using real-time polymerase chain reaction assay. Clinical and pathologic data were gathered by reviewing medical records. There were 78 of the 127 suspected patients (61%) who exhibited Epstein-Barr virus DNAemia and 19 patients had posttransplant lymphoproliferative disorder. The median EBV viral load of posttransplant lymphoproliferative disorder patients was significantly higher than unaffected patients. Posttransplant lymphoproliferative disorder was diagnosed clinically in 34 subjects (4.9%). Estimated mortality rate of posttransplant lymphoproliferative disorder patients was 35% during 1.5-year follow-up after transplant. Monitoring Epstein-Barr virus load may enable detection of Epstein-Barr virus infection/disease in liver transplant patients suspected of having the virus, even several weeks before the onset of any clinical manifestations, especially in pediatric patients who have high incidence and mortality from posttransplant lymphoproliferative disorder.

  19. Aphids preserved in propylene glycol can be used for reverse transcription-polymerase chain reaction detection of Potato virus Y.

    Science.gov (United States)

    Nie, Xianzhou; Pelletier, Yvan; Mason, Nicola; Dilworth, Andrea; Giguère, Marie-Andrée

    2011-08-01

    The effectiveness of propylene glycol on the retention of RNA target of Potato virus Y (PVY), an aphid stylet-borne virus, in Myzus persicae was investigated in comparison to ethanol and liquid nitrogen/-80°C. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the PVY targets from the propylene glycol/ethanol/liquid nitrogen preserved single aphids after a 5min acquisition period from infected potato plants. In the liquid nitrogen/-80°C and 70% ethanol treatments, 55.6% and 38.8% aphids tested PVY-positive, respectively. In the 0-75% propylene glycol treatments, 12.2-44.7% aphids tested PVY-positive. The lowest detection rate was in the 0% (positive rate, 15.2%) and the 10% propylene glycol (positive rate, 12.2%). As the propylene glycol concentration increased to 25%, 29.8% aphids tested positive. A high PVY-positive rate was also found in 35-75% propylene glycol treatments at 44.7% (35% propylene glycol), 36.7% (50% propylene glycol) and 34.8% (75% propylene glycol), which is comparable to the rate shown in 70% ethanol. No significant difference in the positive detection rate was observed in aphids preserved in 50% propylene glycol at room temperature for 2, 4 and 10 days. These results demonstrate that propylene glycol at 25-75% can retain PVY targets effectively in aphids for an extended time period, and thus can be used in aphid traps to preserve viruliferous aphids for later RT-PCR detection of PVY. Crown Copyright © 2011. Published by Elsevier B.V. All rights reserved.

  20. Do Viruses Exchange Genes across Superkingdoms of Life?

    Directory of Open Access Journals (Sweden)

    Shahana S. Malik

    2017-10-01

    Full Text Available Viruses can be classified into archaeoviruses, bacterioviruses, and eukaryoviruses according to the taxonomy of the infected host. The host-constrained perception of viruses implies preference of genetic exchange between viruses and cellular organisms of their host superkingdoms and viral origins from host cells either via escape or reduction. However, viruses frequently establish non-lytic interactions with organisms and endogenize into the genomes of bacterial endosymbionts that reside in eukaryotic cells. Such interactions create opportunities for genetic exchange between viruses and organisms of non-host superkingdoms. Here, we take an atypical approach to revisit virus-cell interactions by first identifying protein fold structures in the proteomes of archaeoviruses, bacterioviruses, and eukaryoviruses and second by tracing their spread in the proteomes of superkingdoms Archaea, Bacteria, and Eukarya. The exercise quantified protein structural homologies between viruses and organisms of their host and non-host superkingdoms and revealed likely candidates for virus-to-cell and cell-to-virus gene transfers. Unexpected lifestyle-driven genetic affiliations between bacterioviruses and Eukarya and eukaryoviruses and Bacteria were also predicted in addition to a large cohort of protein folds that were universally shared by viral and cellular proteomes and virus-specific protein folds not detected in cellular proteomes. These protein folds provide unique insights into viral origins and evolution that are generally difficult to recover with traditional sequence alignment-dependent evolutionary analyses owing to the fast mutation rates of viral gene sequences.

  1. The role of alternative sigma factors of RNA polymerase in regulation of gene expression in Corynebacterium glutamicum

    OpenAIRE

    Šilar, Radoslav

    2016-01-01

    Abstract Regulation of transcription by extracytoplasmic-function (ECF) sigma factors of RNA polymerase is an efficient way of cell adaptation to diverse environmental stresses. Amino acid-producing gram-positive bacterium Corynebacterium glutamicum codes for seven sigma factors: the primary sigma factor SigA, the primary-like sigma factor SigB and five ECF stress- responsive sigma factors (SigC, SigD, SigE, SigH and SigM). The sigH gene encoding SigH sigma factor is located in a gene cluster...

  2. Viral microRNAs targeting virus genes promote virus infection in shrimp in vivo.

    Science.gov (United States)

    He, Yaodong; Yang, Kai; Zhang, Xiaobo

    2014-01-01

    Viral microRNAs (miRNAs), most of which are characterized in cell lines, have been found to play important roles in the virus life cycle to avoid attack by the host immune system or to keep virus in the latency state. Viral miRNAs targeting virus genes can inhibit virus infection. In this study, in vivo findings in Marsupenaeus japonicus shrimp revealed that the viral miRNAs could target virus genes and further promote the virus infection. The results showed that white spot syndrome virus (WSSV)-encoded miRNAs WSSV-miR-66 and WSSV-miR-68 were transcribed at the early stage of WSSV infection. When the expression of WSSV-miR-66 and WSSV-miR-68 was silenced with sequence-specific anti-miRNA oligonucleotides (AMOs), the number of copies of WSSV and the WSSV-infected shrimp mortality were significantly decreased, indicating that the two viral miRNAs had a great effect on virus infection. It was revealed that the WSSV wsv094 and wsv177 genes were the targets of WSSV-miR-66 and that the wsv248 and wsv309 genes were the targets of WSSV-miR-68. The data demonstrate that the four target genes play negative roles in the WSSV infection. The targeting of the four virus genes by WSSV-miR-66 and WSSV-miR-68 led to the promotion of virus infection. Therefore, our in vivo findings show a novel aspect of viral miRNAs in virus-host interactions.

  3. Resistance to tomato spotted wilt virus infection in transgenic tobacco expressing the viral nucleocapsid gene.

    Science.gov (United States)

    MacKenzie, D J; Ellis, P J

    1992-01-01

    A recombinant plasmid containing the entire tomato spotted with virus (TSWV) nucleocapsid gene, with the exception of nucleotide encoding three N-terminal amino acids, was isolated by screening a complementary DNA library, prepared against random primed viral RNA, using a specific monoclonal antibody. The insert contained in plasmid pTSW1 was repaired and amplified by polymerase chain reaction, and the complete nucleocapsid protein gene was introduced into Nicotiana tabacum 'Samsun' by leaf disk transformation using Agrobacterium tumefaciens. Transgenic plants expressing the viral nucleocapsid protein were resistant to subsequent infection following mechanical inoculation with TSWV as indicated by a lack of systemic symptoms and little or no systemic accumulation of virus as determined by double antibody sandwich enzyme-liked immunosorbent assay. These results further extend the applicability of coat protein-mediated resistance, as previously demonstrated for a number of simple plant viruses composed of a positive-sense RNA genome encapsidated with a single species of coat protein, to a membrane-encapsidated, multi-component, negative-sense RNA virus.

  4. Detection of African swine fever virus from formalin fixed and non-fixed tissues by polymerase chain reaction

    Directory of Open Access Journals (Sweden)

    P. D. Luka

    2014-10-01

    Full Text Available Aim: Formalin fixing and paraffin embedding of tissue samples is one of the techniques for preserving the structural integrity of cells for a very long time. However, extraction and analysis of genomic material from formalin fixed tissue (FFT remains a challenge despite numerous attempts to develop a more effective method. The success of polymerase chain reaction (PCR depends on the quality of DNA extract. Materials and Methods: Here we assessed the conventional method of DNA extraction from FFT for African swine fever virus (ASFV detection. The modified conventional method gave a higher quality DNA when compared with commercially available DNA extraction kits (QIAamp® DNA Mini Kit, DNeasy® Blood and Tissue Kit, and ZR Genomic DNA™ Tissue MiniPrep. Results: An average A260/A280 DNA purity of 0.86-1.68 and 3.22-5.32 μg DNA/mg for formalin fixed and non-fixed tissues, respectively using a conventional method. In a reproducible and three times repeat PCR, the ASFV DNA expected product size of 278 bp was obtained from the DNA extract of the conventional method but not from the DNA extract of the commercial kits. Conclusion: The present study has demonstrated that the conventional method extracts ASFV genome better than commercial kit. In summary, the commercial kit extraction appeared not suitable to purify ASFV DNA from FFT. We, therefore, recommend that the use of the conventional method be considered for African swine fever DNA extraction from FFT.

  5. Analysis of Respiratory Syncytial Virus in Clinical Samples by Reverse Transcriptase-Polymerase Chain Reaction Restriction Mapping

    Directory of Open Access Journals (Sweden)

    Angel Valdivia

    1997-05-01

    Full Text Available The aim of this study was to develop a polymerase chain reaction (PCR for the detection of respiratory syncytial virus (RSV genomes. The primers were designed from published sequences and selected from conserved regions of the genome encoding for the N protein of subgroups A and B of RSV. PCR was applied to 20 specimens from children admitted to the respiratory ward of "William Soler" Pediatric Hospital in Havana City with a clinical diagnosis of bronchiolitis. The PCR was compared with viral isolation and with an indirect immunofluorescence technique that employs monoclonal antibodies of subgroups A and B. Of 20 nasopharyngeal exudates, 10 were found positive by the three assayed methods. In only two cases, samples that yielded positive RNA-PCR were found negative by indirect immunofluorescence and cell culture. Considering viral isolation as the "gold standard" technique, RNA-PCR had 100% sensitivity and 80% specificity. RNA-PCR is a specific and sensitive technique for the detection of the RSV genome. Technical advantages are discussed

  6. Analysis of respiratory syncytial virus in clinical samples by reverse transcriptase-polymerase chain reaction restriction mapping.

    Science.gov (United States)

    Valdivia, A; Savón, C; Chacón, D; Sarmiento, L; Morier, L; Otero, A; Soto, Y; Oropesa, S; Goyenechea, A

    1997-01-01

    The aim of this study was to develop a polymerase chain reaction (PCR) for the detection of respiratory syncytial virus (RSV) genomes. The primers were designed from published sequences and selected from conserved regions of the genome encoding for the N protein of subgroups A and B of RSV. PCR was applied to 20 specimens from children admitted to the respiratory ward of "William Soler" Pediatric Hospital in Havana City with a clinical diagnosis of bronchiolitis. The PCR was compared with viral isolation and with an indirect immunofluorescence technique that employs monoclonal antibodies of subgroups A and B. Of 20 nasopharyngeal exudates, 10 were found positive by the three assayed methods. In only two cases, samples that yielded positive RNA-PCR were found negative by indirect immunofluorescence and cell culture. Considering viral isolation as the "gold standard" technique, RNA-PCR had 100% sensitivity and 80% specificity. RNA-PCR is a specific and sensitive technique for the detection of the RSV genome. Technical advantages are discussed.

  7. Direct polymerase chain reaction from blood and tissue samples for rapid diagnosis of bovine leukemia virus infection.

    Science.gov (United States)

    Nishimori, Asami; Konnai, Satoru; Ikebuchi, Ryoyo; Okagawa, Tomohiro; Nakahara, Ayako; Murata, Shiro; Ohashi, Kazuhiko

    2016-06-01

    Bovine leukemia virus (BLV) infection induces bovine leukemia in cattle and causes significant financial harm to farmers and farm management. There is no effective therapy or vaccine; thus, the diagnosis and elimination of BLV-infected cattle are the most effective method to eradicate the infection. Clinical veterinarians need a simpler and more rapid method of diagnosing infection, because both nested polymerase chain reaction (PCR) and real-time PCR are labor intensive, time-consuming, and require specialized molecular biology techniques and expensive equipment. In this study, we describe a novel PCR method for amplifying the BLV provirus from whole blood, thus eliminating the need for DNA extraction. Although the sensitivity of PCR directly from whole blood (PCR-DB) samples as measured in bovine blood containing BLV-infected cell lines was lower than that of nested PCR, the PCR-DB technique showed high specificity and reproducibility. Among 225 clinical samples, 49 samples were positive by nested PCR, and 37 samples were positive by PCR-DB. There were no false positive samples; thus, PCR-DB sensitivity and specificity were 75.51% and 100%, respectively. However, the provirus loads of the samples detected by nested PCR and not PCR-DB were quite low. Moreover, PCR-DB also stably amplified the BLV provirus from tumor tissue samples. PCR-DB method exhibited good reproducibility and excellent specificity and is suitable for screening of thousands of cattle, thus serving as a viable alternative to nested PCR and real-time PCR.

  8. Comparative analysis of hepatitis B virus polymerase sequences required for viral RNA binding, RNA packaging, and protein priming.

    Science.gov (United States)

    Jones, Scott A; Clark, Daniel N; Cao, Feng; Tavis, John E; Hu, Jianming

    2014-02-01

    Hepatitis B virus replicates a DNA genome through reverse transcription of a pregenomic RNA (pgRNA) by using a multifunctional polymerase (HP). A critical function of HP is its specific association with a viral RNA signal, termed ε (Hε), located on pgRNA, which is required for specific packaging of pgRNA into viral nucleocapsids and initiation of viral reverse transcription. HP initiates reverse transcription by using itself as a protein primer (protein priming) and Hε as the obligatory template. HP is made up of four domains, including the terminal protein (TP), the spacer, the reverse transcriptase (RT), and the RNase H domains. A recently developed, Hε-dependent, in vitro protein priming assay was used in this study to demonstrate that almost the entire TP and RT domains and most of the RNase H domain were required for protein priming. Specific residues within TP, RT, and the spacer were identified as being critical for HP-Hε binding and/or protein priming. Comparison of HP sequence requirements for Hε binding, pgRNA packaging, and protein priming allowed the classification of the HP mutants into five groups, each with distinct effects on these complex and related processes. Detailed characterization of HP requirements for these related and essential functions of HP will further elucidate the mechanisms of its multiple functions and aid in the targeting of these functions for antiviral therapy.

  9. Detection of Chloramphenicol Resistance Genes (cat in Clinical Isolates of Pseudomonas aeruginosa with Polymerase Chain Reaction Method

    Directory of Open Access Journals (Sweden)

    Tiana Milanda

    2014-12-01

    Full Text Available Pseudomonas aeruginosa is an opportunistic Gram negative bacteria, which may cause infection in eyes, ears, skin, bones, central nervous system, gastrointestinal tract, circulatory system, heart, respiratory system, and urinary tract. Recently, chloramphenicol is no longer used as the main option of the therapy due of its resistance case. The aim of this research was to detect the presence of gene which is responsible to chloramphenicol resistance in clinical isolates of P.aeruginosa. These bacteria isolated from pus of external otitis patients in Hasan Sadikin Hospital in Bandung City. Polymerase Chain Reaction (PCR method (colony-PCR and DNA-PCR were performed to detect this resistance gene. Electropherogram from PCR products showed that the chloramphenicol resistance in clinical isolates of P. aeruginosa was caused by cat gene (317 bp. Based on this research, cat gene may be used to detect the chloramphenicol resistance in patients with external ostitis.

  10. Rapid detection of cytomegalovirus in bronchoalveolar lavage fluid and serum samples by polymerase chain reaction: correlation of virus isolation and clinical outcome for patients with human immunodeficiency virus infection

    DEFF Research Database (Denmark)

    Hansen, K K; Vestbo, Jørgen; Benfield, T

    1997-01-01

    Bronchoalveolar lavage (BAL) fluids and serum samples from 153 patients with pulmonary symptoms who were infected with human immunodeficiency virus (HIV) and underwent BAL were examined for the presence of cytomegalovirus (CMV) by conventional culture and by polymerase chain reaction (PCR...

  11. Dynamic Blue Light-Inducible T7 RNA Polymerases (Opto-T7RNAPs) for Precise Spatiotemporal Gene Expression Control.

    Science.gov (United States)

    Baumschlager, Armin; Aoki, Stephanie K; Khammash, Mustafa

    2017-11-17

    Light has emerged as a control input for biological systems due to its precise spatiotemporal resolution. The limited toolset for light control in bacteria motivated us to develop a light-inducible transcription system that is independent from cellular regulation through the use of an orthogonal RNA polymerase. Here, we present our engineered blue light-responsive T7 RNA polymerases (Opto-T7RNAPs) that show properties such as low leakiness of gene expression in the dark state, high expression strength when induced with blue light, and an inducible range of more than 300-fold. Following optimization of the system to reduce expression variability, we created a variant that returns to the inactive dark state within minutes once the blue light is turned off. This allows for precise dynamic control of gene expression, which is a key aspect for most applications using optogenetic regulation. The regulators, which only require blue light from ordinary light-emitting diodes for induction, were developed and tested in the bacterium Escherichia coli, which is a crucial cell factory for biotechnology due to its fast and inexpensive cultivation and well understood physiology and genetics. Opto-T7RNAP, with minor alterations, should be extendable to other bacterial species as well as eukaryotes such as mammalian cells and yeast in which the T7 RNA polymerase and the light-inducible Vivid regulator have been shown to be functional. We anticipate that our approach will expand the applicability of using light as an inducer for gene expression independent from cellular regulation and allow for a more reliable dynamic control of synthetic and natural gene networks.

  12. Identification of a new region in the vesicular stomatitis virus L polymerase protein which is essential for mRNA cap methylation.

    Science.gov (United States)

    Grdzelishvili, Valery Z; Smallwood, Sherin; Tower, Dallas; Hall, Richard L; Hunt, D Margaret; Moyer, Sue A

    2006-07-05

    The vesicular stomatitis virus (VSV) L polymerase protein possesses two methyltransferase (MTase) activities, which catalyze the methylation of viral mRNA cap structures at the guanine-N7 and 2'-O-adenosine positions. To identify L sequences required for the MTase activities, we analyzed a host range (hr) and temperature-sensitive (ts) mutant of VSV, hr8, which was defective in mRNA cap methylation. Sequencing hr8 identified five amino acid substitutions, all residing in the L protein. Recombinant VSV were generated with each of the identified L mutations, and the presence of a single G1481R substitution in L, located between conserved domains V and VI, was sufficient to produce a dramatic reduction (about 90%) in overall mRNA methylation. Cap analysis showed residual guanine-N7 methylation and reduced 2'-O-adenosine methylation, identical to that of the original hr8 virus. When recombinant viruses were tested for virus growth under conditions that were permissive and nonpermissive for the hr8 mutant, the same single L mutation, G1481R, was solely responsible for both the hr and ts phenotypes. A spontaneous suppressor mutant of the rG1481R virus that restored both growth on nonpermissive cells and cap methylation was identified and mapped to a single change, L1450I, in L. Site-directed mutagenesis of the region between domains V and VI, amino acids 1419-1672 of L, followed by the rescue of recombinant viruses identified five additional virus mutants, K1468A, R1478A/D1479A, G1481A, G1481N, and G1672A, that were all hr and defective in mRNA cap methylation. Thus, in addition to the previously characterized domain VI [Grdzelishvili, V.Z., Smallwood, S., Tower, D., Hall, R.L., Hunt, D.M., Moyer, S.A., 2005. A single amino acid change in the L-polymerase protein of vesicular stomatitis virus completely abolishes viral mRNA cap methylation. J. Virol. 79, 7327-7337; Li, J., Fontaine-Rodriguez, E.C., Whelan, S.P., 2005. Amino acid residues within conserved domain VI of the

  13. Analysis of E2 gene integrity in HPV16 and HPV58 viruses isolated from women with cervical pathology

    OpenAIRE

    González-Losa,María del R; Puerto-Solis,Marylin; Tenorio Ruiz,Juan; Rosado-López,Ariel I; Hau-Aviles,Oscar; Ayora-Talavera,Guadalupe; Cisneros-Cutz,Isidro; Conde-Ferráez,Laura

    2016-01-01

    Integration of human papillomavirus (HPV) DNA into human cells accompanied by the disruption of the viral genome has been described as a prerequisite for cancer development. This study aimed to investigate E2 gene integrity of HPV16 and HPV58 viruses isolated from infected women with cervical lesions. Forty-two HPV16- and 31 HPV58-positive samples were analysed. E2 integrity was assumed when all fragments covering the E2 gene were amplified with specific polymerase chain reaction primers. Ove...

  14. Effects of poliovirus 2A(pro) on vaccinia virus gene expression.

    Science.gov (United States)

    Feduchi, E; Aldabe, R; Novoa, I; Carrasco, L

    1995-12-15

    The effects of transient expression of poliovirus 2A(pro) on p220 cleavage in COS cells have been analyzed. When 2A(pro) was cloned in plasmid pTM1 and transiently expressed in COS cells, efficient cleavage of p220 occurred after infection of these cells with a recombinant vaccinia virus bearing phage T7 RNA polymerase. High numbers of COS cells were transfected with pTM1-2A, as judged by p220 cleavage, thereby allowing an analysis of the effects of poliovirus 2A(pro) on vaccinia virus gene expression. A 40-50% cleavage of p220 by transfected poliovirus 2A(pro) was observed ten hours post infection and cleavage was almost complete (80-90%) 20-25 hours post infection with vaccinia virus. Profound inhibition of vaccinia virus protein synthesis was detectable ten hours post infection and was maximal 20-25 hours post infection. This inhibition resulted from neither a blockade of transcription of vaccinia virus nor a lack of translatability of the mRNAs present in cells that synthesize poliovirus 2A(pro). Addition of ara-C inhibited the replication of vaccinia virus and allowed the continued synthesis of cellular proteins. Under these conditions, 2A(pro) is expressed and blocks cellular translation. Finally, p220 cleavage by 2A(pro) did not inhibit the translation of a mRNA encoding poliovirus protein 2C, as directed by the 5' leader sequences of encephalomiocarditis virus. Therefore, these findings show a correlation between p220 cleavage and inhibition of translation from newly made mRNAs. Our results are discussed in the light of present knowledge of p220 function, and new approaches are considered that might provide further insights into the function(s) of initiation factor eIF-4F.

  15. Expression of VP60 gene from rabbit haemorrhagic disease virus ...

    African Journals Online (AJOL)

    The VP60 gene from rabbit haemorrhagic disease virus (RHDV) YL strain in Northeast of China, under control of the ats1A promoter from Rubisco small subunit genes of Arabidopsis thaliana, was introduced into the transfer deoxyribonucleic acid (T-DNA) region of plant transfer vector pCAMBIA1300 and transferred to ...

  16. Nucleotide sequence of the coat protein gene of the Skierniewice isolate of plum pox virus (PPV)

    Energy Technology Data Exchange (ETDEWEB)

    Wypijewski, K.; Musial, W.; Augustyniak, J. [Uniwersytet Adama Mickiewicza, Poznan (Poland); Malinowski, T. [Research Institute of Pomology and Floriculture, Skierniewice (Poland)

    1994-12-31

    The coat protein (CP) gene of the Skierniewice isolate of plum pox virus (PPV-S) has been amplified using the reverse transcription - polymerase chain reaction (RT-PCR), cloned and sequenced. The nucleotide sequence of the gene and the deduced amino-acid sequences of PPV-S CP were compared with those of other PPV strains. The nucleotide sequence showed very high homology to most of the published sequences. The motif: Asp-Ala-Gly (DAG), important for the aphid transmissibility, was present in the amino-acid sequence. Our isolate did not react in ELISA with monoclonal antibodies MAb06 supposed to be specific for PPV-D. (author). 32 refs, 1 fig., 2 tabs.

  17. Identification of Propionibacterium acnes by polymerase chain reaction for amplification of 16S ribosomal RNA and lipase genes.

    Science.gov (United States)

    Nakamura, Masahiko; Kametani, Ikuyo; Higaki, Shuichi; Yamagishi, Takayoshi

    2003-02-01

    Propionibacterium acnes belongs to the cutaneous flora and is present in sebaceous follicles. The fatty acids that are released from sebum triglycerides by the action of this bacterial lipase play an important role in the pathogenesis of acne vulgaris. P. acnes is also involved in postoperative disorders and opportunistic infections in immunosuppressed hosts. Recently, it has been proposed that P. acnes causes sarcoidosis. Therefore, rapid isolation and identification of P. acnes is important. This study evaluated the polymerase chain reaction (PCR) for the detection of the 16S rRNA and lipase genes of P. acnes. The PCR used to detect the 16S rRNA gene could amplify the gene of P. acnes, but not the genes of the other tested strains of P. avidum, P. granulosum, P. lymphophilum, P. jensenii, P. acidipropionici and P. thoenii. The PCR to detect the lipase gene of P. acnes, however, could amplify not only the gene of P. acnes but also that of P. avidum. The PCR product of this lipase gene was not found in the strains of the other species tested. Therefore, the organism that has both the 16S rRNA gene and lipase gene was identified as P. acnes, while the strain with the lipase gene but not the 16S rRNA gene of P. acnes was characterized as P. avidum. These findings were confirmed by the conventional biochemical tests including lipase activity. Furthermore, out of the seven clinical isolates from acne vulgaris, four were identified as P. acnes and three as P. avidum by the PCR method and biochemical tests. The combination of two PCR, one for the detection of the 16S rRNA and the other of lipase genes was shown to be an easier, faster and more accurate method to identify P. acnes and P. avidum than conventional methods.

  18. Whole blood gene expression in infants with respiratory syncytial virus bronchiolitis

    Directory of Open Access Journals (Sweden)

    Skjaeret Camilla

    2006-12-01

    Full Text Available Abstract Background Respiratory syncytial virus (RSV is a major cause of viral bronchiolitis in infants worldwide, and environmental, viral and host factors are all of importance for disease susceptibility and severity. To study the systemic host response to this disease we used the microarray technology to measure mRNA gene expression levels in whole blood of five male infants hospitalised with acute RSV, subtype B, bronchiolitis versus five one year old male controls exposed to RSV during infancy without bronchiolitis. The gene expression levels were further evaluated in a new experiment using quantitative real-time polymerase chain reaction (QRT-PCR both in the five infants selected for microarray and in 13 other infants hospitalised with the same disease. Results Among the 30 genes most differentially expressed by microarray nearly 50% were involved in immunological processes. We found the highly upregulated interferon, alpha-inducible protein 27 (IFI27 and the highly downregulated gene Charcot-Leyden crystal protein (CLC to be the two most differentially expressed genes in the microarray study. When performing QRT-PCR on these genes IFI27 was upregulated in all but one infant, and CLC was downregulated in all 18 infants, and similar to that given by microarray. Conclusion The gene IFI27 is upregulated and the gene CLC is downregulated in whole blood of infants hospitalised with RSV, subtype B, bronchiolitis and is not reported before. More studies are needed to elucidate the specificity of these gene expressions in association with host response to this virus in bronchiolitis of moderate severity.

  19. The Vpr gene polymorphism of human immunodeficiency virus type 1 in China and its clinical significance.

    Science.gov (United States)

    Chen, Xia; Zheng, Yuhuang; Li, Hui; Mamadou, Diallo; Zhang, Chunying; He, Yan; Zhou, Huaying; Chen, Zi; Liu, Meng

    2011-07-01

    Recent studies have explored that mutated Human immunodeficiency virus type 1(HIV-1) Vpr genes likely influence clinical manifestations of HIV infected patients. However, the relationship between the mutation sites on HIV Vpr gene and subsequent function changes is still not clear. In this study we investigated such relationship in analyzing the Vpr genes of HIV-1 viruses isolated from 208 HIV-1 infected patients from different regions in China. Reverse transcription polymerase chain reaction (RT-PCR) and nested PCR were used to amplify HIV-1 Vpr gene extracted from plasma of 208 HIV-1 infected patients and 153 isolates displayed the target gene sequences. Biological analysis software analyzed the deduced amino acid sequence, and identified the characteristics of the polymorphism of HIV-1 Vpr gene and its clinical significance. Results show the sequence subtypes as follows: CRF01-AE is 51.63%, subtype C is 24.84%, ubtype B is 17.65%, CRF03-AB is 3.92% and CRF08-BC is 1.31%. This paper revealed for the first time the HIV-1 Vpr gene polymorphism in HIV-1 positive individuals in China.: the subtype CRF01-AE is the main Vpr gene subtype in this region. The mutations in the C-terminal were more obvious than those observed in the N-terminal. It was also discovered that in the 77th position, 84.3% of the 153 amino acid sequences were glutamine (Q), which differ from overseas reports. Our data showed that the mutations 63, 70, 85, 86, 89 and 94 of the Vpr gene were possibly correlated with the clinical manifestations of the HIV-1 infected individuals.

  20. Establishment of Vero cell RNA polymerase I-driven reverse genetics for Influenza A virus and its application for pandemic (H1N1) 2009 influenza virus vaccine production.

    Science.gov (United States)

    Song, Min-Suk; Baek, Yun Hee; Pascua, Philippe Noriel Q; Kwon, Hyeok-Il; Park, Su-Jin; Kim, Eun-Ha; Lim, Gyo-Jin; Choi, Young-Ki

    2013-06-01

    The constant threat of newly emerging influenza viruses with pandemic potential requires the need for prompt vaccine production. Here, we utilized the Vero cell polymerase I (PolI) promoter, rather than the commonly used human PolI promoter, in an established reverse-genetics system to rescue viable influenza viruses in Vero cells, an approved cell line for human vaccine production. The Vero PolI promoter was more efficient in Vero cells and demonstrated enhanced transcription levels and virus rescue rates commensurate with that of the human RNA PolI promoter in 293T cells. These results appeared to be associated with more efficient generation of A(H1N1)pdm09- and H5N1-derived vaccine seed viruses in Vero cells, whilst the rescue rates in 293T cells were comparable. Our study provides an alternative means for improving vaccine preparation by using a novel reverse-genetics system for generating influenza A viruses.

  1. Reference gene selection for quantitative real-time PCR analysis in virus infected cells: SARS corona virus, Yellow fever virus, Human Herpesvirus-6, Camelpox virus and Cytomegalovirus infections

    Directory of Open Access Journals (Sweden)

    Müller Marcel A

    2005-02-01

    Full Text Available Abstract Ten potential reference genes were compared for their use in experiments investigating cellular mRNA expression of virus infected cells. Human cell lines were infected with Cytomegalovirus, Human Herpesvirus-6, Camelpox virus, SARS coronavirus or Yellow fever virus. The expression levels of these genes and the viral replication were determined by real-time PCR. Genes were ranked by the BestKeeper tool, the GeNorm tool and by criteria we reported previously. Ranking lists of the genes tested were tool dependent. However, over all, β-actin is an unsuitable as reference gene, whereas TATA-Box binding protein and peptidyl-prolyl-isomerase A are stable reference genes for expression studies in virus infected cells.

  2. Collaborative ring trial of the papaya endogenous reference gene and its polymerase chain reaction assays for genetically modified organism analysis.

    Science.gov (United States)

    Wei, Jiaojun; Li, Feiwu; Guo, Jinchao; Li, Xiang; Xu, Junfeng; Wu, Gang; Zhang, Dabing; Yang, Litao

    2013-11-27

    The papaya (Carica papaya L.) Chymopapain (CHY) gene has been reported as a suitable endogenous reference gene for genetically modified (GM) papaya detection in previous studies. Herein, we further validated the use of the CHY gene and its qualitative and quantitative polymerase chain reaction (PCR) assays through an interlaboratory collaborative ring trial. A total of 12 laboratories working on detection of genetically modified organisms participated in the ring trial and returned test results. Statistical analysis of the returned results confirmed the species specificity, low heterogeneity, and single-copy number of the CHY gene among different papaya varieties. The limit of detection of the CHY qualitative PCR assay was 0.1%, while the limit of quantification of the quantitative PCR assay was ∼25 copies of haploid papaya genome with acceptable PCR efficiency and linearity. The differences between the tested and true values of papaya content in 10 blind samples ranged from 0.84 to 6.58%. These results indicated that the CHY gene was suitable as an endogenous reference gene for the identification and quantification of GM papaya.

  3. Distinct co-evolution patterns of genes associated to DNA polymerase III DnaE and PolC

    Directory of Open Access Journals (Sweden)

    Engelen Stefan

    2012-02-01

    Full Text Available Abstract Background Bacterial genomes displaying a strong bias between the leading and the lagging strand of DNA replication encode two DNA polymerases III, DnaE and PolC, rather than a single one. Replication is a highly unsymmetrical process, and the presence of two polymerases is therefore not unexpected. Using comparative genomics, we explored whether other processes have evolved in parallel with each polymerase. Results Extending previous in silico heuristics for the analysis of gene co-evolution, we analyzed the function of genes clustering with dnaE and polC. Clusters were highly informative. DnaE co-evolves with the ribosome, the transcription machinery, the core of intermediary metabolism enzymes. It is also connected to the energy-saving enzyme necessary for RNA degradation, polynucleotide phosphorylase. Most of the proteins of this co-evolving set belong to the persistent set in bacterial proteomes, that is fairly ubiquitously distributed. In contrast, PolC co-evolves with RNA degradation enzymes that are present only in the A+T-rich Firmicutes clade, suggesting at least two origins for the degradosome. Conclusion DNA replication involves two machineries, DnaE and PolC. DnaE co-evolves with the core functions of bacterial life. In contrast PolC co-evolves with a set of RNA degradation enzymes that does not derive from the degradosome identified in gamma-Proteobacteria. This suggests that at least two independent RNA degradation pathways existed in the progenote community at the end of the RNA genome world.

  4. Pathogenicity and transmissibility of novel reassortant H3N2 influenza viruses with 2009 pandemic H1N1 genes in pigs.

    Science.gov (United States)

    Ma, Jingjiao; Shen, Huigang; Liu, Qinfang; Bawa, Bhupinder; Qi, Wenbao; Duff, Michael; Lang, Yuekun; Lee, Jinhwa; Yu, Hai; Bai, Jianfa; Tong, Guangzhi; Hesse, Richard A; Richt, Jürgen A; Ma, Wenjun

    2015-03-01

    At least 10 different genotypes of novel reassortant H3N2 influenza viruses with 2009 pandemic H1N1 [A(H1N1)pdm09] gene(s) have been identified in U.S. pigs, including the H3N2 variant with a single A(H1N1)pdm09 M gene, which has infected more than 300 people. To date, only three genotypes of these viruses have been evaluated in animal models, and the pathogenicity and transmissibility of the other seven genotype viruses remain unknown. Here, we show that three H3N2 reassortant viruses that contain 3 (NP, M, and NS) or 5 (PA, PB2, NP, M, and NS) genes from A(H1N1)pdm09 were pathogenic in pigs, similar to the endemic H3N2 swine virus. However, the reassortant H3N2 virus with 3 A(H1N1)pdm09 genes and a recent human influenza virus N2 gene was transmitted most efficiently among pigs, whereas the reassortant H3N2 virus with 5 A(H1N1)pdm09 genes was transmitted less efficiently than the endemic H3N2 virus. Interestingly, the polymerase complex of reassortant H3N2 virus with 5 A(H1N1)pdm09 genes showed significantly higher polymerase activity than those of endemic and reassortant H3N2 viruses with 3 A(H1N1)pdm09 genes. Further studies showed that an avian-like glycine at position 228 at the hemagglutinin (HA) receptor binding site is responsible for inefficient transmission of the reassortant H3N2 virus with 5 A(H1N1)pdm09 genes. Taken together, our results provide insights into the pathogenicity and transmissibility of novel reassortant H3N2 viruses in pigs and suggest that a mammalian-like serine at position 228 in the HA is critical for the transmissibility of these reassortant H3N2 viruses. Swine influenza is a highly contagious zoonotic disease that threatens animal and public health. Introduction of 2009 pandemic H1N1 virus [A(H1N1)pdm09] into swine herds has resulted in novel reassortant influenza viruses in swine, including H3N2 and H1N2 variants that have caused human infections in the United States. We showed that reassortant H3N2 influenza viruses with 3 or 5

  5. Sequence and Structure Analysis of Distantly-Related Viruses Reveals Extensive Gene Transfer between Viruses and Hosts and among Viruses

    Science.gov (United States)

    Caprari, Silvia; Metzler, Saskia; Lengauer, Thomas; Kalinina, Olga V.

    2015-01-01

    The origin and evolution of viruses is a subject of ongoing debate. In this study, we provide a full account of the evolutionary relationships between proteins of significant sequence and structural similarity found in viruses that belong to different classes according to the Baltimore classification. We show that such proteins can be found in viruses from all Baltimore classes. For protein families that include these proteins, we observe two patterns of the taxonomic spread. In the first pattern, they can be found in a large number of viruses from all implicated Baltimore classes. In the other pattern, the instances of the corresponding protein in species from each Baltimore class are restricted to a few compact clades. Proteins with the first pattern of distribution are products of so-called viral hallmark genes reported previously. Additionally, this pattern is displayed by the envelope glycoproteins from Flaviviridae and Bunyaviridae and helicases of superfamilies 1 and 2 that have homologs in cellular organisms. The second pattern can often be explained by horizontal gene transfer from the host or between viruses, an example being Orthomyxoviridae and Coronaviridae hemagglutinin esterases. Another facet of horizontal gene transfer comprises multiple independent introduction events of genes from cellular organisms into otherwise unrelated viruses. PMID:26492264

  6. Transcriptional organization and in vivo role of the Escherichia coli rsd gene, encoding the regulator of RNA polymerase sigma D.

    Science.gov (United States)

    Jishage, M; Ishihama, A

    1999-06-01

    The regulator of sigma D (Rsd) was identified as an RNA polymerase sigma70-associated protein in stationary-phase Escherichia coli with the inhibitory activity of sigma70-dependent transcription in vitro (M. Jishage and A. Ishihama, Proc. Natl. Acad. Sci. USA 95:4953-4958, 1998). Primer extension analysis of rsd mRNA indicated the presence of two promoters, sigmaS-dependent P1 and sigma70-dependent P2 with the gearbox sequence. To get insight into the in vivo role of Rsd, the expression of a reporter gene fused to either the sigma70- or sigmaS-dependent promoter was analyzed in the absence of Rsd or the presence of overexpressed Rsd. In the rsd null mutant, the sigma70- and sigmaS-dependent gene expression was increased or decreased, respectively. On the other hand, the sigma70- or sigmaS-dependent transcription was reduced or enhanced, respectively, after overexpression of Rsd. The repression of the sigmaS-dependent transcription in the rsd mutant is overcome by increased production of the sigmaS subunit. Together these observations support the prediction that Rsd is involved in replacement of the RNA polymerase sigma subunit from sigma70 to sigmaS during the transition from exponential growth to the stationary phase.

  7. Mumps virus F gene and HN gene sequencing as a molecular tool to study mumps virus transmission.

    Science.gov (United States)

    Gouma, Sigrid; Cremer, Jeroen; Parkkali, Saara; Veldhuijzen, Irene; van Binnendijk, Rob S; Koopmans, Marion P G

    2016-11-01

    Various mumps outbreaks have occurred in the Netherlands since 2004, particularly among persons who had received 2 doses of measles, mumps, and rubella (MMR) vaccination. Genomic typing of pathogens can be used to track outbreaks, but the established genotyping of mumps virus based on the small hydrophobic (SH) gene sequences did not provide sufficient resolution. Therefore, we expanded the sequencing to include fusion (F) gene and haemagglutinin-neuraminidase (HN) gene sequences in addition to the SH gene sequences from 109 mumps virus genotype G strains obtained between 2004 and mid 2015 in the Netherlands. When the molecular information from these 3 genes was combined, we were able to identify separate mumps virus clusters and track mumps virus transmission. The analyses suggested that multiple mumps virus introductions occurred in the Netherlands between 2004 and 2015 resulting in several mumps outbreaks throughout this period, whereas during some local outbreaks the molecular data pointed towards endemic circulation. Combined analysis of epidemiological data and sequence data collected in 2015 showed good support for the phylogenetic clustering. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Moloney murine sarcoma virus MuSVts110 DNA: cloning, nucleotide sequence, and gene expression.

    Science.gov (United States)

    Huai, L; Chiocca, S M; Gilbreth, M A; Ainsworth, J R; Bishop, L A; Murphy, E C

    1992-09-01

    We have cloned Moloney murine sarcoma virus (MuSV) MuSVts110 DNA by assembly of polymerase chain reaction (PCR)-amplified segments of integrated viral DNA from infected NRK cells (6m2 cells) and determined its complete sequence. Previously, by direct sequencing of MuSVts110 RNA transcribed in 6m2 cells, we established that the thermosensitive RNA splicing phenotype uniquely characteristic of MuSVts110 results from a deletion of 1,487 nucleotides of progenitor MuSV-124 sequences. As anticipated, the sequence obtained in this study contained precisely this same deletion. In addition, several other unexpected sequence differences were found between MuSVts110 and MuSV-124. For example, in the noncoding region upstream of the gag gene, MuSVts110 DNA contained a 52-nucleotide tract typical of murine leukemia virus rather than MuSV-124, suggesting that MuSVts110 originated as a MuSV-helper murine leukemia virus recombinant during reverse transcription rather than from a straightforward deletion within MuSV-124. In addition, both MuSVts110 long terminal repeats contained head-to-tail duplications of eight nucleotides in the U3 region. Finally, seven single-nucleotide substitutions were found scattered throughout MuSVts110 DNA. Three of the nucleotide substitutions were in the gag gene, resulting in one coding change in p15 and one in p30. All of the remaining nucleotide changes were found in the noncoding region between the 5' long terminal repeat and the gag gene. In NIH 3T3 cells transfected with the cloned MuSVts110 DNA, the pattern of viral RNA expression conformed with that observed in cells infected with authentic MuSVts110 virus in that viral RNA splicing was 30 to 40% efficient at growth temperatures between 28 and 33 degrees C but reduced to trace levels above 37 degrees C.

  9. The immunomodulatory gene products of myxoma virus

    Indian Academy of Sciences (India)

    Unknown

    vity regulates innate immunity. Moreover, TNF-α is in- volved in the regulation of cell differentiation, prolifera- tion and apoptosis. Rabbit RL-5 T lymphocyte cells in- fected with an M-T2 knock-out myxoma virus undergo apoptosis, resulting in an aborted infection (Macen et al. 1996). These results suggest that the binding of ...

  10. Prevalence of Tobacco mosaic virus in Iran and Evolutionary Analyses of the Coat Protein Gene

    Directory of Open Access Journals (Sweden)

    Athar Alishiri

    2013-09-01

    Full Text Available The incidence and distribution of Tobacco mosaic virus (TMV and related tobamoviruses was determined using an enzyme-linked immunosorbent assay on 1,926 symptomatic horticultural crops and 107 asymptomatic weed samples collected from 78 highly infected fields in the major horticultural crop-producing areas in 17 provinces throughout Iran. The results were confirmed by host range studies and reverse transcription-polymerase chain reaction. The overall incidence of infection by these viruses in symptomatic plants was 11.3%. The coat protein (CP gene sequences of a number of isolates were determined and disclosed to be a high identity (up to 100% among the Iranian isolates. Phylogenetic analysis of all known TMV CP genes showed three clades on the basis of nucleotide sequences with all Iranian isolates distinctly clustered in clade II. Analysis using the complete CP amino acid sequence showed one clade with two subgroups, IA and IB, with Iranian isolates in both subgroups. The nucleotide diversity within each sub-group was very low, but higher between the two clades. No correlation was found between genetic distance and geographical origin or host species of isolation. Statistical analyses suggested a negative selection and demonstrated the occurrence of gene flow from the isolates in other clades to the Iranian population.

  11. Analyzing the gene expression profile of anaplastic histology Wilms' tumor with real-time polymerase chain reaction arrays.

    Science.gov (United States)

    Lu, Jun; Tao, Yan-Fang; Li, Zhi-Heng; Cao, Lan; Hu, Shao-Yan; Wang, Na-Na; Du, Xiao-Juan; Sun, Li-Chao; Zhao, Wen-Li; Xiao, Pei-Fang; Fang, Fang; Xu, Li-Xiao; Li, Yan-Hong; Li, Gang; Zhao, He; Ni, Jian; Wang, Jian; Feng, Xing; Pan, Jian

    2015-01-01

    Wilms' tumor (WT) is one of the most common malignant neoplasms of the urinary tract in children. Anaplastic histology (unfavorable histology) accounts for about 10% of whole WTs, and it is the single most important histologic predictor of treatment response and survival in patients with WT; however, until now the molecular basis of this phenotype is not very clearly. A real-time polymerase chain reaction (PCR) array was designed and tested. Next, the gene expression profile of pediatric anaplastic histology WT and normal adjacent tissues were analyzed. These expression data were anlyzed with Multi Experiment View (MEV) cluster software further. Datasets representing genes with altered expression profiles derived from cluster analyses were imported into the Ingenuity Pathway Analysis Tool (IPA). 88 real-time PCR primer pairs for quantitative gene expression analysis of key genes involved in pediatric anaplastic histology WT were designed and tested. The gene expression profile of pediatric anaplastic histology WT is significantly different from adjacent normal controls; we identified 15 genes that are up-regulated and 16 genes that are down-regulated in the former. To investigate biological interactions of these differently regulated genes, datasets representing genes with altered expression profiles were imported into the IPA for further analysis, which revealed three significant networks: Cancer, Hematological Disease, and Gene Expression, which included 27 focus molecules and a significance score of 43. The IPA analysis also grouped the differentially expressed genes into biological mechanisms related to Cell Death and Survival 1.15E(-12), Cellular Development 2.84E(-11), Cellular Growth and Proliferation 2.84E(-11), Gene Expression 4.43E(-10), and DNA Replication, Recombination, and Repair 1.39E(-07). The important upstream regulators of pediatric anaplastic histology WT were TP53 and TGFβ1 signaling (P = 1.15E(-14) and 3.79E(-13), respectively). Our study

  12. Complete nucleotide sequence analysis of Cymbidium mosaic virus ...

    Indian Academy of Sciences (India)

    Phylogenetic analyses on the basis of RNA-dependent RNA polymerase (RdRp), triple gene block protein and coat protein (CP) amino acid sequences revealed that CymMV is closely related to the Narcissus mosaic virus (NMV), Scallion virus X (SVX), Pepino mosaic virus (PepMV) and Potato aucuba mosaic virus (PAMV).

  13. Comparison of the effect of Carbovir, AZT, and dideoxynucleoside triphosphates on the activity of human immunodeficiency virus reverse transcriptase and selected human polymerases.

    Science.gov (United States)

    White, E L; Parker, W B; Macy, L J; Shaddix, S C; McCaleb, G; Secrist, J A; Vince, R; Shannon, W M

    1989-06-15

    Carbocylic 2',3'-didehydro-2',3'-dideoxyguanosine (Carbovir; NSC 614846) is an antiretroviral agent which may be useful in the treatment of AIDS. We have synthesized the 5'-triphosphate of Carbovir and examined its ability to inhibit human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (EC 2.7.7.49) and other retroviral reverse transcriptases, as well as human DNA polymerases alpha, beta, gamma (EC 2.7.7.7) and DNA primase (EC 2.7.7.6). Carbovir triphosphate emerges as a highly selective inhibitor of reverse transcriptases with little, if any, effect on the cellular enzymes. 3'-Azido-2',3'-dideoxythymidine (AZT) triphosphate and the two dideoxynucleoside triphosphates, ddTTP and ddGTP, inhibited HIV-1 reverse transcriptase to the same degree as Carbovir triphosphate, but were less selective in that they also inhibited DNA polymerases beta and gamma. We conclude that Carbovir is a highly selective antiretroviral agent.

  14. 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.

  15. Gene molecular study of biofilm of Staphylococcus aureus isolated from fresh milk using multiplex polymerase chain reaction

    Directory of Open Access Journals (Sweden)

    Atefeh Mahmoudi-Kojedi

    2017-08-01

    Full Text Available Background: Staphylococcus aureus is one of the main causes of food poisoning in the world. This pathogen has the ability to create biofilms that can lead to food contamination. The presence of biofilm genes in bacteria is very important. The aim of this study was to identify sticky genes (eno, cna, ebp, bbp that play an important role in virulence and pathogenicity of the bacteria and even prevent the penetration of antibiotics in pathogenicity time. Materials and Methods: A total of 100 samples of fresh milk were collected from live animals and 60 isolates were selected to identify sticky genes (eno, cna, ebp, bbp in the production of biofilm of S. aureus using the multiplex polymerase chain reaction method. In addition, the frequency rates of S. aureus strains resistant and susceptible to antibiotics such as methicillin, vancomycin, and clindamycin were determined among the samples. Results: From a total of 60 isolates of fresh milk, 43.4% of the colonies had laminin-binding protein gene or eno gene. Also, 90% of the isolates were sensitive to vancomycin, 50% sensitive to clindamycin and 43.4% sensitive to methicillin. Distribution rates of other sticky genes including ebp, cna, bbp were 11.6%, 20% and 25%, respectively. Molecular study results showed that the highest and lowest percentages of genes were related to the eno and bbp genes, respectively. Conclusion: The present study shows that the maximum sensitivity of the samples (90% was related to vancomycin and the least amount of sensitivity (43.3% was related to methicillin.

  16. Evaluation of different embryonating bird eggs and cell cultures for isolation efficiency of avian influenza A virus and avian paramyxovirus serotype 1 from real-time reverse transcription polymerase chain reaction--positive

    Science.gov (United States)

    Two hundred samples collected from Anseriformes, Charadriiformes, Gruiformes, and Galliformes were assayed using real-time reverse transcriptase polymerase chain reaction (RRT-PCR) for presence of avian influenza virus and avian paramyxovirus-1. Virus isolation using embryonating chicken eggs, embr...

  17. Zn(2+ inhibits coronavirus and arterivirus RNA polymerase activity in vitro and zinc ionophores block the replication of these viruses in cell culture.

    Directory of Open Access Journals (Sweden)

    Aartjan J W te Velthuis

    Full Text Available Increasing the intracellular Zn(2+ concentration with zinc-ionophores like pyrithione (PT can efficiently impair the replication of a variety of RNA viruses, including poliovirus and influenza virus. For some viruses this effect has been attributed to interference with viral polyprotein processing. In this study we demonstrate that the combination of Zn(2+ and PT at low concentrations (2 µM Zn(2+ and 2 µM PT inhibits the replication of SARS-coronavirus (SARS-CoV and equine arteritis virus (EAV in cell culture. The RNA synthesis of these two distantly related nidoviruses is catalyzed by an RNA-dependent RNA polymerase (RdRp, which is the core enzyme of their multiprotein replication and transcription complex (RTC. Using an activity assay for RTCs isolated from cells infected with SARS-CoV or EAV--thus eliminating the need for PT to transport Zn(2+ across the plasma membrane--we show that Zn(2+ efficiently inhibits the RNA-synthesizing activity of the RTCs of both viruses. Enzymatic studies using recombinant RdRps (SARS-CoV nsp12 and EAV nsp9 purified from E. coli subsequently revealed that Zn(2+ directly inhibited the in vitro activity of both nidovirus polymerases. More specifically, Zn(2+ was found to block the initiation step of EAV RNA synthesis, whereas in the case of the SARS-CoV RdRp elongation was inhibited and template binding reduced. By chelating Zn(2+ with MgEDTA, the inhibitory effect of the divalent cation could be reversed, which provides a novel experimental tool for in vitro studies of the molecular details of nidovirus replication and transcription.

  18. Zn(2+) inhibits coronavirus and arterivirus RNA polymerase activity in vitro and zinc ionophores block the replication of these viruses in cell culture.

    Science.gov (United States)

    te Velthuis, Aartjan J W; van den Worm, Sjoerd H E; Sims, Amy C; Baric, Ralph S; Snijder, Eric J; van Hemert, Martijn J

    2010-11-04

    Increasing the intracellular Zn(2+) concentration with zinc-ionophores like pyrithione (PT) can efficiently impair the replication of a variety of RNA viruses, including poliovirus and influenza virus. For some viruses this effect has been attributed to interference with viral polyprotein processing. In this study we demonstrate that the combination of Zn(2+) and PT at low concentrations (2 µM Zn(2+) and 2 µM PT) inhibits the replication of SARS-coronavirus (SARS-CoV) and equine arteritis virus (EAV) in cell culture. The RNA synthesis of these two distantly related nidoviruses is catalyzed by an RNA-dependent RNA polymerase (RdRp), which is the core enzyme of their multiprotein replication and transcription complex (RTC). Using an activity assay for RTCs isolated from cells infected with SARS-CoV or EAV--thus eliminating the need for PT to transport Zn(2+) across the plasma membrane--we show that Zn(2+) efficiently inhibits the RNA-synthesizing activity of the RTCs of both viruses. Enzymatic studies using recombinant RdRps (SARS-CoV nsp12 and EAV nsp9) purified from E. coli subsequently revealed that Zn(2+) directly inhibited the in vitro activity of both nidovirus polymerases. More specifically, Zn(2+) was found to block the initiation step of EAV RNA synthesis, whereas in the case of the SARS-CoV RdRp elongation was inhibited and template binding reduced. By chelating Zn(2+) with MgEDTA, the inhibitory effect of the divalent cation could be reversed, which provides a novel experimental tool for in vitro studies of the molecular details of nidovirus replication and transcription.

  19. Zn2+ Inhibits Coronavirus and Arterivirus RNA Polymerase Activity In Vitro and Zinc Ionophores Block the Replication of These Viruses in Cell Culture

    Science.gov (United States)

    te Velthuis, Aartjan J. W.; van den Worm, Sjoerd H. E.; Sims, Amy C.; Baric, Ralph S.; Snijder, Eric J.; van Hemert, Martijn J.

    2010-01-01

    Increasing the intracellular Zn2+ concentration with zinc-ionophores like pyrithione (PT) can efficiently impair the replication of a variety of RNA viruses, including poliovirus and influenza virus. For some viruses this effect has been attributed to interference with viral polyprotein processing. In this study we demonstrate that the combination of Zn2+ and PT at low concentrations (2 µM Zn2+ and 2 µM PT) inhibits the replication of SARS-coronavirus (SARS-CoV) and equine arteritis virus (EAV) in cell culture. The RNA synthesis of these two distantly related nidoviruses is catalyzed by an RNA-dependent RNA polymerase (RdRp), which is the core enzyme of their multiprotein replication and transcription complex (RTC). Using an activity assay for RTCs isolated from cells infected with SARS-CoV or EAV—thus eliminating the need for PT to transport Zn2+ across the plasma membrane—we show that Zn2+ efficiently inhibits the RNA-synthesizing activity of the RTCs of both viruses. Enzymatic studies using recombinant RdRps (SARS-CoV nsp12 and EAV nsp9) purified from E. coli subsequently revealed that Zn2+ directly inhibited the in vitro activity of both nidovirus polymerases. More specifically, Zn2+ was found to block the initiation step of EAV RNA synthesis, whereas in the case of the SARS-CoV RdRp elongation was inhibited and template binding reduced. By chelating Zn2+ with MgEDTA, the inhibitory effect of the divalent cation could be reversed, which provides a novel experimental tool for in vitro studies of the molecular details of nidovirus replication and transcription. PMID:21079686

  20. Virus infection triggers widespread silencing of host genes by a distinct class of endogenous siRNAs in Arabidopsis.

    Science.gov (United States)

    Cao, Mengji; Du, Peng; Wang, Xianbing; Yu, Yun-Qi; Qiu, Yan-Hong; Li, Wanxiang; Gal-On, Amit; Zhou, Changyong; Li, Yi; Ding, Shou-Wei

    2014-10-07

    Antiviral immunity controlled by RNA interference (RNAi) in plants and animals is thought to specifically target only viral RNAs by the virus-derived small interfering RNAs (siRNAs). Here we show that activation of antiviral RNAi in Arabidopsis plants is accompanied by the production of an abundant class of endogenous siRNAs mapped to the exon regions of more than 1,000 host genes and rRNA. These virus-activated siRNAs (vasiRNAs) are predominantly 21 nucleotides long with an approximately equal ratio of sense and antisense strands. Genetically, vasiRNAs are distinct from the known plant endogenous siRNAs characterized to date and instead resemble viral siRNAs by requiring Dicer-like 4 and RNA-dependent RNA polymerase 1 (RDR1) for biogenesis. However, loss of exoribonuclease4/thylene-insensitive5 enhances vasiRNA biogenesis and virus resistance without altering the biogenesis of viral siRNAs. We show that vasiRNAs are active in directing widespread silencing of the target host genes and that Argonaute-2 binds to and is essential for the silencing activity of vasiRNAs. Production of vasiRNAs is readily detectable in Arabidopsis after infection by viruses from two distinct supergroups of plant RNA virus families and is targeted for inhibition by the silencing suppressor protein 2b of Cucumber mosaic virus. These findings reveal RDR1 production of Arabidopsis endogenous siRNAs and identify production of vasiRNAs to direct widespread silencing of host genes as a conserved response of plants to infection by diverse viruses. A possible function for vasiRNAs to confer broad-spectrum antiviral activity distinct to the virus-specific antiviral RNAi by viral siRNAs is discussed.

  1. H9N2 influenza A virus isolated from a Greater White-fronted wild goose (Anser albifrons) in Alaska has a mutation in the PB2 gene, which is associated with pathogenicity in human pandemic 2009 H1N1

    Science.gov (United States)

    Reeves, Andrew; Ip, Hon S.

    2016-01-01

    We report here the genomic sequence of an H9N2 influenza A virus [A/greater white-fronted goose/Alaska/81081/2008 (H9N2)]. This virus shares ≥99.8% identity with a previously reported virus. Both strains contain a G590S mutation in the polymerase basic 2 (PB2) gene, which is a pathogenicity marker in the pandemic 2009 H1N1 virus when combined with R591.

  2. The development of a real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay using TaqMan technology for the pan detection of bluetongue virus (BTV).

    Science.gov (United States)

    Mulholland, Catherine; McMenamy, Michael J; Hoffmann, Bernd; Earley, Bernadette; Markey, Bryan; Cassidy, Joseph; Allan, Gordon; Welsh, Michael D; McKillen, John

    2017-07-01

    Bluetongue virus (BTV) is an infectious, non-contagious viral disease of domestic and wild ruminants that is transmitted by adult females of certain Culicoides species. Since 2006, several serotypes including BTV-1, 2, 4, 6, 8, 9 and 16, have spread from the Mediterranean basin into Northern Europe for the first time. BTV-8 in particular, caused a major epidemic in northern Europe. As a result, it is evident that most European countries are at risk of BTV infection. The objective of this study was to develop and validate a real-time reverse transcriptase-polymerase chain reaction (rRT-PCR) assay based on TaqMan technology for the detection of representative strains of all BTV serotypes. Primers and probes were based on genome segment 10 of the virus, the NS3 gene. The assay was tested for sensitivity, and specificity. The analytical sensitivity of the rRT-PCR assay was 200 copies of RNA per reaction. The assay did not amplify the closely related orbivirus epizootic hemorrhagic disease virus (EHDV) but successfully detected all BTV reference strains including clinical samples from animals experimentally infected with BTV-8. This real time RT-PCR assay offers a sensitive, specific and rapid alternative assay for the pan detection of BTV that could be used as part of a panel of diagnostic assays for the detection of all serotypes of BTV. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

  3. Virus-induced gene silencing in Catharanthus roseus by biolistic inoculation of tobacco rattle virus vectors.

    Science.gov (United States)

    Carqueijeiro, I; Masini, E; Foureau, E; Sepúlveda, L J; Marais, E; Lanoue, A; Besseau, S; Papon, N; Clastre, M; Dugé de Bernonville, T; Glévarec, G; Atehortùa, L; Oudin, A; Courdavault, V

    2015-11-01

    Catharanthus roseus constitutes the unique source of several valuable monoterpenoid indole alkaloids, including the antineoplastics vinblastine and vincristine. These alkaloids result from a complex biosynthetic pathway encompassing between 30 and 50 enzymatic steps whose characterisation is still underway. The most recent identifications of genes from this pathway relied on a tobacco rattle virus-based virus-induced gene silencing (VIGS) approach, involving an Agrobacterium-mediated inoculation of plasmids encoding the two genomic components of the virus. As an alternative, we developed a biolistic-mediated approach of inoculation of virus-encoding plasmids that can be easily performed by a simple bombardment of young C. roseus plants. After optimisation of the transformation conditions, we showed that this approach efficiently silenced the phytoene desaturase gene, leading to strong and reproducible photobleaching of leaves. This biolistic transformation was also used to silence a previously characterised gene from the alkaloid biosynthetic pathway, encoding iridoid oxidase. Plant bombardment caused down-regulation of the targeted gene (70%), accompanied by a correlated decreased in MIA biosynthesis (45-90%), similar to results obtained via agro-transformation. Thus, the biolistic-based VIGS approach developed for C. roseus appears suitable for gene function elucidation and can readily be used instead of the Agrobacterium-based approach, e.g. when difficulties arise with agro-inoculations or when Agrobacterium-free procedures are required to avoid plant defence responses. © 2015 German Botanical Society and The Royal Botanical Society of the Netherlands.

  4. Comparison of FilmArray Respiratory Panel and laboratory-developed real-time reverse transcription-polymerase chain reaction assays for respiratory virus detection.

    Science.gov (United States)

    Renaud, Christian; Crowley, Janet; Jerome, Keith R; Kuypers, Jane

    2012-12-01

    The FilmArray Respiratory Panel (Idaho Technology) is a highly multiplexed respiratory virus real-time polymerase chain reaction (PCR) assay. Eighty-four respiratory viruses identified by laboratory-developed real-time reverse transcription-PCR assays (LDA) or by viral cultures were mixed and tested by FilmArray to assess its performance. FilmArray identified 72 (90%) of 80 viruses also detected by LDA. Six of the 8 viruses not detected by FilmArray had PCR cycle threshold values >35. Compared to LDA, FilmArray showed comparable sensitivity when used to test serial dilutions of virus mixtures and good agreement with negative samples. With the use of 1 FilmArray instrument, 7 clinical samples could be analyzed and reported in an 8-h shift compared to 20 using LDA and 1 real-time detection instrument. While the FilmArray was rapid and easy to use, its low throughput and qualitative results may be a disadvantage in some clinical settings. Copyright © 2012 Elsevier Inc. All rights reserved.

  5. Detection of human papillomavirus and Epstein-Barr virus DNA sequences in oral mucosa of HIV-infected patients by the polymerase chain reaction.

    Science.gov (United States)

    Snijders, P. J.; Schulten, E. A.; Mullink, H.; ten Kate, R. W.; Jiwa, M.; van der Waal, I.; Meijer, C. J.; Walboomers, J. M.

    1990-01-01

    The presence of human papillomavirus (HPV) and Epstein-Barr virus (EBV) was analyzed in 21 oral biopsy specimens of HIV-infected patients using the polymerase chain reaction (PCR) method. Biopsies were categorized as hairy leukoplakia (HL) (n = 12), candidiasis (n = 3), oral warts (n = 2), and clinically normal epithelium (n = 4). For HPV detection a modified general primer-mediated PCR method (GP-PCR), which detects a broad spectrum of HPV genotypes at sub-picogram levels, was used. Human papillomavirus DNA was only found in two oral warts and was identified as HPV type 32. Epstein-Barr virus DNA was detected in 16 biopsy specimens, including the 12 HLs, 2 cases of candidiasis, and 2 samples of normal epithelium. Epstein-Barr virus positivity in HL could be confirmed by Southern blot analysis and DNA in situ hybridization using biotinylated DNA probes (bio-DISH). Epstein-Barr virus bio-DISH was also positive in one sample of normal epithelium from a patient with HL. The results indicate that HL is strongly associated with EBV and not with any of the common HPV types that react with general HPV primers in the PCR. However the detection of EBV in normal oral epithelium by PCR and bio-DISH suggests that the presence of this virus is not exclusively related to HL. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:2169191

  6. Virus-Induced Gene Silencing in Maize with a Foxtail mosaic virus Vector.

    Science.gov (United States)

    Mei, Yu; Whitham, Steven A

    2018-01-01

    Virus-induced gene silencing (VIGS) is a powerful technology for rapidly and transiently knocking down the expression of plant genes to study their functions. A VIGS vector for maize derived from Foxtail mosaic virus (FoMV), a positive-sense single-stranded RNA virus, was recently developed. A cloning site created near the 3' end of the FoMV genome enables insertion of 200-400 nucleotide fragments of maize genes targeted for silencing. The recombinant FoMV clones are inoculated into leaves of maize seedlings by biolistic particle delivery, and silencing is typically observed within 2 weeks after inoculation. This chapter provides a protocol for constructing FoMV VIGS clones and inoculating them into maize seedlings.

  7. A study of variability of capsid protein genes of Radish mosaic virus

    OpenAIRE

    HOLÁ, Marcela

    2008-01-01

    The part of RNA2 genome segment of several isolates of Radish mosaic virus (RaMV) including capsid protein genes was sequenced. Variability of capsid protein genes among the isolates of Radish mosaic virus was studied.

  8. Discrimination between dog-related and vampire bat-related rabies viruses in Brazil by strain-specific reverse transcriptase-polymerase chain reaction and restriction fragment length polymorphism analysis.

    Science.gov (United States)

    Ito, Mikako; Itou, Takuya; Shoji, Youko; Sakai, Takeo; Ito, Fumio H; Arai, Yohko T; Takasaki, Tomohiko; Kurane, Ichiro

    2003-04-01

    There is a geographical overlap between the two main rabies epidemiological cycles maintained by dogs and vampire bats in Latin America. The geographical and temporal coincidence of rabies outbreaks of respective origins is not unusual in rural areas of Latin America. These circumstances make it difficult to discriminate the intraspecies and interspecies transmission pathways of rabies. This study was conducted to develop techniques to discriminate dog-related and vampire bat-related rabies virus isolates (DRRV and VRRV, respectively) in Brazil. The 1396 nucleotides of the nucleoprotein gene of a total of 27 DRRV and VRRV were sequenced. Strain-specific (SS) primers were developed based on these sequences. Forty-nine rabies virus strains isolated from animals and humans in several parts of Brazil were examined by reverse transcriptase-polymerase chain reaction (RT-PCR) with SS primers. These rabies viruses were also amplified by RT-PCR with general rabies primers and the PCR products were cut by three restriction enzymes, Blp I, Bsu36 I and BspE I. All the DRRV and VRRV were distinguished by RT-PCR with SS primers. The PCR products obtained from DRRV were cut at one site by Blp I, but not by Bsu36 I. The PCR products obtained from VRRV were cut at one or two sites by Bsu36 I, but not by Blp I. Blp I and Bsu36 I clearly discriminated DRRV and VRRV in restriction fragment length polymorphysim (RFLP) assays. The results of SS RT-PCR and RFLP were consistent. SS RT-PCR and RFLP assays have been developed for determining the origins of rabies virus isolates in Brazil. These assays are simple and rapid, and will be useful for identifying the rabies virus reservoirs of field isolates in Brazil, especially when used together.

  9. Immunostimulatory Gene Therapy Using Oncolytic Viruses as Vehicles

    Directory of Open Access Journals (Sweden)

    Angelica Loskog

    2015-11-01

    Full Text Available Immunostimulatory gene therapy has been developed during the past twenty years. The aim of immunostimulatory gene therapy is to tilt the suppressive tumor microenvironment to promote anti-tumor immunity. Hence, like a Trojan horse, the gene vehicle can carry warriors and weapons into enemy territory to combat the tumor from within. The most promising immune stimulators are those activating and sustaining Th1 responses, but even if potent effects were seen in preclinical models, many clinical trials failed to show objective responses in cancer patients. However, with new tools to control ongoing immunosuppression in cancer patients, immunostimulatory gene therapy is now emerging as an interesting option. In parallel, oncolytic viruses have been shown to be safe in patients. To prolong immune stimulation and to increase efficacy, these two fields are now merging and oncolytic viruses are armed with immunostimulatory transgenes. These novel agents are racing towards approval as established cancer immunotherapeutics.

  10. Immunostimulatory Gene Therapy Using Oncolytic Viruses as Vehicles.

    Science.gov (United States)

    Loskog, Angelica

    2015-11-06

    Immunostimulatory gene therapy has been developed during the past twenty years. The aim of immunostimulatory gene therapy is to tilt the suppressive tumor microenvironment to promote anti-tumor immunity. Hence, like a Trojan horse, the gene vehicle can carry warriors and weapons into enemy territory to combat the tumor from within. The most promising immune stimulators are those activating and sustaining Th1 responses, but even if potent effects were seen in preclinical models, many clinical trials failed to show objective responses in cancer patients. However, with new tools to control ongoing immunosuppression in cancer patients, immunostimulatory gene therapy is now emerging as an interesting option. In parallel, oncolytic viruses have been shown to be safe in patients. To prolong immune stimulation and to increase efficacy, these two fields are now merging and oncolytic viruses are armed with immunostimulatory transgenes. These novel agents are racing towards approval as established cancer immunotherapeutics.

  11. Virus-induced gene silencing in diverse maize lines using the Brome Mosaic virus-based silencing vector

    Science.gov (United States)

    Virus-induced gene silencing (VIGS) is a widely used tool for gene function studies in many plant species, though its use in monocots has been limited. Using a Brome mosaic virus (BMV) vector designed to silence the maize phytoene desaturase gene, a genetically diverse set of maize inbred lines was ...

  12. Selection of relatively exact reference genes for gene expression studies in flixweed (Descurainia sophia) by quantitative real-time polymerase chain reaction.

    Science.gov (United States)

    Xu, Xian; Liu, Xiaomin; Chen, Silong; Li, Binghua; Wang, Xiaoyun; Fan, Cuiqin; Wang, Guiqi; Ni, Hanwen

    2016-02-01

    The reliable gene expression analysis of a reverse transcription quantitative polymerase chain reaction (qRT-PCR) mainly depends on selecting suitable reference genes that can express stably under different experimental conditions. Thus far, no reference genes have been identified in flixweed. In this paper, 7 supposed reference genes were selected to evaluate their expression stabilities by qRT-PCR in flixweed under three conditions including different sampling times after tribenuron treatment, different organs, and different growth stages using geNorm, NormFinder, and BestKeeper statistical algorithms. The results showed that ACT7, UBC and 18SrRNA were the stable reference genes in all of the tested samples. ACT7 and UBC showed high stability in different sampling times after the tribenuron treatment. UBC and 18SrRNA were the most suitable genes for different organs and growth stages. This work confirmed the suitable reference genes of flixweed for a relatively accurate gene expression analysis under different experimental conditions. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. Discovery of Novel Hepatitis C Virus NS5B Polymerase Inhibitors by Combining Random Forest, Multiple e-Pharmacophore Modeling and Docking.

    Directory of Open Access Journals (Sweden)

    Yu Wei

    Full Text Available The NS5B polymerase is one of the most attractive targets for developing new drugs to block Hepatitis C virus (HCV infection. We describe the discovery of novel potent HCV NS5B polymerase inhibitors by employing a virtual screening (VS approach, which is based on random forest (RB-VS, e-pharmacophore (PB-VS, and docking (DB-VS methods. In the RB-VS stage, after feature selection, a model with 16 descriptors was used. In the PB-VS stage, six energy-based pharmacophore (e-pharmacophore models from different crystal structures of the NS5B polymerase with ligands binding at the palm I, thumb I and thumb II regions were used. In the DB-VS stage, the Glide SP and XP docking protocols with default parameters were employed. In the virtual screening approach, the RB-VS, PB-VS and DB-VS methods were applied in increasing order of complexity to screen the InterBioScreen database. From the final hits, we selected 5 compounds for further anti-HCV activity and cellular cytotoxicity assay. All 5 compounds were found to inhibit NS5B polymerase with IC50 values of 2.01-23.84 μM and displayed anti-HCV activities with EC50 values ranging from 1.61 to 21.88 μM, and all compounds displayed no cellular cytotoxicity (CC50 > 100 μM except compound N2, which displayed weak cytotoxicity with a CC50 value of 51.3 μM. The hit compound N2 had the best antiviral activity against HCV, with a selective index of 32.1. The 5 hit compounds with new scaffolds could potentially serve as NS5B polymerase inhibitors through further optimization and development.

  14. Investigation of the mode of binding of a novel series of N-benzyl-4-heteroaryl-1-(phenylsulfonyl)piperazine-2-carboxamides to the hepatitis C virus polymerase

    Energy Technology Data Exchange (ETDEWEB)

    Gentles, Robert G.; Sheriff, Steven; Beno, Brett R.; Wan, Changhong; Kish, Kevin; Ding, Min; Zheng, Xiaofan; Chupak, Louis; Poss, Michael A.; Witmer, Mark R.; Morin, Paul; Wang, Ying-Kai; Rigat, Karen; Lemm, Julie; Voss, Stacey; Liu, Mengping; Pelosi, Lenore; Roberts, Susan B.; Gao, Min; Kadow, John F. (BMS)

    2013-11-20

    Structure based rationales for the activities of potent N-benzyl-4-heteroaryl-1-(phenylsulfonyl)piperazine-2-carboxamide inhibitors of the hepatitis C viral polymerase are described herein. These compounds bind to the hepatitis C virus non-structural protein 5B (NS5B), and co-crystal structures of select examples from this series with NS5B are reported. Comparison of co-crystal structures of a potent analog with both NS5B genotype 1a and genotype 1b provides a possible explanation for the genotype-selectivity observed with this compound class and suggests opportunities for the further optimization of the series.

  15. Detection of human papillomavirus and Epstein-Barr virus DNA sequences in oral mucosa of HIV-infected patients by the polymerase chain reaction.

    OpenAIRE

    Snijders, P. J.; Schulten, E. A.; Mullink, H.; ten Kate, R. W.; Jiwa, M.; van der Waal, I.; Meijer, C. J.; Walboomers, J. M.

    1990-01-01

    The presence of human papillomavirus (HPV) and Epstein-Barr virus (EBV) was analyzed in 21 oral biopsy specimens of HIV-infected patients using the polymerase chain reaction (PCR) method. Biopsies were categorized as hairy leukoplakia (HL) (n = 12), candidiasis (n = 3), oral warts (n = 2), and clinically normal epithelium (n = 4). For HPV detection a modified general primer-mediated PCR method (GP-PCR), which detects a broad spectrum of HPV genotypes at sub-picogram levels, was used. Human pa...

  16. Porcine reproductive and respiratory syndrome virus: Interlaboratory ring trial to evaluate real-time reverse transcription polymerase chain reaction detection methods

    DEFF Research Database (Denmark)

    Wernike, Kerstin; Bonilauri, Paolo; Dauber, Malte

    2012-01-01

    To compare the real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays used for the diagnosis of Porcine reproductive and respiratory syndrome virus (PRRSV), a Europe-wide interlaboratory ring trial was conducted. A variety of PRRSV strains including North American...... (NA) and European (EU) genotype isolates were analyzed by the participants. Great differences regarding qualitative diagnostics as well as analytical sensitivity were observed between the individual RT-qPCR systems, especially when investigating strains from the EU genotype. None of the assays...

  17. Adeno-associated virus inverted terminal repeats stimulate gene editing

    OpenAIRE

    Hirsch, ML

    2014-01-01

    Advancements in genome editing have relied on technologies to specifically damage DNA which, in turn, stimulates DNA repair including homologous recombination (HR). As off-target concerns complicate the therapeutic translation of site-specific DNA endonucleases, an alternative strategy to stimulate gene editing based on fragile DNA was investigated. To do this, an episomal gene-editing reporter was generated by a disruptive insertion of the adeno-associated virus (AAV) inverted terminal repea...

  18. A universal RNA polymerase II CTD cycle is orchestrated by complex interplays between kinase, phosphatase, and isomerase enzymes along genes.

    Science.gov (United States)

    Bataille, Alain R; Jeronimo, Célia; Jacques, Pierre-Étienne; Laramée, Louise; Fortin, Marie-Ève; Forest, Audrey; Bergeron, Maxime; Hanes, Steven D; Robert, François

    2012-01-27

    Transcription by RNA polymerase II (RNAPII) is coupled to mRNA processing and chromatin modifications via the C-terminal domain (CTD) of its largest subunit, consisting of multiple repeats of the heptapeptide YSPTSPS. Pioneering studies showed that CTD serines are differentially phosphorylated along genes in a prescribed pattern during the transcription cycle. Genome-wide analyses challenged this idea, suggesting that this cycle is not uniform among different genes. Moreover, the respective role of enzymes responsible for CTD modifications remains controversial. Here, we systematically profiled the location of the RNAPII phosphoisoforms in wild-type cells and mutants for most CTD modifying enzymes. Together with results of in vitro assays, these data reveal a complex interplay between the modifying enzymes, and provide evidence that the CTD cycle is uniform across genes. We also identify Ssu72 as the Ser7 phosphatase and show that proline isomerization is a key regulator of CTD dephosphorylation at the end of genes. Copyright © 2012 Elsevier Inc. All rights reserved.

  19. Influenza A Virus with a Human-Like N2 Gene Is Circulating in Pigs

    DEFF Research Database (Denmark)

    Breum, Solvej Østergaard; Hjulsager, Charlotte Kristiane; Trebbien, Ramona

    2013-01-01

    A novel reassortant influenza A virus, H1avN2hu, has been found in Danish swine. The virus contains an H1 gene similar to the hemagglutinin (HA) gene of H1N1 avian-like swine viruses and an N2 gene most closely related to the neuraminidase (NA) gene of human H3N2 viruses from the mid-1990s....

  20. Chlorella viruses contain genes encoding a complete polyamine biosynthetic pathway

    Science.gov (United States)

    Baumann, Sascha; Sander, Adrianne; Gurnon, James R.; Yanai-Balser, Giane; VanEtten, James L.; Piotrowski, Markus

    2007-01-01

    Two genes encoding the putative polyamine biosynthetic enzymes agmatine iminohydrolase (AIH) and N-carbamoylputrescine amidohydrolase (CPA) were cloned from the chloroviruses PBCV-1, NY-2A and MT325. They were expressed in Escherichia coli to form C-terminal (His)6-tagged proteins and the recombinant proteins were purified by Ni2+- binding affinity chromatography. The biochemical properties of the two enzymes are similar to AIH and CPA enzymes from Arabidopsis thaliana and Pseudomonas aeruginosa. Together with the previously known virus genes encoding ornithine/arginine decarboxlyase (ODC/ADC) and homospermidine synthase, the chloroviruses have genes that encode a complete set of functional enzymes that synthesize the rare polyamine homospermidine from arginine via agmatine, N-carbamoylputrescine and putrescine. The PBCV-1 aih and cpa genes are expressed early during virus infection together with the odc/adc gene, suggesting that biosynthesis of putrescine is important in early stages of viral replication. The aih and cpa genes are widespread in the chlorella viruses. PMID:17101165

  1. Molecular characterization of capsid protein gene of potato virus X ...

    African Journals Online (AJOL)

    Molecular characterization of capsid protein gene of potato virus X from Pakistan. Arshad Jamal, Idrees Ahmad Nasir, Bushra Tabassum, Muhammad Tariq, Abdul Munim Farooq, Zahida Qamar, Mohsin Ahmad Khan, Nadeem Ahmad, Muhammad Shafiq, Muhammad Saleem Haider, M. Arshad Javed, Tayyab Husnain ...

  2. Selection of reference genes for analysis of stress-responsive genes after challenge with viruses and temperature changes in the silkworm Bombyx mori.

    Science.gov (United States)

    Guo, Huizhen; Jiang, Liang; Xia, Qingyou

    2016-04-01

    Viruses and high temperature (HT) are the primary threats to silkworms. Changes in the expression of stress-response genes can be measured using quantitative polymerase chain reaction (qPCR) after exposure to viruses or HT. However, appropriate reference genes (RGs) for qPCR data normalization have not been established in this organism. In this study, we summarized the RGs used in the previous silkworm studies after infection with Bombyx mori nucleopolyhedrovirus (BmNPV), B. mori cytoplasmic polyhedrosis virus (BmCPV), or B. mori densovirus (BmDNV) or after HT treatment. The expression levels of these RGs were extracted from silkworm transcriptome data to screen for candidate RGs that were unaffected by the experimental conditions. Actin-1 (A1), actin-3 (A3), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and translation initiation factor 4a (TIF-4A) were selected for further qPCR verification. The results of RNA-seq and qPCR showed that GAPDH and TIF-4A were suitable RGs after BmNPV challenge or HT stress, whereas TIF-4A was an appropriate RG for BmCPV or BmDNV-Z challenge in silkworms. These results suggested that TIF-4A may be the most appropriate RG for gene expression analysis after challenge with viruses or HT in silkworms.

  3. Detection of infectious bursal disease virus in various lymphoid tissues of experimentally infected specific pathogen free chickens by different reverse transcription polymerase chain reaction assays

    DEFF Research Database (Denmark)

    Kabell, Susanne; Handberg, Kurt; Kusk, Mette

    2005-01-01

    transcription polymerase chain reaction (RT-PCR) assays, including two recently developed strain-specific assays, were employed for detection of ribonucleic acid (RNA) from three different IBDV strains in bursa tissue samples from experimentally infected specific pathogen free chickens. The virus strains...... included vaccine strain D78, classical strain Faragher 52/70, and the very virulent Danish strain DK01 The presence of the virus infection was confirmed by histopathologic evaluation of bursa lesions. The largest number of positive samples was obtained with a strain-specific two-step multiplex (MPX) RT...... of the IBDV strains used were detected in bursa tissues, whereas only the two virulent strains were detected in bone marrow, spleen, and thymus....

  4. Direct amplification of a single dissected chromosomal segment by polymerase chain reaction: a human brain sodium channel gene is on chromosome 2q22-q23.

    Science.gov (United States)

    Han, J A; Lu, C M; Brown, G B; Rado, T A

    1991-01-01

    We have devised a general strategy for gene mapping based upon the direct amplification of a target sequence within a single microdissected Giemsa-banded chromosomal segment using the polymerase chain reaction. The usefulness of this approach was demonstrated by mapping a cloned human brain sodium channel (alpha subunit) gene sequence to chromosome 2q22-q23. When DNA from single, dissected chromosome segments 2q21-qter and 2q24-pter were used as templates, a sodium channel-specific 172-base-pair polymerase chain reaction product was obtained. This product was not synthesized when segments 2q21-pter and 2q24-qter were used. Chromosome microdissection-polymerase chain reaction is not only a simple, fast, and accurate method for gene mapping but also may offer significant advantages for other applications, such as cancer cytogenetics and linkage analysis. Images PMID:1846440

  5. High-resolution structure of the N-terminal endonuclease domain of the Lassa virus L polymerase in complex with magnesium ions.

    Directory of Open Access Journals (Sweden)

    Gregor D Wallat

    Full Text Available Lassa virus (LASV causes deadly hemorrhagic fever disease for which there are no vaccines and limited treatments. LASV-encoded L polymerase is required for viral RNA replication and transcription. The functional domains of L-a large protein of 2218 amino acid residues-are largely undefined, except for the centrally located RNA-dependent RNA polymerase (RdRP motif. Recent structural and functional analyses of the N-terminal region of the L protein from lymphocytic choriomeningitis virus (LCMV, which is in the same Arenaviridae family as LASV, have identified an endonuclease domain that presumably cleaves the cap structures of host mRNAs in order to initiate viral transcription. Here we present a high-resolution crystal structure of the N-terminal 173-aa region of the LASV L protein (LASV L173 in complex with magnesium ions at 1.72 Å. The structure is highly homologous to other known viral endonucleases of arena- (LCMV NL1, orthomyxo- (influenza virus PA, and bunyaviruses (La Crosse virus NL1. Although the catalytic residues (D89, E102 and K122 are highly conserved among the known viral endonucleases, LASV L endonuclease structure shows some notable differences. Our data collected from in vitro endonuclease assays and a reporter-based LASV minigenome transcriptional assay in mammalian cells confirm structural prediction of LASV L173 as an active endonuclease. The high-resolution structure of the LASV L endonuclease domain in complex with magnesium ions should aid the development of antivirals against lethal Lassa hemorrhagic fever.

  6. Analysis of E2 gene integrity in HPV16 and HPV58 viruses isolated from women with cervical pathology

    Directory of Open Access Journals (Sweden)

    María del R González-Losa

    Full Text Available Integration of human papillomavirus (HPV DNA into human cells accompanied by the disruption of the viral genome has been described as a prerequisite for cancer development. This study aimed to investigate E2 gene integrity of HPV16 and HPV58 viruses isolated from infected women with cervical lesions. Forty-two HPV16- and 31 HPV58-positive samples were analysed. E2 integrity was assumed when all fragments covering the E2 gene were amplified with specific polymerase chain reaction primers. Overall, in 59% of the samples, at least one fragment was not amplified in HPV16- (57% and HPV58-positive samples (61%. Samples from high-grade squamous intraepithelial lesions had the highest frequency of E2 gene disruptions (73%, followed by samples from low-grade squamous intraepithelial lesions (63% and, finally, samples from invasive cervical cancer (35%. Association between the integrity status of the E2 gene, and lesion grade was assessed by the chi-squared test applied to the combined set of viruses (p = 0.6555 or to populations of the same virus type (HPV58, p = 0.3101; HPV16, p = 0.3024. In conclusion, in this study, no association was found between the presence of E2 gene disruptions and the grade of cervical lesions caused by HPV16 and HPV58.

  7. Analysis of E2 gene integrity in HPV16 and HPV58 viruses isolated from women with cervical pathology.

    Science.gov (United States)

    González-Losa, María Del R; Puerto-Solis, Marylin; Tenorio Ruiz, Juan; Rosado-López, Ariel I; Hau-Aviles, Oscar; Ayora-Talavera, Guadalupe; Cisneros-Cutz, Isidro; Conde-Ferráez, Laura

    2016-12-01

    Integration of human papillomavirus (HPV) DNA into human cells accompanied by the disruption of the viral genome has been described as a prerequisite for cancer development. This study aimed to investigate E2 gene integrity of HPV16 and HPV58 viruses isolated from infected women with cervical lesions. Forty-two HPV16- and 31 HPV58-positive samples were analysed. E2 integrity was assumed when all fragments covering the E2 gene were amplified with specific polymerase chain reaction primers. Overall, in 59% of the samples, at least one fragment was not amplified in HPV16- (57%) and HPV58-positive samples (61%). Samples from high-grade squamous intraepithelial lesions had the highest frequency of E2 gene disruptions (73%), followed by samples from low-grade squamous intraepithelial lesions (63%) and, finally, samples from invasive cervical cancer (35%). Association between the integrity status of the E2 gene, and lesion grade was assessed by the chi-squared test applied to the combined set of viruses (p = 0.6555) or to populations of the same virus type (HPV58, p = 0.3101; HPV16, p = 0.3024). In conclusion, in this study, no association was found between the presence of E2 gene disruptions and the grade of cervical lesions caused by HPV16 and HPV58.

  8. Human mitochondrial ribosomal protein MRPL12 interacts directly with mitochondrial RNA polymerase to modulate mitochondrial gene expression.

    Science.gov (United States)

    Wang, Zhibo; Cotney, Justin; Shadel, Gerald S

    2007-04-27

    The core human mitochondrial transcription machinery comprises a single subunit bacteriophage-related RNA polymerase, POLRMT, the high mobility group box DNA-binding protein h-mtTFA/TFAM, and two transcriptional co-activator proteins, h-mtTFB1 and h-mtTFB2 that also have rRNA methyltransferase activity. Recapitulation of specific initiation of transcription in vitro can be achieved by a complex of POL-RMT, h-mtTFA, and either h-mtTFB1 or h-mtTFB2. However, the nature of mitochondrial transcription complexes in vivo and the potential involvement of additional proteins in the transcription process in human mitochondria have not been extensively investigated. In Saccharomyces cerevisiae, transcription and translation are physically coupled via the formation of a multiprotein complex nucleated by the binding of Nam1p to the amino-terminal domain of mtRNA polymerase (Rpo41p). This model system paradigm led us to search for proteins that interact with POLRMT to regulate mitochondrial gene expression in humans. Using an affinity capture strategy to identify POL-RMT-binding proteins, we identified mitochondrial ribosomal protein L7/L12 (MRPL12) as a protein in HeLa mitochondrial extracts that interacts specifically with POLRMT in vitro. Purified recombinant MRPL12 binds to POLRMT and stimulates mitochondrial transcription activity in vitro, demonstrating that this interaction is both direct and functional. Finally, from HeLa cells that overexpress FLAG epitope-tagged MRPL12, increased steady-state levels of mtDNA-encoded transcripts are observed and MRPL12-POLRMT complexes can be co-immunoprecipitated, providing strong evidence that this interaction enhances mitochondrial transcription or RNA stability in vivo. We speculate that the MRPL12 interaction with POLRMT is likely part of a novel regulatory mechanism that coordinates mitochondrial transcription with translation and/or ribosome biogenesis during human mitochondrial gene expression.

  9. Detection of Tomato spotted wilt virus in its vector Frankliniella occidentalis by reverse transcription-polymerase chain reaction.

    Science.gov (United States)

    Mason, Giovanna; Roggero, Piero; Tavella, Luciana

    2003-04-01

    A method for rapid and reliable detection of Tomato spotted wilt virus (TSWV) (Tospovirus, Bunyaviridae) in its vector Frankliniella occidentalis (Thysanoptera Thripidae) would be a useful tool for studying the epidemiology of this virus. A RT-PCR method developed for this purpose is reported. The method was tested on thrips involved in laboratory transmission trials and on thrips collected in the field, whose capability to transmit TSWV was checked previously by leaf disk assays. The RT-PCR results were consistent with the results obtained by the leaf disk assays. Among thrips involved in laboratory experiments, 97% of the adults that transmitted TSWV were positive by RT-PCR; as did some non-transmitter adults reacted, whereas among field-collected thrips only the individuals able to transmit were positive by RT-PCR. In addition, healthy thrips were allowed to feed as adults on virus-infected leaves for 48 h, and then examined by RT-PCR immediately or after starving or feeding on virus-free plants for various times, to determine if virus ingested (but not transmissible) was also detectable. The virus was detectable immediately after the feed or within 12 and 24 h for individuals starved or fed on virus-free plants, respectively, but not after those periods. Thus, the method could detect rapidly and reliably the virus in vectors from the field, providing 24 h of starving to avoid positive RT-PCR results from thrips simply carrying the virus.

  10. Molecular characterisation of lumpy skin disease virus and sheeppox virus based on P32 gene

    Directory of Open Access Journals (Sweden)

    P.M.A.Rashid

    2017-06-01

    Full Text Available Lumpy skin disease virus (LSDV and sheeppox virus (SPV have a considerable economic impact on the cattle and small ruminant industry. They are listed in group A of contagious disease by the World Organization for Animal Health (OIE. This study addressed molecular characterisation of first LSDV outbreak and an endemic SPV in Kurdistan region of Iraq based on P32 gene. The results indicated that P32 gene can be successfully used for diagnosis of LSDV. The phylogenic and molecular analysis showed that there may be a new LSDV isolate circulating in Kurdistan which uniquely shared the same characteristic amino acid sequence with SPV and GPV, leucine at amino acid position 51 in P32 gene as well as few genetically distinct SPV causing pox disease in Kurdistan sheep. This study provided sequence information of P32 gene for several LSDV isolates, which positively affects the epidemiological study of Capripoxvirus

  11. Characterization of the ptfA gene of avian Pasteurella multocida strains by allele-specific polymerase chain reaction.

    Science.gov (United States)

    Sellyei, Boglárka; Bányai, Krisztián; Magyar, Tibor

    2010-07-01

    Pasteurella multocida is the causative agent of fowl cholera in domesticated and wild birds. The disease outcome is affected by various host- and pathogen-specific determinants. Several putative virulence factors have been proposed to play a key role in this interaction, including the ptfA gene, the products of which assemble to form type 4 fimbriae on the bacterial surface. One way to understand more precisely how ptfA contributes to pathogenesis is to gather molecular features of this gene in circulating avian P. multocida strains. Therefore, molecular characterization of the ptfA gene of P. multocida strains isolated from domestic poultry was performed using the combination of nucleotide sequence analysis and a newly developed allele-specific polymerase chain reaction assay. Two major ptfA alleles were identified among 31 strains, representing various serogroups and somatic serotypes. It was noteworthy that allele specificity and case severity of a subset of strains correlated with the available gross pathology data. Therefore, the acquisition of comprehensive clinical and epidemiological data together with molecular characteristics of individual strains will help to design and implement adequate preventive and intervention strategies.

  12. Identification of nickel response genes in abnormal early developments of sea urchin by differential display polymerase chain reaction.

    Science.gov (United States)

    Ryu, Tae Kwon; Lee, Gunsup; Rhee, Yong; Park, Heung-Sik; Chang, Man; Lee, Sukchan; Lee, Jaean; Lee, Taek-Kyun

    2012-10-01

    Bioassays and biomarkers have been previously developed to assess the effects of heavy metal contaminants on the early life stages of the sea urchin. In this study, malformation in the early developmental processes was observed in sea urchin (Strongylocentrotus intermedius) larvae exposed to 10 ppm Ni for over 30 h. The most critical stage at which the triggering of nickel effects takes place is thought to be the blastula stage, which occurs after fertilization in larval development. To investigate the molecular-level responses of sea urchin exposed to heavy metal stress and to explore the differentially expressed genes that are induced or repressed by nickel, differential display polymerase chain reaction (DD-PCR) was used with sea urchin mRNAs. The malformation-related genes expressed in the early life stages of the sea urchin were cloned from larvae exposed to 10 ppm of nickel for 15 h, and accessed via DD-PCR. Sequence analysis results revealed that each of the genes evidenced high homology with EGF2, PCSK9, serine/threonine protein kinase, apolipophorin precursor protein, and MGC80921 protein/transcript variant 2. This result may prove useful in the development of novel biomarkers for the assessment of heavy metal stresses on sea urchin embryos. Copyright © 2012 Elsevier Inc. All rights reserved.

  13. Adeno-associated virus for cystic fibrosis gene therapy

    Directory of Open Access Journals (Sweden)

    S.V. Martini

    2011-11-01

    Full Text Available Gene therapy is an alternative treatment for genetic lung disease, especially monogenic disorders such as cystic fibrosis. Cystic fibrosis is a severe autosomal recessive disease affecting one in 2500 live births in the white population, caused by mutation of the cystic fibrosis transmembrane conductance regulator (CFTR. The disease is classically characterized by pancreatic enzyme insufficiency, an increased concentration of chloride in sweat, and varying severity of chronic obstructive lung disease. Currently, the greatest challenge for gene therapy is finding an ideal vector to deliver the transgene (CFTR to the affected organ (lung. Adeno-associated virus is the most promising viral vector system for the treatment of respiratory disease because it has natural tropism for airway epithelial cells and does not cause any human disease. This review focuses on the basic properties of adeno-associated virus and its use as a vector for cystic fibrosis gene therapy.

  14. Foamy virus for efficient gene transfer in regeneration studies.

    Science.gov (United States)

    Khattak, Shahryar; Sandoval-Guzmán, Tatiana; Stanke, Nicole; Protze, Stephanie; Tanaka, Elly M; Lindemann, Dirk

    2013-05-03

    Molecular studies of appendage regeneration have been hindered by the lack of a stable and efficient means of transferring exogenous genes. We therefore sought an efficient integrating virus system that could be used to study limb and tail regeneration in salamanders. We show that replication-deficient foamy virus (FV) vectors efficiently transduce cells in two different regeneration models in cell culture and in vivo. Injection of EGFP-expressing FV but not lentivirus vector particles into regenerating limbs and tail resulted in widespread expression that persisted throughout regeneration and reamputation pointing to the utility of FV for analyzing adult phenotypes in non-mammalian models. Furthermore, tissue specific transgene expression is achieved using FV vectors during limb regeneration. FV vectors are efficient mean of transferring genes into axolotl limb/tail and infection persists throughout regeneration and reamputation. This is a nontoxic method of delivering genes into axolotls in vivo/ in vitro and can potentially be applied to other salamander species.

  15. Characterizing the roles of Cryphonectria parasitica RNA-dependent RNA polymerase-like genes in antiviral defense, viral recombination and transposon transcript accumulation.

    Directory of Open Access Journals (Sweden)

    Dong-Xiu Zhang

    Full Text Available An inducible RNA-silencing pathway, involving a single Dicer protein, DCL2, and a single Argonaute protein, AGL2, was recently shown to serve as an effective antiviral defense response in the chestnut blight fungus Cryphonectria parasitica. Eukaryotic RNA-dependent RNA polymerases (RdRPs are frequently involved in transcriptional and posttranscriptional gene silencing and antiviral defense. We report here the identification and characterization of four RdRP genes (rdr1-4 in the C. parasitica genome. Sequence relationships with other eukaryotic RdRPs indicated that RDR1 and RDR2 were closely related to QDE-1, an RdRP involved in RNA silencing ("quelling" in Neurospora crassa, whereas RDR3 was more closely related to the meiotic silencing gene SAD-1 in N. crassa. The RdRP domain of RDR4, related to N. crassa RRP-3 of unknown function, was truncated and showed evidence of alternative splicing. Similar to reports for dcl2 and agl2, the expression levels for rdr3 and rdr4 increased after hypovirus CHV-1/EP713 infection, while expression levels of rdr1 and rdr2 were unchanged. The virus-responsive induction patterns for rdr3 and rdr4 were altered in the Δdcl2 and Δagl2 strains, suggesting some level of interaction between rdr3 and rdr4 and the dcl2/agl2 silencing pathway. Single rdr gene knockouts Δrdr1-4, double knockouts Δrdr1/2, Δrdr2/3, Δrdr1/3, and a triple knockout, Δrdr1/2/3, were generated and evaluated for effects on fungal phenotype, the antiviral defense response, viral RNA recombination activity and transposon expression. None of the single or multiple rdr knockout strains displayed any phenotypic differences from the parental strains with or without viral infection or any significant changes in viral RNA accumulation or recombination activity or transposon RNA accumulation, indicating no detectable contribution by the C. parasitica rdr genes to these processes.

  16. Lung epithelial cells have virus-specific and shared gene expression responses to infection by diverse respiratory viruses.

    Science.gov (United States)

    VanLeuven, James T; Ridenhour, Benjamin J; Gonzalez, Andres J; Miller, Craig R; Miura, Tanya A

    2017-01-01

    The severity of respiratory viral infections is partially determined by the cellular response mounted by infected lung epithelial cells. Disease prevention and treatment is dependent on our understanding of the shared and unique responses elicited by diverse viruses, yet few studies compare host responses to viruses from different families while controlling other experimental parameters. Murine models are commonly used to study the pathogenesis of respiratory viral infections, and in vitro studies using murine cells provide mechanistic insight into the pathogenesis observed in vivo. We used microarray analysis to compare changes in gene expression of murine lung epithelial cells infected individually by three respiratory viruses causing mild (rhinovirus, RV1B), moderate (coronavirus, MHV-1), and severe (influenza A virus, PR8) disease in mice. RV1B infection caused numerous gene expression changes, but the differential effect peaked at 12 hours post-infection. PR8 altered an intermediate number of genes whose expression continued to change through 24 hours. MHV-1 had comparatively few effects on host gene expression. The viruses elicited highly overlapping responses in antiviral genes, though MHV-1 induced a lower type I interferon response than the other two viruses. Signature genes were identified for each virus and included host defense genes for PR8, tissue remodeling genes for RV1B, and transcription factors for MHV-1. Our comparative approach identified universal and specific transcriptional signatures of virus infection that can be used to distinguish shared and virus-specific mechanisms of pathogenesis in the respiratory tract.

  17. CAG repeat length variation in the polymerase gamma (POLG) gene: effect on semen quality

    NARCIS (Netherlands)

    Westerveld, G. H.; Kaaij-Visser, L.; Tanck, M.; van der Veen, F.; Repping, S.

    2008-01-01

    Several case-control studies have investigated the effect of CAG repeat length variation in the POLG gene on male fertility and semen quality. Some described an association between the homozygous not10 CAG-repeat genotype and male subfertility and/or reduced semen quality, whereas others did not.

  18. Direct detection of diarrheagenic Aeromonas from faeces by polymerase chain reaction (PCR) targeting aerolysin toxin gene.

    Science.gov (United States)

    Kannan, S; Suresh Kanna, P; Karkuzhali, K; Chattopadhyay, U K; Pal, D

    2001-01-01

    Detection of diarrheagenic Aeromonas specific aerolysin toxin (Aer) gene by PCR based assay and isolation, identification of diarrhea causing Aeromonas from faeces by culture methods were carried out in the Division of Active Surveillance, National Institute of Cholera and Enteric Diseases (NICED), Kolkata, India for a period of 12 months. Out of 602 faecal samples collected from patients with acute diarrhea admitted in Infectious Diseases (ID) Hospital, Kolkata, 68 (11.29%) samples were found to be possessing Aer gene by PCR technique. The conventional culture methods using selective media yielded only 64 (10.6%) Aeromonas strains from the same faecal samples. The different Aeromonas species possessing Aer gene identified by PCR based technique include A. hydrophila (55.8%), A. caviae (17.6%), A. veronii (10.2%), A. schubertii (4.4%), A. jandaei (2.9%) and A. trota (8.8%). The isolation and identification of Aeromonas by routine culture did not detect enterotoxigenic A. trota present in four diarrheal faecal samples. The failure of the growth of enterotoxigenic A. trota on selective media may be attributed to the ampicillin susceptibility of those strains. The quality control studies revealed that PCR method for the direct detection of Aer gene from the faeces has the sensitivity of 100% and specificity of 98%.

  19. Pyviko: an automated Python tool to design gene knockouts in complex viruses with overlapping genes.

    Science.gov (United States)

    Taylor, Louis J; Strebel, Klaus

    2017-01-07

    Gene knockouts are a common tool used to study gene function in various organisms. However, designing gene knockouts is complicated in viruses, which frequently contain sequences that code for multiple overlapping genes. Designing mutants that can be traced by the creation of new or elimination of existing restriction sites further compounds the difficulty in experimental design of knockouts of overlapping genes. While software is available to rapidly identify restriction sites in a given nucleotide sequence, no existing software addresses experimental design of mutations involving multiple overlapping amino acid sequences in generating gene knockouts. Pyviko performed well on a test set of over 240,000 gene pairs collected from viral genomes deposited in the National Center for Biotechnology Information Nucleotide database, identifying a point mutation which added a premature stop codon within the first 20 codons of the target gene in 93.2% of all tested gene-overprinted gene pairs. This shows that Pyviko can be used successfully in a wide variety of contexts to facilitate the molecular cloning and study of viral overprinted genes. Pyviko is an extensible and intuitive Python tool for designing knockouts of overlapping genes. Freely available as both a Python package and a web-based interface ( http://louiejtaylor.github.io/pyViKO/ ), Pyviko simplifies the experimental design of gene knockouts in complex viruses with overlapping genes.

  20. GeneChip Resequencing of the Smallpox Virus Genome Can Identify Novel Strains: a Biodefense Application▿

    Science.gov (United States)

    Sulaiman, Irshad M.; Tang, Kevin; Osborne, John; Sammons, Scott; Wohlhueter, Robert M.

    2007-01-01

    We developed a set of seven resequencing GeneChips, based on the complete genome sequences of 24 strains of smallpox virus (variola virus), for rapid characterization of this human-pathogenic virus. Each GeneChip was designed to analyze a divergent segment of approximately 30,000 bases of the smallpox virus genome. This study includes the hybridization results of 14 smallpox virus strains. Of the 14 smallpox virus strains hybridized, only 7 had sequence information included in the design of the smallpox virus resequencing GeneChips; similar information for the remaining strains was not tiled as a reference in these GeneChips. By use of variola virus-specific primers and long-range PCR, 22 overlapping amplicons were amplified to cover nearly the complete genome and hybridized with the smallpox virus resequencing GeneChip set. These GeneChips were successful in generating nucleotide sequences for all 14 of the smallpox virus strains hybridized. Analysis of the data indicated that the GeneChip resequencing by hybridization was fast and reproducible and that the smallpox virus resequencing GeneChips could differentiate the 14 smallpox virus strains characterized. This study also suggests that high-density resequencing GeneChips have potential biodefense applications and may be used as an alternate tool for rapid identification of smallpox virus in the future. PMID:17182757

  1. Detection and identification of dengue virus isolates from Brazil by a simplified reverse transcription - polymerase chain reaction (RT-PCR method

    Directory of Open Access Journals (Sweden)

    FIGUEIREDO Luiz Tadeu Moraes

    1997-01-01

    Full Text Available We show here a simplified RT-PCR for identification of dengue virus types 1 and 2. Five dengue virus strains, isolated from Brazilian patients, and yellow fever vaccine 17DD as a negative control, were used in this study. C6/36 cells were infected and supernatants were collected after 7 days. The RT-PCR, done in a single reaction vessel, was carried out following a 1/10 dilution of virus in distilled water or in a detergent mixture containing Nonidet P40. The 50 µl assay reaction mixture included 50 pmol of specific primers amplifying a 482 base pair sequence for dengue type 1 and 210 base pair sequence for dengue type 2. In other assays, we used dengue virus consensus primers having maximum sequence similarity to the four serotypes, amplifying a 511 base pair sequence. The reaction mixture also contained 0.1 mM of the four deoxynucleoside triphosphates, 7.5 U of reverse transcriptase, 1U of thermostable Taq DNA polymerase. The mixture was incubated for 5 minutes at 37ºC for reverse transcription followed by 30 cycles of two-step PCR amplification (92ºC for 60 seconds, 53ºC for 60 seconds with slow temperature increment. The PCR products were subjected to 1.7% agarose gel electrophoresis and visualized by UV light after staining with ethidium bromide solution. Low virus titer around 10 3, 6 TCID50/ml was detected by RT-PCR for dengue type 1. Specific DNA amplification was observed with all the Brazilian dengue strains by using dengue virus consensus primers. As compared to other RT-PCRs, this assay is less laborious, done in a shorter time, and has reduced risk of contamination

  2. Sheeppox Virus Kelch-Like Gene SPPV-019 Affects Virus Virulence▿

    Science.gov (United States)

    Balinsky, C. A.; Delhon, G.; Afonso, C. L.; Risatti, G. R.; Borca, M. V.; French, R. A.; Tulman, E. R.; Geary, S. J.; Rock, D. L.

    2007-01-01

    Sheeppox virus (SPPV), a member of the Capripoxvirus genus of the Poxviridae, is the etiologic agent of a significant disease of sheep in the developing world. Genomic analysis of pathogenic and vaccine capripoxviruses identified genes with potential roles in virulence and host range, including three genes with similarity to kelch-like genes of other poxviruses and eukaryotes. Here, a mutant SPPV with a deletion in the SPPV-019 kelch-like gene, ΔKLP, was derived from the pathogenic strain SPPV-SA. ΔKLP exhibited in vitro growth characteristics similar to those of SPPV-SA and revertant virus (RvKLP). ΔKLP-infected cells exhibited a reduction in Ca2+-independent cell adhesion, suggesting that SPPV-019 may modulate cellular adhesion. When inoculated in sheep by the intranasal or intradermal routes, ΔKLP was markedly attenuated, since all ΔKLP-infected lambs survived infection. In contrast, SPPV-SA and RvKLP induced mortality approaching 100%. Lambs inoculated with ΔKLP exhibited marked reduction or delay in fever response, gross lesions, viremia, and virus shedding compared to parental and revertant viruses. Together, these findings indicate that SPPV-019 is a significant SPPV virulence determinant in sheep. PMID:17686843

  3. Sheeppox virus kelch-like gene SPPV-019 affects virus virulence.

    Science.gov (United States)

    Balinsky, C A; Delhon, G; Afonso, C L; Risatti, G R; Borca, M V; French, R A; Tulman, E R; Geary, S J; Rock, D L

    2007-10-01

    Sheeppox virus (SPPV), a member of the Capripoxvirus genus of the Poxviridae, is the etiologic agent of a significant disease of sheep in the developing world. Genomic analysis of pathogenic and vaccine capripoxviruses identified genes with potential roles in virulence and host range, including three genes with similarity to kelch-like genes of other poxviruses and eukaryotes. Here, a mutant SPPV with a deletion in the SPPV-019 kelch-like gene, DeltaKLP, was derived from the pathogenic strain SPPV-SA. DeltaKLP exhibited in vitro growth characteristics similar to those of SPPV-SA and revertant virus (RvKLP). DeltaKLP-infected cells exhibited a reduction in Ca(2+)-independent cell adhesion, suggesting that SPPV-019 may modulate cellular adhesion. When inoculated in sheep by the intranasal or intradermal routes, DeltaKLP was markedly attenuated, since all DeltaKLP-infected lambs survived infection. In contrast, SPPV-SA and RvKLP induced mortality approaching 100%. Lambs inoculated with DeltaKLP exhibited marked reduction or delay in fever response, gross lesions, viremia, and virus shedding compared to parental and revertant viruses. Together, these findings indicate that SPPV-019 is a significant SPPV virulence determinant in sheep.

  4. Involvement of the rabies virus phosphoprotein gene in neuroinvasiveness.

    Science.gov (United States)

    Yamaoka, Satoko; Ito, Naoto; Ohka, Seii; Kaneda, Shohei; Nakamura, Hiroko; Agari, Takahiro; Masatani, Tatsunori; Nakagawa, Keisuke; Okada, Kazuma; Okadera, Kota; Mitake, Hiromichi; Fujii, Teruo; Sugiyama, Makoto

    2013-11-01

    Rabies virus (RABV), which is transmitted via a bite wound caused by a rabid animal, infects peripheral nerves and then spreads to the central nervous system (CNS) before causing severe neurological symptoms and death in the infected individual. Despite the importance of this ability of the virus to spread from a peripheral site to the CNS (neuroinvasiveness) in the pathogenesis of rabies, little is known about the mechanism underlying the neuroinvasiveness of RABV. In this study, to obtain insights into the mechanism, we conducted comparative analysis of two fixed RABV strains, Nishigahara and the derivative strain Ni-CE, which cause lethal and asymptomatic infections, respectively, in mice after intramuscular inoculation. Examination of a series of chimeric viruses harboring the respective genes from Nishigahara in the genetic background of Ni-CE revealed that the Nishigahara phosphoprotein (P) gene plays a major role in the neuroinvasiveness by mediating infection of peripheral nerves. The results obtained from both in vivo and in vitro experiments strongly suggested that the Nishigahara P gene, but not the Ni-CE P gene, is important for stable viral replication in muscle cells. Further investigation based on the previous finding that RABV phosphoprotein counteracts the host interferon (IFN) system demonstrated that the Nishigahara P gene, but not the Ni-CE P gene, functions to suppress expression of the beta interferon (IFN-β) gene (Ifn-β) and IFN-stimulated genes in muscle cells. In conclusion, we provide the first data strongly suggesting that RABV phosphoprotein assists viral replication in muscle cells by counteracting the host IFN system and, consequently, enhances infection of peripheral nerves.

  5. Polymerase chain reaction detection of small round-structured viruses from two related hospital outbreaks of gastroenteritis using inosine-containing primers.

    Science.gov (United States)

    Green, S M; Lambden, P R; Deng, Y; Lowes, J A; Lineham, S; Bushell, J; Rogers, J; Caul, E O; Ashley, C R; Clarke, I N

    1995-02-01

    Two outbreaks of gastroenteritis in the UK which occurred nine days apart at Lymington and Southampton hospitals were investigated. The clinical and epidemiological features of both outbreaks were characteristic of small round-structured virus (SRSV) infection with rapid onset of diarrhoea and/or nausea and vomiting and propagation of the outbreaks by secondary spread. SRSV particles were observed by immune electron microscopy (EM) in 60% of faecal samples from both outbreaks and no other pathogens were detected. The index case for the second outbreak was a patient who was admitted with diarrhoea and vomiting after being discharged from Lymington hospital during the first outbreak. The possibility that the two outbreaks were caused by the same strain of SRSV was investigated by the polymerase chain reaction (PCR). New inosine-containing PCR primers were designed to amplify the RNA polymerase region of SRSV cDNA from genetic groups I and II. The PCR using the group II primers achieved a higher detection rate for SRSVs in faecal samples (68% of samples positive from both outbreaks) than immune EM. SRSVs were not detected using the group I primers or using conventional degenerate PCR primers. The nucleotide sequences of PCR amplicons from both outbreaks were identical providing molecular epidemiological evidence for the involvement of a single SRSV strain. Comparison of the RNA polymerase region of this virus with the equivalent regions of genetic group I (69.4-75.0% amino acid identify) and genetic group II (88.9-100% amino acid and 77.1-88.1% nucleotide identity) SRSVs revealed that the causative SRSV was a distinct member of genetic group II.

  6. Real-Time qPCR Identifies Suitable Reference Genes for Borna Disease Virus-Infected Rat Cortical Neurons

    Directory of Open Access Journals (Sweden)

    Lujun Zhang

    2014-11-01

    Full Text Available Quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR is the most commonly-used technique to identify gene expression profiles. The selection of stably expressed reference genes is a prerequisite to properly evaluating gene expression. Here, the suitability of commonly-used reference genes in normalizing RT-qPCR assays of mRNA expression in cultured rat cortical neurons infected with Borna disease virus (BDV was assessed. The expressions of eight commonly-used reference genes were comparatively analyzed in BDV-infected rat cortical neurons and non-infected control neurons mainly across 9 and 12 days post-infection. These reference genes were validated by RT-qPCR and separately ranked by four statistical algorithms: geNorm, NormFinder, BestKeeper and the comparative delta-Ct method. Then, the RankAggreg package was used to construct consensus rankings. ARBP was found to be the most stable internal control gene at Day 9, and ACTB at Day 12. As the assessment of the validity of the selected reference genes confirms the suitability of applying a combination of the two most stable references genes, combining the two most stable genes for normalization of RT-qPCR studies in BDV-infected rat cortical neurons is recommended at each time point. This study can contribute to improving BDV research by providing the means by which to obtain more reliable and accurate gene expression measurements.

  7. Real-time qPCR identifies suitable reference genes for Borna disease virus-infected rat cortical neurons.

    Science.gov (United States)

    Zhang, Lujun; Liu, Siwen; Zhang, Liang; You, Hongmin; Huang, Rongzhong; Sun, Lin; He, Peng; Chen, Shigang; Zhang, Hong; Xie, Peng

    2014-11-26

    Quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) is the most commonly-used technique to identify gene expression profiles. The selection of stably expressed reference genes is a prerequisite to properly evaluating gene expression. Here, the suitability of commonly-used reference genes in normalizing RT-qPCR assays of mRNA expression in cultured rat cortical neurons infected with Borna disease virus (BDV) was assessed. The expressions of eight commonly-used reference genes were comparatively analyzed in BDV-infected rat cortical neurons and non-infected control neurons mainly across 9 and 12 days post-infection. These reference genes were validated by RT-qPCR and separately ranked by four statistical algorithms: geNorm, NormFinder, BestKeeper and the comparative delta-Ct method. Then, the RankAggreg package was used to construct consensus rankings. ARBP was found to be the most stable internal control gene at Day 9, and ACTB at Day 12. As the assessment of the validity of the selected reference genes confirms the suitability of applying a combination of the two most stable references genes, combining the two most stable genes for normalization of RT-qPCR studies in BDV-infected rat cortical neurons is recommended at each time point. This study can contribute to improving BDV research by providing the means by which to obtain more reliable and accurate gene expression measurements.

  8. Complete genome sequence of Fer-de-Lance Virus reveals a novel gene in reptilian Paramyxoviruses

    Science.gov (United States)

    Kurath, G.; Batts, W.N.; Ahne, W.; Winton, J.R.

    2004-01-01

    The complete RNA genome sequence of the archetype reptilian paramyxovirus, Fer-de-Lance virus (FDLV), has been determined. The genome is 15,378 nucleotides in length and consists of seven nonoverlapping genes in the order 3??? N-U-P-M-F-HN-L 5???, coding for the nucleocapsid, unknown, phospho-, matrix, fusion, hemagglutinin-neuraminidase, and large polymerase proteins, respectively. The gene junctions contain highly conserved transcription start and stop signal sequences and tri-nucleotide intergenic regions similar to those of other Paramyxoviridae. The FDLV P gene expression strategy is like that of rubulaviruses, which express the accessory V protein from the primary transcript and edit a portion of the mRNA to encode P and I proteins. There is also an overlapping open reading frame potentially encoding a small basic protein in the P gene. The gene designated U (unknown), encodes a deduced protein of 19.4 kDa that has no counterpart in other paramyxoviruses and has no similarity with sequences in the National Center for Biotechnology Information database. Active transcription of the U gene in infected cells was demonstrated by Northern blot analysis, and bicistronic N-U mRNA was also evident. The genomes of two other snake paramyxovirus genotypes were also found to have U genes, with 11 to 16% nucleotide divergence from the FDLV U gene. Pairwise comparisons of amino acid identities and phylogenetic analyses of all deduced FDLV protein sequences with homologous sequences from other Paramyxoviridae indicate that FDLV represents a new genus within the subfamily Paramyxovirinae. We suggest the name Ferlavirus for the new genus, with FDLV as the type species.

  9. Characterization of an Avipoxvirus From a Bald Eagle ( Haliaeetus leucocephalus ) Using Novel Consensus PCR Protocols for the rpo147 and DNA-Dependent DNA Polymerase Genes.

    Science.gov (United States)

    Stephen, Alexa A; Leone, Angelique M; Toplon, David E; Archer, Linda L; Wellehan, James F X

    2016-12-01

    A juvenile female bald eagle ( Haliaeetus leucocephalus ) was presented with emaciation and proliferative periocular lesions. The eagle did not respond to supportive therapy and was euthanatized. Histopathologic examination of the skin lesions revealed plaques of marked epidermal hyperplasia parakeratosis, marked acanthosis and spongiosis, and eosinophilic intracytoplasmic inclusion bodies. Novel polymerase chain reaction (PCR) assays were done to amplify and sequence DNA polymerase and rpo147 genes. The 4b gene was also analyzed by a previously developed assay. Bayesian and maximum likelihood phylogenetic analyses of the obtained sequences found it to be poxvirus of the genus Avipoxvirus and clustered with other raptor isolates. Better phylogenetic resolution was found in rpo147 rather than the commonly used DNA polymerase. The novel consensus rpo147 PCR assay will create more accurate phylogenic trees and allow better insight into poxvirus history.

  10. Detection of hblA and bal Genes in Bacillus cereus Isolates From Cheese Samples Using the Polymerase Chain Reaction

    Directory of Open Access Journals (Sweden)

    Molayi Kohneshahri

    2016-03-01

    Full Text Available Background Bacillus cereus is a Gram-positive spore-forming bacterium, which causes food poisoning. Spores enable the persistence of B. cereus in the environment, and B. cereus strains can tolerate adverse environmental conditions, such as temperature and insufficient nutrients. B. cereus causes food poisoning via the production of two enterotoxins. Most isolates produce toxins leading to diarrhea (enterotoxins and vomiting (emetic forms. Diarrhea is caused by the production of three different heat-labile enterotoxins: HBL, NHE, and cytotoxin K. A heat-stable toxin, cereulide, is responsible for emesis. Objectives This study aimed to detect enterotoxigenic B. cereus isolates in cheese samples using the polymerase chain reaction (PCR. Materials and Methods Two-hundred pasteurized (n = 100 and nonpasteurized (n = 100 cheese samples were collected. The initial isolation was performed on PEMBA specific medium. Antibiotic susceptibility testing was performed using several antibiotic disks, according to the guidelines of the Clinical Laboratory and Standards Institute. Specific primers amplifying the hblA enterotoxin-encoding gene and bal hemolysin-encoding gene were used for the molecular detection of the toxins. Results Ten samples were positive for the presence of B. cereus, with both Gram staining and biochemical reactions. All the isolates were resistant to penicillin and ampicillin but susceptible to vancomycin, erythromycin, and ciprofloxacin. Six and three isolates were resistant to tetracycline and trimethoprim-sulfamethoxazole, respectively. The hblA and bal genes were amplified in all the B. cereus isolates. Conclusions The prevalence of B. cereus among the cheese samples was low. All the isolates were positive for genes encoding the hblA enterotoxin and bal toxin.

  11. Development of Virus-Induced Gene Expression and Silencing Vector Derived from Grapevine Algerian Latent Virus.

    Science.gov (United States)

    Park, Sang-Ho; Choi, Hoseong; Kim, Semin; Cho, Won Kyong; Kim, Kook-Hyung

    2016-08-01

    Grapevine Algerian latent virus (GALV) is a member of the genus Tombusvirus in the Tombusviridae and infects not only woody perennial grapevine plant but also herbaceous Nicotiana benthamiana plant. In this study, we developed GALV-based gene expression and virus-induced gene silencing (VIGS) vectors in N. benthamiana. The GALV coat protein deletion vector, pGMG, was applied to express the reporter gene, green fluorescence protein (GFP), but the expression of GFP was not detected due to the necrotic cell death on the infiltrated leaves. The p19 silencing suppressor of GALV was engineered to inactivate its expression and GFP was successfully expressed with unrelated silencing suppressor, HC-Pro, from soybean mosaic virus. The pGMG vector was used to knock down magnesium chelatase (ChlH) gene in N. benthamaina and the silencing phenotype was clearly observed on systemic leaves. Altogether, the GALV-derived vector is expected to be an attractive tool for useful gene expression and VIGS vectors in grapevine as well as N. benthamiana.

  12. Detection of Ampicillin Resistance Genes (bla in Clinical Isolates of Escherichia coli with Polymerase Chain Reaction Method

    Directory of Open Access Journals (Sweden)

    Tiana Milanda

    2014-09-01

    Full Text Available Escherichia coli is a rod negative Gram which could be pathogenic, if its value increases or located in outer gastrointestinal tract. Pathogenic E. coli will produce enterotoxin which will cause diarrhoea or infection in urine tract. Ampicilin was one of particular antibiotics to overcome infection. Ampicilin nowadays is no longer used as primary medicine, because of its resistance case. The aim of this research is to detect the presence of gene which is responsible to ampicilin resistant E. coli. We used isolated midstream urine from cystitis object in Hasan Sadikin Hospital (RSHS as samples. Polymerase Chain Reaction (PCR method (colony-PCR and DNA-PCR were done to invenstigate the antibiotic resistency. Based on the result of antibiotic susceptibility testing to ampicillin, E. coli samples were resistant to ampicilin. Elektroforegram products of colony-PCR and DNA-PCR showed that the resistance case of ampicilin caused by bla gene (199 bp. Selective and rational antibiotic treatment is required to prevent ampicillin resistance in patients with symptoms

  13. Duplication and Loss of Function of Genes Encoding RNA Polymerase III Subunit C4 Causes Hybrid Incompatibility in Rice

    Directory of Open Access Journals (Sweden)

    Giao Ngoc Nguyen

    2017-08-01

    Full Text Available Reproductive barriers are commonly observed in both animals and plants, in which they maintain species integrity and contribute to speciation. This report shows that a combination of loss-of-function alleles at two duplicated loci, DUPLICATED GAMETOPHYTIC STERILITY 1 (DGS1 on chromosome 4 and DGS2 on chromosome 7, causes pollen sterility in hybrid progeny derived from an interspecific cross between cultivated rice, Oryza sativa, and an Asian annual wild rice, O. nivara. Male gametes carrying the DGS1 allele from O. nivara (DGS1-nivaras and the DGS2 allele from O. sativa (DGS2-T65s were sterile, but female gametes carrying the same genotype were fertile. We isolated the causal gene, which encodes a protein homologous to DNA-dependent RNA polymerase (RNAP III subunit C4 (RPC4. RPC4 facilitates the transcription of 5S rRNAs and tRNAs. The loss-of-function alleles at DGS1-nivaras and DGS2-T65s were caused by weak or nonexpression of RPC4 and an absence of RPC4, respectively. Phylogenetic analysis demonstrated that gene duplication of RPC4 at DGS1 and DGS2 was a recent event that occurred after divergence of the ancestral population of Oryza from other Poaceae or during diversification of AA-genome species.

  14. Identification of Aeromonas trota (hybridization group 13) by amplification of the aerolysin gene using polymerase chain reaction.

    Science.gov (United States)

    Khan, A A; Nawaz, M S; Khan, S A; Cerniglia, C E

    1999-04-01

    Aeromonas trota is recognized as an important enteropathogen, and its haemolysin (aerolysin) is purported to be one of the virulence factors. Rapid detection and identification of A. trota is important for early and specific diagnosis of the infectious diseases that it causes. Synthetic oligonucleotide primers were used in a polymerase chain reaction (PCR) technique to amplify a species-specific sequence of the aerA gene, which encodes the aerolysin of A. trota. A DNA fragment of 622 bp was amplified from both lysed cells and isolated DNA from A. trota. The identity of the amplified 622 bp fragment was confirmed by digestion with BamH I restriction endonuclease, which produced the predicted 557 and 65 bp fragments. The lower limit for detection of the aerA gene by PCR amplification was 10 pg of total DNA or 10-15 cells ml-1. Primer specificity for A. trota was determined by the PCR assay with cells of 55 strains of Aeromonas sppincluding all of the 14 currently recognized DNA hybridization groups. A strain of Aeromonas enteropelogenes that had been reclassified as A. trota was also PCR positive. The method described here can be used to detect aerolysin-producing A. trota (hybridization group 13) strains from environmental and clinical samples without the use of selective media or additional biochemical tests. Copyright 1999 Academic Press.

  15. Evaluation of G2677T/A polymorphism of MDR1 gene by polymerase chain reaction in Mazandaran province, Iran

    Directory of Open Access Journals (Sweden)

    Razieh Keshavarz-Maleki

    2015-06-01

    Full Text Available The human MDR1 gene encodes for a P-glycoprotein (PGP, which acts as an efflux pump that transports a large variety of substrates from the inside of cells to the outside until protection against xenobiotics. The G2677T/A polymorphism in exon 21 is associated with PGP expression and function in humans. The present study was aimed to determine the frequencies of this polymorphism in a healthy population from Mazandaran province of Iran. A total of 120 unrelated healthy subjects from Mazandaran province, residing in Sari, coming for blood donating at Sari Blood Transfusion Center were enrolled. Genomic DNA was extracted from the peripheral blood lymphocytes of each subject. All subjects were genotyped for G2677T/A polymorphism by polymerase chain reaction-restriction fragment length polymorphism method. The genotype frequencies were G2677G (65%, G2677T (20.83%, G2677A (14.17% and TT, AA, TA genotypes were not observed. Moreover, frequency of G allele (82.5% was significantly (p ˂ 0.05 higher than the T (10.42% and A (7.08%. This is the first study to investigate the G2677T/A polymorphism of MDR1 gene in population from Mazandaran province of Iran. These data may be relevant for dose recommendation of PGP substrate drugs and can help for individualizing drug therapy of organ transplantation and important diseases such as cancer and AIDS, congestive heart failure and etc.

  16. Arabidopsis DNA polymerase ϵ recruits components of Polycomb repressor complex to mediate epigenetic gene silencing.

    Science.gov (United States)

    Del Olmo, Iván; López, Juan A; Vázquez, Jesús; Raynaud, Cécile; Piñeiro, Manuel; Jarillo, José A

    2016-07-08

    Arabidopsis ESD7 locus encodes the catalytic subunit of the DNA Pol ϵ involved in the synthesis of the DNA leading strand and is essential for embryo viability. The hypomorphic allele esd7-1 is viable but displays a number of pleiotropic phenotypic alterations including an acceleration of flowering time. Furthermore, Pol ϵ is involved in the epigenetic silencing of the floral integrator genes FT and SOC1, but the molecular nature of the transcriptional gene silencing mechanisms involved remains elusive. Here we reveal that ESD7 interacts with components of the PRC2 such as CLF, EMF2 and MSI1, and that mutations in ESD7 cause a decrease in the levels of the H3K27me3 mark present in the chromatin of FT and SOC1 We also demonstrate that a domain of the C-terminal region of ESD7 mediates the binding to the different PRC2 components and this interaction is necessary for the proper recruitment of PRC2 to FT and SOC1 chromatin. We unveil the existence of interplay between the DNA replication machinery and the PcG complexes in epigenetic transcriptional silencing. These observations provide an insight into the mechanisms ensuring that the epigenetic code at pivotal loci in developmental control is faithfully transmitted to the progeny of eukaryotic cells. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  17. Association of the polymorphism of the CAG repeat in the mitochondrial DNA polymerase gamma gene (POLG) with testicular germ-cell cancer

    DEFF Research Database (Denmark)

    Blomberg Jensen, M; Leffers, H; Petersen, J H

    2008-01-01

    BACKGROUND: A possible association between the polymorphic CAG repeat in the DNA polymerase gamma (POLG) gene and the risk of testicular germ-cell tumours (TGCT) was investigated in this study. The hypothesis was prompted by an earlier preliminary study proposing an association of the absence...

  18. Primers and polymerase chain reaction conditions for DNA barcoding teleost fish based on the mitochondrial cytochrome b and nuclear rhodopsin genes

    NARCIS (Netherlands)

    Sevilla, R.G.; Diez, A.; Noren, M.; Mouchel, O.; Jerome, M.; Verrez-Bagnis, V.; Pelt-Heerschap, van H.M.L.

    2007-01-01

    This report describes a set of 21 polymerase chain reaction primers and amplification conditions developed to barcode practically any teleost fish species according to their mitochondrial cytochrome b and nuclear rhodopsin gene sequences. The method was successfully tested in more than 200 marine

  19. Characterisation of Toxoplasma gondii isolates using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) of the non-coding Toxoplasma gondii (TGR)-gene sequences

    DEFF Research Database (Denmark)

    Høgdall, Estrid; Vuust, Jens; Lind, Peter

    2000-01-01

    of using TGR gene variants as markers to distinguish among T. gondii isolates from different animals and different geographical sources. Based on the band patterns obtained by restriction fragment length polymorphism (RFLP) analysis of the polymerase chain reaction (PCR) amplified TGR sequences, the T...

  20. Development, Evaluation, and Integration of a Quantitative Reverse-Transcription Polymerase Chain Reaction Diagnostic Test for Ebola Virus on a Molecular Diagnostics Platform.

    Science.gov (United States)

    Cnops, Lieselotte; Van den Eede, Peter; Pettitt, James; Heyndrickx, Leo; De Smet, Birgit; Coppens, Sandra; Andries, Ilse; Pattery, Theresa; Van Hove, Luc; Meersseman, Geert; Van Den Herrewegen, Sari; Vergauwe, Nicolas; Thijs, Rein; Jahrling, Peter B; Nauwelaers, David; Ariën, Kevin K

    2016-10-15

     The 2013-2016 Ebola epidemic in West Africa resulted in accelerated development of rapid diagnostic tests for emergency outbreak preparedness. We describe the development and evaluation of the Idylla™ prototype Ebola virus test, a fully automated sample-to-result molecular diagnostic test for rapid detection of Zaire ebolavirus (EBOV) and Sudan ebolavirus (SUDV).  The Idylla™ prototype Ebola virus test can simultaneously detect EBOV and SUDV in 200 µL of whole blood. The sample is directly added to a disposable cartridge containing all reagents for sample preparation, RNA extraction, and amplification by reverse-transcription polymerase chain reaction analysis. The performance was evaluated with a variety of sample types, including synthetic constructs and whole blood samples from healthy volunteers spiked with viral RNA, inactivated virus, and infectious virus.  The 95% limits of detection for EBOV and SUDV were 465 plaque-forming units (PFU)/mL (1010 copies/mL) and 324 PFU/mL (8204 copies/mL), respectively. In silico and in vitro analyses demonstrated 100% correct reactivity for EBOV and SUDV and no cross-reactivity with relevant pathogens. The diagnostic sensitivity was 97.4% (for EBOV) and 91.7% (for SUDV), the specificity was 100%, and the diagnostic accuracy was 95.9%.  The Idylla™ prototype Ebola virus test is a fast, safe, easy-to-use, and near-patient test that meets the performance criteria to detect EBOV in patients with suspected Ebola. © 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.

  1. Variations of Human DNA Polymerase Genes as Biomarkers of Prostate Cancer Progression

    Science.gov (United States)

    2011-07-01

    IRG); Grant number: 58-007-45. Current affiliation for Lúcio Fábio Caldas Ferraz: Centro de Biologia Molecular e Engenharia Genética, University of...8-9 References…………………………………………………………………………….9 Appendices………………………………………………………………………10-29 4 INTRODUCTION The early molecular ...Picopure® DNA Extraction Kit ( Molecular Devices; Sunnyvale, CA), and PCR amplified using the same primers that identified the POLB mutations shown on Table

  2. Adeno-associated virus inverted terminal repeats stimulate gene editing.

    Science.gov (United States)

    Hirsch, M L

    2015-02-01

    Advancements in genome editing have relied on technologies to specifically damage DNA which, in turn, stimulates DNA repair including homologous recombination (HR). As off-target concerns complicate the therapeutic translation of site-specific DNA endonucleases, an alternative strategy to stimulate gene editing based on fragile DNA was investigated. To do this, an episomal gene-editing reporter was generated by a disruptive insertion of the adeno-associated virus (AAV) inverted terminal repeat (ITR) into the egfp gene. Compared with a non-structured DNA control sequence, the ITR induced DNA damage as evidenced by increased gamma-H2AX and Mre11 foci formation. As local DNA damage stimulates HR, ITR-mediated gene editing was investigated using DNA oligonucleotides as repair substrates. The AAV ITR stimulated gene editing >1000-fold in a replication-independent manner and was not biased by the polarity of the repair oligonucleotide. Analysis of additional human DNA sequences demonstrated stimulation of gene editing to varying degrees. In particular, inverted yet not direct, Alu repeats induced gene editing, suggesting a role for DNA structure in the repair event. Collectively, the results demonstrate that inverted DNA repeats stimulate gene editing via double-strand break repair in an episomal context and allude to efficient gene editing of the human chromosome using fragile DNA sequences.

  3. Detection of herpes simplex-1 and -2 and varicella zoster virus by quantitative real-time polymerase chain reaction in corneas from patients with bacterial keratitis.

    Science.gov (United States)

    Nascimento, Heloisa; Watanabe, Aripuanã; Vieira, Ana Carolina Cabreira; Pelegrini, Andrea; Yu, Maria Cecília; Bispo, Paulo José Martins; Granato, Celso Francisco Hernandes; Höfling-Lima, Ana Luisa

    2017-01-01

    Bacterial keratitis occurs worldwide, and despite recent developments, it remains a potentially blinding condition. This study assesses the presence of herpes simplex virus (HSV-1 and -2) and varicella zoster virus (VZV) by quantitative real-time polymerase chain reaction (qPCR) in corneal scrapings from patients with bacterial keratitis. A total of 65 patients with clinical diagnoses of infectious corneal ulcers prospectively underwent clinical eye examinations. Corneal scrapings were investigated by Gram staining, Giemsa staining, culture, and qPCR (the study group). Risk factors and epidemiological data were recorded. The control group comprising 25 eyes with typical herpes dendritic keratitis was also analyzed by qPCR. From the study group (n=65), nine patients (13.8%) had negative smears, cultures, and qPCR findings. Fifty-six (86.2%) patients had positive cultures: 51 for bacteria, 4 for fungi, and 1 for amoebae. Of the patients who had positive bacterial cultures, qPCR identified 10 patients who were also positive for virus: one for VZV and nine for HSV-1. Of the 25 patients in the control group, 21 tested positive for HSV-1 by qPCR analysis. Herpes may be present in patients with bacterial corneal ulcers, and qPCR may be useful in its detection.

  4. LINGUISTIC ANALYSIS OF THE NUCLEOPROTEIN GENE OF INFLUENZA A VIRUS

    Energy Technology Data Exchange (ETDEWEB)

    A. SKOURIKHINE; T. BURR

    2000-05-01

    We applied linguistic analysis approach, specifically N-grams, to classify nucleotide and amino acids sequences of nucleoprotein (NP) gene of the Influenza A virus isolated from a range of hosts and geographic regions. We considered letter frequency (1-grams), letter pairs frequency (2-grams) and triplets' frequency (3-grams). Classification trees based on 1,2,3-grams variables were constructed for the same NP nucleotide and amino acids strains and their classification efficiency were compared with the clustering obtained using phylogenetic analysis. The results have shown that disregarding positional information for a NP gene can provide the same level of recognition accuracy like alternative more complex classification techniques.

  5. Engineering adeno-associated viruses for clinical gene therapy.

    Science.gov (United States)

    Kotterman, Melissa A; Schaffer, David V

    2014-07-01

    Clinical gene therapy has been increasingly successful owing both to an enhanced molecular understanding of human disease and to progressively improving gene delivery technologies. Among these technologies, delivery vectors based on adeno-associated viruses (AAVs) have emerged as safe and effective and, in one recent case, have led to regulatory approval. Although shortcomings in viral vector properties will render extension of such successes to many other human diseases challenging, new approaches to engineer and improve AAV vectors and their genetic cargo are increasingly helping to overcome these barriers.

  6. Efficient Virus-Induced Gene Silencing in Solanum rostratum

    Science.gov (United States)

    Meng, Lan-Huan; Wang, Rui-Heng; Zhu, Ben-Zhong; Zhu, Hong-Liang; Luo, Yun-Bo; Fu, Da-Qi

    2016-01-01

    Solanum rostratum is a “super weed” that grows fast, is widespread, and produces the toxin solanine, which is harmful to both humans and other animals. To our knowledge, no study has focused on its molecular biology owing to the lack of available transgenic methods and sequence information for S. rostratum. Virus-induced gene silencing (VIGS) is a powerful tool for the study of gene function in plants; therefore, in the present study, we aimed to establish tobacco rattle virus (TRV)-derived VIGS in S. rostratum. The genes for phytoene desaturase (PDS) and Chlorophyll H subunit (ChlH) of magnesium protoporphyrin chelatase were cloned from S. rostratum and used as reporters of gene silencing. It was shown that high-efficiency VIGS can be achieved in the leaves, flowers, and fruit of S. rostratum. Moreover, based on our comparison of three different types of infection methods, true leaf infection was found to be more efficient than cotyledon and sprout infiltration in long-term VIGS in multiple plant organs. In conclusion, the VIGS technology and tomato genomic sequences can be used in the future to study gene function in S. rostratum. PMID:27258320

  7. Efficient Virus-Induced Gene Silencing in Solanum rostratum.

    Directory of Open Access Journals (Sweden)

    Lan-Huan Meng

    Full Text Available Solanum rostratum is a "super weed" that grows fast, is widespread, and produces the toxin solanine, which is harmful to both humans and other animals. To our knowledge, no study has focused on its molecular biology owing to the lack of available transgenic methods and sequence information for S. rostratum. Virus-induced gene silencing (VIGS is a powerful tool for the study of gene function in plants; therefore, in the present study, we aimed to establish tobacco rattle virus (TRV-derived VIGS in S. rostratum. The genes for phytoene desaturase (PDS and Chlorophyll H subunit (ChlH of magnesium protoporphyrin chelatase were cloned from S. rostratum and used as reporters of gene silencing. It was shown that high-efficiency VIGS can be achieved in the leaves, flowers, and fruit of S. rostratum. Moreover, based on our comparison of three different types of infection methods, true leaf infection was found to be more efficient than cotyledon and sprout infiltration in long-term VIGS in multiple plant organs. In conclusion, the VIGS technology and tomato genomic sequences can be used in the future to study gene function in S. rostratum.

  8. Rapid detection and quantification of Ebola Zaire virus by one-step real-time quantitative reverse transcription-polymerase chain reaction.

    Science.gov (United States)

    Ro, Young-Tae; Ticer, Anysha; Carrion, Ricardo; Patterson, Jean L

    2017-04-01

    Given that Ebola virus causes severe hemorrhagic fever in humans with mortality rates as high as 90%, rapid and accurate detection of this virus is essential both for controlling infection and preventing further transmission. Here, a one-step qRT-PCR assay for rapid and quantitative detection of an Ebola Zaire strain using GP, VP24 or VP40 genes as a target is introduced. Routine assay conditions for hydrolysis probe detection were established from the manufacturer's protocol used in the assays. The analytical specificity and sensitivity of each assay was evaluated using in vitro synthesized viral RNA transcripts. The assays were highly specific for the RNA transcripts, no cross-reactivity being observed among them. The limits of detection of the assays ranged from 102 to 103 copies per reaction. The assays were also evaluated using viral RNAs extracted from cell culture-propagated viruses (Ebola Zaire, Sudan and Reston strains), confirming that they are gene- and strain-specific. The RT-PCR assays detected viral RNAs in blood samples from virus-infected animal, suggesting that they can be also a useful method for identifying Ebola virus in clinical samples. © 2017 The Societies and John Wiley & Sons Australia, Ltd.

  9. Optimization of the elution buffer and concentration method for detecting hepatitis E virus in swine liver using a nested reverse transcription-polymerase chain reaction and real-time reverse transcription-polymerase chain reaction.

    Science.gov (United States)

    Son, Na Ry; Seo, Dong Joo; Lee, Min Hwa; Seo, Sheungwoo; Wang, Xiaoyu; Lee, Bog-Hieu; Lee, Jeong-Su; Joo, In-Sun; Hwang, In-Gyun; Choi, Changsun

    2014-09-01

    The aim of this study was to develop an optimal technique for detecting hepatitis E virus (HEV) in swine livers. Here, three elution buffers and two concentration methods were compared with respect to enhancing recovery of HEV from swine liver samples. Real-time reverse transcription-polymerase chain reaction (RT-PCR) and nested RT-PCR were performed to detect HEV RNA. When phosphate-buffered saline (PBS, pH 7.4) was used to concentrate HEV in swine liver samples using ultrafiltration, real-time RT-PCR detected HEV in 6 of the 26 samples. When threonine buffer was used to concentrate HEV using polyethylene glycol (PEG) precipitation and ultrafiltration, real-time RT-PCR detected HEV in 1 and 3 of the 26 samples, respectively. When glycine buffer was used to concentrate HEV using ultrafiltration and PEG precipitation, real-time RT-PCR detected HEV in 1 and 3 samples of the 26 samples, respectively. When nested RT-PCR was used to detect HEV, all samples tested negative regardless of the type of elution buffer or concentration method used. Therefore, the combination of real-time RT-PCR and ultrafiltration with PBS buffer was the most sensitive and reliable method for detecting HEV in swine livers. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. [Experimental study on hepatitis B-virus X gene expression in adenoid cystic carcinoma].

    Science.gov (United States)

    Xie, Ling; Wang, Weihong; Xu, Biao; Liu, Yu

    2014-08-01

    To explore the expression of hepatitis B-virus X gene (HBX) in adenoid cystic carcinoma (ACC) and determine its clinical significance. Between June 2008 and October 2012, in-hospital patients with salivary gland tumors who were treated at the Department of Oral and Maxillofacial Surgery, Affiliated Stomatology Hospital of Kunming Medical University, were enrolled to this study. HBeAb-positive patients were defined as those exposed to hepatitis B virus (HBV) or harboring persistent HBV infection regardless of being HBeAg positive or negative. According to the pathological results, all patients were divided into ACC group and control group. Immunohistochemical staining and polymerase chain reaction (PCR) were used to detect HBX expression in ACC group and control group. HBX expression was mostly detected in the cytoplasm of ACC cells. Minimal HBX expression was detected in the nucleus. HBX expression was significantly higher in ACC than in Warthin's tumor. A significant difference was observed between the two groups. HBX is expressed in ACC and may be associated with the development of ACC. HBX might serve important functions in the carcinogenesis and development of ACC.

  11. A Multiplex Real-time Reverse Transcription Polymerase Chain Reaction Assay for Detection and Differentiation of Bluetongue Virus and Epizootic Hemorrhagic Disease Virus Serogroups

    Science.gov (United States)

    Bluetongue virus (BTV) causes disease in domestic and wild ruminants resulting in significant economic loss. The closely related Epizootic hemorrhagic diseases virus (EHDV) has been associated with bluetongue-like disease in cattle. Although US EHDV strains have not been experimentally proven to cau...

  12. E2F mediates induction of the Sp1-controlled promoter of the human DNA polymerase ɛ B-subunit gene POLE2

    Science.gov (United States)

    Huang, Deqi; Jokela, Maarit; Tuusa, Jussi; Skog, Sven; Poikonen, Kari; Syväoja, Juhani E.

    2001-01-01

    The B-subunits of replicative DNA polymerases from Archaea to humans belong to the same protein family, suggesting that they share a common fundamental function. We report here the gene structure for the B-subunit of human DNA polymerase ɛ (POLE2), whose expression and transcriptional regulation is typical for replication proteins with some unique features. The 75 bp core promoter region, located within exon 1, contains an Sp1 element that is a critical determinant of promoter activity as shown by the luciferase reporter, electrophoretic mobility shift and DNase I footprinting assays. Two overlapping E2F elements adjacent to the Sp1 element are essential for full promoter activity and serum response. Binding sites for E2F1 and NF-1 reside immediately downstream from the core promoter region. Our results suggest that human POLE2 is regulated by two E2F–pocket protein complexes, one associated with Sp1 and the other with NF-1. So far, only one replicative DNA polymerase B-subunit gene promoter, POLA2 encoding the B-subunit of DNA polymerase α, has been characterized. Mitogenic activation of the POLE2 promoter by an E2F-mediated mechanism resembles that of POLA2, but the regulation of basal promoter activity is different between these two genes. PMID:11433027

  13. Gene silencing and gene expression in phytopathogenic fungi using a plant virus vector.

    Science.gov (United States)

    Mascia, Tiziana; Nigro, Franco; Abdallah, Alì; Ferrara, Massimo; De Stradis, Angelo; Faedda, Roberto; Palukaitis, Peter; Gallitelli, Donato

    2014-03-18

    RNA interference (RNAi) is a powerful approach for elucidating gene functions in a variety of organisms, including phytopathogenic fungi. In such fungi, RNAi has been induced by expressing hairpin RNAs delivered through plasmids, sequences integrated in fungal or plant genomes, or by RNAi generated in planta by a plant virus infection. All these approaches have some drawbacks ranging from instability of hairpin constructs in fungal cells to difficulties in preparing and handling transgenic plants to silence homologous sequences in fungi grown on these plants. Here we show that RNAi can be expressed in the phytopathogenic fungus Colletotrichum acutatum (strain C71) by virus-induced gene silencing (VIGS) without a plant intermediate, but by using the direct infection of a recombinant virus vector based on the plant virus, tobacco mosaic virus (TMV). We provide evidence that a wild-type isolate of TMV is able to enter C71 cells grown in liquid medium, replicate, and persist therein. With a similar approach, a recombinant TMV vector carrying a gene for the ectopic expression of the green fluorescent protein (GFP) induced the stable silencing of the GFP in the C. acutatum transformant line 10 expressing GFP derived from C71. The TMV-based vector also enabled C. acutatum to transiently express exogenous GFP up to six subcultures and for at least 2 mo after infection, without the need to develop transformation technology. With these characteristics, we anticipate this approach will find wider application as a tool in functional genomics of filamentous fungi.

  14. Quantitative fucK gene polymerase chain reaction on sputum and nasopharyngeal secretions to detect Haemophilus influenzae pneumonia.

    Science.gov (United States)

    Abdeldaim, Guma M K; Strålin, Kristoffer; Olcén, Per; Blomberg, Jonas; Mölling, Paula; Herrmann, Björn

    2013-06-01

    A quantitative polymerase chain reaction (PCR) for the fucK gene was developed for specific detection of Haemophilus influenzae. The method was tested on sputum and nasopharyngeal aspirate (NPA) from 78 patients with community-acquired pneumonia (CAP). With a reference standard of sputum culture and/or serology against the patient's own nasopharyngeal isolate, H. influenzae etiology was detected in 20 patients. Compared with the reference standard, fucK PCR (using the detection limit 10(5) DNA copies/mL) on sputum and NPA showed a sensitivity of 95.0% (19/20) in both cases, and specificities of 87.9% (51/58) and 89.5% (52/58), respectively. In a receiver operating characteristic curve analysis, sputum fucK PCR was found to be significantly superior to sputum P6 PCR for detection of H. influenzae CAP. NPA fucK PCR was positive in 3 of 54 adult controls without respiratory symptoms. In conclusion, quantitative fucK real-time PCR provides a sensitive and specific identification of H. influenzae in respiratory secretions. Copyright © 2013 Elsevier Inc. All rights reserved.

  15. The hepatitis C virus replicon presents a higher barrier to resistance to nucleoside analogs than to nonnucleoside polymerase or protease inhibitors.

    Science.gov (United States)

    McCown, Matthew F; Rajyaguru, Sonal; Le Pogam, Sophie; Ali, Samir; Jiang, Wen-Rong; Kang, Hyunsoon; Symons, Julian; Cammack, Nick; Najera, Isabel

    2008-05-01

    Specific inhibitors of hepatitis C virus (HCV) replication that target the NS3/4A protease (e.g., VX-950) or the NS5B polymerase (e.g., R1479/R1626, PSI-6130/R7128, NM107/NM283, and HCV-796) have advanced into clinical development. Treatment of patients with VX-950 or HCV-796 rapidly selected for drug-resistant variants after a 14-day monotherapy treatment period. However, no viral resistance was identified after monotherapy with R1626 (prodrug of R1479) or NM283 (prodrug of NM107) after 14 days of monotherapy. Based upon the rapid selection of resistance to the protease and nonnucleoside inhibitors during clinical trials and the lack of selection of resistance to the nucleoside inhibitors, we used the replicon system to determine whether nucleoside inhibitors demonstrate a higher genetic barrier to resistance than protease and nonnucleoside inhibitors. Treatment of replicon cells with nucleoside inhibitors at 10 and 15 times the 50% effective concentration resulted in clearance of the replicon, while treatment with a nonnucleoside or protease inhibitor selected resistant colonies. In combination, the presence of a nucleoside inhibitor reduced the frequency of colonies resistant to the other classes of inhibitors. These results indicate that the HCV replicon presents a higher barrier to the selection of resistance to nucleoside inhibitors than to nonnucleoside or protease inhibitors. Furthermore, the combination of a nonnucleoside or protease inhibitor with a nucleoside polymerase inhibitor could have a clear clinical benefit through the delay of resistance emergence.

  16. Studies on Parameters Influencing the Performance of Reverse Transcriptase Polymerase Chain Reaction (RT-PCR in Detecting Prunus Necrotic Ringpot Virus (PNRSV

    Directory of Open Access Journals (Sweden)

    M. Usta

    2005-08-01

    Full Text Available In order to have a more detailed understanding of the various factors influencing a reverse transcriptase polymerase chain reaction (RT-PCR, a number of important parameters such as Mg+2, primer, enzyme concentration and others were optimized for the detection of Prunus necrotic ringspot virus (PNRSV. Using a PNRSV isolate with a pair of primers, complementary DNA of viral genome as template, and an appropriate enzyme together with magnesium chloride, the following optimal conditions were identified: primer concentration between 0.2 and 0.0002 pmol µl-1 and 0.06–2 units µl-1 for Taq DNA polymerase enzyme for a 50 µl reaction volume when other parameters were optimum; magnesium chloride concentration less than 2.5 mM; dNTP concentration between 1 and 10 mM. The optimum cDNA amount should be ~360 ng for a 50 µl reaction mixture. When these optimized concentrations and/or values of the main PCR parameters were brought together for a new RT-PCR, a clear and a reliable PNRSV detection having no background was performed from both growth-chamber and field-grown PNRSV-infected plants.

  17. AMINO-TERMINUS OF POLYMERASE BASIC-2 OF AVIAN INFLUENZA VIRUS OF H5N1 SUBTYPE ISOLATED FROM VARIOUS ANIMAL SPECIES IN INDONESIA

    Directory of Open Access Journals (Sweden)

    Gusti Ayu Yuniati Kencana

    2008-09-01

    Full Text Available The information on pathogenicity and adaptation factors of avian influenza virus (AIV in mammalsis very inportant in an effort to reduce the risk of avian influenza (AI pandemic in the future. Polymerasegene complex appears to be the major factors for adaptation of AIV to certain animal species. A preliminarystudy on role of non-coding region (NCR and amino-terminus of polymerase-basic 2 (PB2 is presented.Purified viral RNA of AIV isolated from chicken, duck, pig, and quail of Bali and Yogyakarta was reversetranscribed into cDNA and amplified using reverse transcriptase-polymerase chain reaction (RT-PCRusing PB-2 universal forward primer and specifically designed backward primer. The result showed thatall AIV’s H5N1 isolated from chicken, duck, quail, and pig, posed PB2 amino-terminus typical for IndonesianAIV H5N1. However, polymorphic amino acids of the protein fragment did not show any species specificmotive, with the exception of the pig isolate Sw/Tabanan/2006 which had specific substitution of D16E,H17Q, M40I, and H124Y.

  18. Initiation of RNA Synthesis by the Hepatitis C Virus RNA-Dependent RNA Polymerase Is Affected by the Structure of the RNA Template

    Science.gov (United States)

    2015-01-01

    The hepatitis C virus (HCV) RNA-dependent RNA polymerase NS5B is a central enzyme of the intracellular replication of the viral (+)RNA genome. Here, we studied the individual steps of NS5B-catalyzed RNA synthesis by a combination of biophysical methods, including real-time 1D 1H NMR spectroscopy. NS5B was found to bind to a nonstructured and a structured RNA template in different modes. Following NTP binding and conversion to the catalysis-competent ternary complex, the polymerase revealed an improved affinity for the template. By monitoring the folding/unfolding of 3′(−)SL by 1H NMR, the base pair at the stem’s edge was identified as the most stable component of the structure. 1H NMR real-time analysis of NS5B-catalyzed RNA synthesis on 3′(−)SL showed that a pronounced lag phase preceded the processive polymerization reaction. The presence of the double-stranded stem with the edge base pair acting as the main energy barrier impaired RNA synthesis catalyzed by NS5B. Our observations suggest a crucial role of RNA-modulating factors in the HCV replication process. PMID:25310724

  19. Effect of dicer-like proteins2 and 4 and RNA-dependent RNA polymerase1 as RNA silencing components on cyclic mosaic symptom development in tobacco infected with the Cucumber mosaic virus

    Directory of Open Access Journals (Sweden)

    Anurag Sunpapao

    2014-02-01

    Full Text Available The Nicotiana tabacum genome contains four Dicer-like proteins (DCLs and six RNA-dependent RNA polymerase (RDR homologues involved in the RNA silencing mechanism employed against viral infection. DCL1 synthesizes 18-21 nt-long microRNA, whereas DCL2, DCL3 and DCL4 produce 22 nt, 24 nt and 21 nt-long siRNA, respectively, in the RNA silencing process. This study aimed to clarify which components among these are involved in changes in the amount of virus and the development of symptoms in Cucumber mosaic virus (CMV-infected tobacco. Infected transgenic tobacco lines with a single down-regulation of DCL2, DCL4, RDR1 or a double down-regulation of both DCL2 and 4 were analyzed. The amounts of viral RNA in young developing leaves in transgenic tobacco lines were examined by Northern blot analysis. Most transgenic plants inoculated with CMV Pepo, a virulent strain, exhibited cyclic mosaic symptoms. The amount of viral RNA in single down-regulated lines varied based on leaf position in a similar manner to that noted in non-transgenic tobacco, while that of the double down-regulated line did not. Furthermore, the expression of RNA-silencing-related genes during high and low CMV infection did not differ among the transgenic plants. These results suggested that (i changes in the amounts of the virus in the developing leaves of all the single down-regulated lines were associated with cyclic symptom expression in fully expanded leaves, and (ii the lower expression of DCL2, DCL4 and RDR1 may be sufficient to establish cyclic symptom development.

  20. Reverse Genetics Plasmid for Cloning Unstable Influenza A Virus Gene Segments

    OpenAIRE

    Zhou, Bin; Jerzak, Greta; Scholes, Derek T.; Donnelly, Matthew E.; Li, Yan; Wentworth, David E.

    2011-01-01

    Reverse genetics approaches that enable the generation of recombinant influenza A viruses entirely from plasmids are invaluable for studies on virus replication, morphogenesis, pathogenesis, or transmission. Furthermore, influenza virus reverse genetics is now critical for the development of new vaccines for this human and animal pathogen. Periodically, influenza gene segments are unstable within plasmids in bacteria. The PB2 gene segment of a highly pathogenic avian H5 influenza virus A/Turk...

  1. Rapid detection of avian influenza virus in chicken fecal samples by immunomagnetic capture reverse transcriptase–polymerase chain reaction assay

    DEFF Research Database (Denmark)

    Dhumpa, Raghuram; Handberg, Kurt; Jørgensen, Poul Henrik

    2011-01-01

    Avian influenza virus (AIV) causes great economic losses for the poultry industry worldwide and threatens the human population with a pandemic. The conventional detection method for AIV involves sample preparation of viral RNA extraction and purification from raw sample such as bird droppings...

  2. A Single Amino Acid Mutation (I1012F) of the RNA Polymerase of Marine Viral Hemorrhagic Septicemia Virus Changes In Vitro Virulence to Rainbow Trout Gill Epithelial Cells

    DEFF Research Database (Denmark)

    Kim, Sung-Hyun; Thu, Beate J.; Skall, Helle Frank

    2014-01-01

    . Otte, and N. J. Olesen, Virus Res. 63: 95-106, 1999]) and IVa (JF-09). DK-3592 and NO/650/07 were pathogenic to GECs, while marine strains 1p8 and JF-09 were nonpathogenic to GECs. Eight conserved amino acid substitutions contrasting high-and low-virulence strains were identified, and reverse genetics...... was used in a gain-of-virulence approach based on the JF-09 backbone. Mutations were introduced into the G, NV, and L genes, and seven different virus clones were obtained. For the first time, we show that a single amino acid mutation in conserved region IV of the L protein, I1012F, rendered the virus able...

  3. Sequencing and sequence analysis of partial nucleoprotein (N) gene and phylogenetic analysis of rabies virus field isolates from Gujarat state, India.

    Science.gov (United States)

    Vagheshwari, Dhaval H; Bhanderi, Bharat B; Mathakiya, Rafyuddin A; Jhala, Mayurdhvaj K

    2017-09-01

    The present study was undertaken with an aim of characterization of rabies virus (genus Lyssavirus of the family Rhabdoviridae under the order Mononegavirales) by sequencing of partial nucleoprotein (N) gene of rabies virus and phylogenetic analysis to know the genotype and lineage of rabies virus present in Gujarat state of India. A total of 32 samples (18 brain samples and 14 saliva samples) were aseptically collected from live and dead animals (viz. dog, buffalo, cow, goat, donkey and hyena) for rabies virus detection. Out of 32 samples, 24 samples were found positive by Reverse Transcriptase Polymerase Chain Reactions and from these 24 positive samples, 20 samples were selected for sequencing having good concentration of gene product. ClustalW alignment of nucleotide sequences and amino acid sequences of field rabies isolates revealed 95.20-100 and 97.95-100% similarity among themselves, respectively. Multiple sequence alignment of field rabies isolates and reference vaccine strains [Pasteur strain and Challenge Virus Strain (CVS)] indicated single nucleotide mutations at total 91 positions and amino acid mutations at total 17 different positions. Phylogenetic analysis of N gene sequences using our 20 field rabies isolates and 21 other reported isolates in Genbank resulted in 3 phylogenetic clusters. All the field rabies isolates showed same genetic lineage among themselves and with other earlier reported Indian rabies isolates placing them in Arctic like lineage of Genotype 1 Rabies virus. However, they were at genetic distance with reference Pasteur and CVS strains, which grouped in different phylogenetic cluster.

  4. The viral tropism of two distinct oncolytic viruses, reovirus and myxoma virus, is modulated by cellular tumor suppressor gene status.

    Science.gov (United States)

    Kim, M; Williamson, C T; Prudhomme, J; Bebb, D G; Riabowol, K; Lee, P W K; Lees-Miller, S P; Mori, Y; Rahman, M M; McFadden, G; Johnston, R N

    2010-07-08

    Replication-competent oncolytic viruses hold great potential for the clinical treatment of many cancers. Importantly, many oncolytic virus candidates, such as reovirus and myxoma virus, preferentially infect cancer cells bearing abnormal cellular signaling pathways. Reovirus and myxoma virus are highly responsive to activated Ras and Akt signaling pathways, respectively, for their specificity for viral oncolysis. However, considering the complexity of cancer cell populations, it is possible that other tumor-specific signaling pathways may also contribute to viral discrimination between normal versus cancer cells. Because carcinogenesis is a multistep process involving the accumulation of both oncogene activations and the inactivation of tumor suppressor genes, we speculated that not only oncogenes but also tumor suppressor genes may have an important role in determining the tropism of these viruses for cancer cells. It has been previously shown that many cellular tumor suppressor genes, such as p53, ATM and Rb, are important for maintaining genomic stability; dysfunction of these tumor suppressors may disrupt intact cellular antiviral activity due to the accumulation of genomic instability or due to interference with apoptotic signaling. Therefore, we speculated that cells with dysfunctional tumor suppressors may display enhanced susceptibility to challenge with these oncolytic viruses, as previously seen with adenovirus. We report here that both reovirus and myxoma virus preferentially infect cancer cells bearing dysfunctional or deleted p53, ATM and Rb tumor suppressor genes compared to cells retaining normal counterparts of these genes. Thus, oncolysis by these viruses may be influenced by both oncogenic activation and tumor suppressor status.

  5. Guanine α-carboxy nucleoside phosphonate (G-α-CNP) shows a different inhibitory kinetic profile against the DNA polymerases of human immunodeficiency virus (HIV) and herpes viruses.

    Science.gov (United States)

    Balzarini, Jan; Menni, Michael; Das, Kalyan; van Berckelaer, Lizette; Ford, Alan; Maguire, Nuala M; Liekens, Sandra; Boehmer, Paul E; Arnold, Eddy; Götte, Matthias; Maguire, Anita R

    2017-07-15

    α-Carboxy nucleoside phosphonates (α-CNPs) are modified nucleotides that represent a novel class of nucleotide-competing reverse transcriptase (RT) inhibitors (NcRTIs). They were designed to act directly against HIV-1 RT without the need for prior activation (phosphorylation). In this respect, they differ from the nucleoside or nucleotide RTIs [N(t)RTIs] that require conversion to their triphosphate forms before being inhibitory to HIV-1 RT. The guanine derivative (G-α-CNP) has now been synthesized and investigated for the first time. The (L)-(+)-enantiomer of G-α-CNP directly and competitively inhibits HIV-1 RT by interacting with the substrate active site of the enzyme. The (D)-(-)-enantiomer proved inactive against HIV-1 RT. In contrast, the (+)- and (-)-enantiomers of G-α-CNP inhibited herpes (i.e. HSV-1, HCMV) DNA polymerases in a non- or uncompetitive manner, strongly indicating interaction of the (L)-(+)- and the (D)-(-)-G-α-CNPs at a location different from the polymerase substrate active site of the herpes enzymes. Such entirely different inhibition profile of viral polymerases is unprecedented for a single antiviral drug molecule. Moreover, within the class of α-CNPs, subtle differences in their sensitivity to mutant HIV-1 RT enzymes were observed depending on the nature of the nucleobase in the α-CNP molecules. The unique properties of the α-CNPs make this class of compounds, including G-α-CNP, direct acting inhibitors of multiple viral DNA polymerases. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. The Uneven Rate of the Molecular Evolution of Gene Sequences of DNA-Dependent RNA Polymerase I of the Genus Lamium L

    Science.gov (United States)

    Krawczyk, Katarzyna; Sawicki, Jakub

    2013-01-01

    RNA polymerase type I (plastid-encoded polymerase, PEP) is one of the key chloroplast enzymes. However, the rpo genes that encode its subunits (rpoA, rpoB, rpoC1 and rpoC2) are relatively rapidly evolving sequences. The aim of this study was to investigate the rate of the molecular evolution of rpo genes and to evaluate them as phylogenetic markers on the example of the genus Lamium L. (Lamiaceae). The analyzed genes were shown to differ in the level of variation, rate of intragenic mutations, phylogenetic informativeness, and in the impact of these mutations on the properties of encoded peptides. Destabilizing effects of the positive pressure were observed in all genes examined coding for PEP enzyme. We have demonstrated the relationship between mutations fixed by positive selection and the separation of phylogenetic lines within the genus Lamium. The study showed also that the rpo genes were reliable phylogenetic markers, useful in the reconstruction of interconnections of species belonging to the same genus. Of the four tested genes, the most promising phylogenetic marker was rpoA gene, while the least useful gene appeared to be rpoC1. PMID:23759988

  7. Classical swine fever virus detection: results of a real-time reverse transcription polymerase chain reaction ring trial conducted in the framework of the European network of excellence for epizootic disease diagnosis and control.

    NARCIS (Netherlands)

    Hoffmann, B.; Blome, S.; Bonulauri, P.; Fernández-Pinero, J.; Greiser-Wilke, I.; Haegeman, A.; Isaksson, M.; Koenen, F.; Leblanc, N.; Leifer, I.; Potier, Le M.F.; Loeffen, W.; Rasmussen, T.B.; Stadejek, T.; Stahl, K.; Tignon, M.; Uttenthal, A.; Poel, van der W.H.M.

    2011-01-01

    The current study reports on a real-time reverse transcription polymerase chain reaction (real-time RT-PCR) ring trial for the detection of Classical swine fever virus (CSFV) genomic RNA undertaken by 10 European laboratories. All laboratories were asked to use their routine in-house real-time

  8. Crystal structure of the vaccinia virus DNA polymerase holoenzyme subunit D4 in complex with the A20 N-terminal domain.

    Directory of Open Access Journals (Sweden)

    Céline Contesto-Richefeu

    2014-03-01

    Full Text Available Vaccinia virus polymerase holoenzyme is composed of the DNA polymerase E9, the uracil-DNA glycosylase D4 and A20, a protein with no known enzymatic activity. The D4/A20 heterodimer is the DNA polymerase co-factor whose function is essential for processive DNA synthesis. Genetic and biochemical data have established that residues located in the N-terminus of A20 are critical for binding to D4. However, no information regarding the residues of D4 involved in A20 binding is yet available. We expressed and purified the complex formed by D4 and the first 50 amino acids of A20 (D4/A20₁₋₅₀. We showed that whereas D4 forms homodimers in solution when expressed alone, D4/A20₁₋₅₀ clearly behaves as a heterodimer. The crystal structure of D4/A20₁₋₅₀ solved at 1.85 Å resolution reveals that the D4/A20 interface (including residues 167 to 180 and 191 to 206 of D4 partially overlaps the previously described D4/D4 dimer interface. A20₁₋₅₀ binding to D4 is mediated by an α-helical domain with important leucine residues located at the very N-terminal end of A20 and a second stretch of residues containing Trp43 involved in stacking interactions with Arg167 and Pro173 of D4. Point mutations of the latter residues disturb D4/A20₁₋₅₀ formation and reduce significantly thermal stability of the complex. Interestingly, small molecule docking with anti-poxvirus inhibitors selected to interfere with D4/A20 binding could reproduce several key features of the D4/A20₁₋₅₀ interaction. Finally, we propose a model of D4/A20₁₋₅₀ in complex with DNA and discuss a number of mutants described in the literature, which affect DNA synthesis. Overall, our data give new insights into the assembly of the poxvirus DNA polymerase cofactor and may be useful for the design and rational improvement of antivirals targeting the D4/A20 interface.

  9. Interleukin-10 and Interferon Gamma Gene Polymorphisms and Hepatitis C Virus-Related Liver Cirrhosis Risk.

    Science.gov (United States)

    Sheneef, Abeer; Esmat, Mamdouh M; Mohammad, Asmaa N; Mahmoud, Aida A; Moghazy, Hoda M; Noureldin, Amal K

    2017-04-01

    The aim of the study was to evaluate the association between the gene polymorphisms in interleukin-10 (IL-10) and interferon gamma (IFN-γ) genes with susceptibility and severity of hepatitis C virus (HCV) infection among Egyptian patients. Interleukin-10 -592 A/C, -1082 G/A and IFN-γ +874 T/A genotypes were determined in 100 chronic HCV patients and 50 healthy controls using restriction fragment length polymorphism (RFLP) and the amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) respectively. IL-10 -592 A/C polymorphism genotyping revealed that the frequency of CC genotype was significantly higher in chronic HCV patients than in controls (58% versus 30%, P IL-10 -1082 G/A polymorphism genotyping, a higher frequency of GG genotype was found in chronic HCV patients compared to controls (31% versus 10%, P IL-10 -592 A/C, -1082 G/A, and IFN-γ +874 T/A polymorphisms had a strong association with susceptibility to HCV infection. However, no significant association was observed between the cytokines (IL-10 and IFN-γ) genotypes profile and HCV-liver cirrhosis risk in the studied population, except for the high frequency of IFN-γ +874 T allele in cirrhotic patients.

  10. Detection Of Hepatitis B Virus DNA In Moroccan Patients With Epithelial Ovarian Carcinoma EOC By Polymerase Chain Reaction

    Directory of Open Access Journals (Sweden)

    Mustapha Benhessou

    2015-08-01

    Full Text Available Abstract Epithelial ovarian cancer EOC is the most common type of ovarian cancer representing 90 of all ovarian cancers. The viruses are known as human malignancies agents. We tried to analyze the presence of Hepatitis B Virus infection in women with epithelial ovarian carcinoma. PCR-based detection of HBV infections was carried out on 50 tissue samples from patients with histologically proven EOC using consensus primers. The samples analyzed showed 8 450 positivity for HBV-DNA in cancerous ovarian tissues. All of the positive patients had serous adenocarcinoma and advanced stage disease. The results of this study suggest that hepatitis B could play a major role in the etiology of ovarian cancer.

  11. Rapid diagnostic detection of plum pox virus in Prunus plants by isothermal AmplifyRP(®) using reverse transcription-recombinase polymerase amplification.

    Science.gov (United States)

    Zhang, Shulu; Ravelonandro, Michel; Russell, Paul; McOwen, Nathan; Briard, Pascal; Bohannon, Seven; Vrient, Albert

    2014-10-01

    Plum pox virus (PPV) causes the most destructive viral disease known as plum pox or Sharka disease in stone fruit trees. As an important regulated pathogen, detection of PPV is thus of critical importance to quarantine and eradication of the spreading disease. In this study, the innovative development of two AmplifyRP(®) tests is reported for a rapid isothermal detection of PPV using reverse transcription-recombinase polymerase amplification. In an AmplifyRP(®) test, all specific recombination and amplification reactions occur at a constant temperature without thermal cycling and the test results are either recorded in real-time with a portable fluorescence reader or displayed using a lateral flow strip contained inside an amplicon detection chamber. The major improvement of this assay is that the entire test from sample preparation to result can be completed in as little as 20min and can be performed easily both in laboratories and in the field. The results from this study demonstrated the ability of the AmplifyRP(®) technique to detect all nine PPV strains (An, C, CR, D, EA, M, Rec, T, or W). Among the economic benefits to pathogen surveys is the higher sensitivity of the AmplifyRP(®) to detect PPV when compared to the conventional ELISA and ImmunoStrip(®) assays. This is the first report describing the use of such an innovative technique to detect rapidly plant viruses affecting perennial crops. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. Genetic polymorphism of toll-like receptors 4 gene by polymerase chain reaction-restriction fragment length polymorphisms, polymerase chain reaction-single-strand conformational polymorphism to correlate with mastitic cows

    Directory of Open Access Journals (Sweden)

    Pooja H. Gupta

    2015-05-01

    Full Text Available Aim: An attempt has been made to study the toll-like receptors 4 (TLR4 gene polymorphism from cattle DNA to correlate with mastitis cows. Materials and Methods: In present investigation, two fragments of TLR4 gene named T4CRBR1 and T4CRBR2 of a 316 bp and 382 bp were amplified by polymerase chain reaction (PCR, respectively from Kankrej (22 and Triple cross (24 cattle. The genetic polymorphisms in the two populations were detected by a single-strand conformational polymorphism in the first locus and by digesting the fragments with restriction endonuclease Alu I in the second one. Results: Results showed that both alleles (A and B of two loci were found in all the two populations and the value of polymorphism information content indicated that these were highly polymorphic. Statistical results of χ2 test indicated that two polymorphism sites in the two populations fit with Hardy–Weinberg equilibrium (p˂0.05. Meanwhile, the effect of polymorphism of TLR4 gene on the somatic cell score (SCS indicated the cattle with allele a in T4CRBR1 showed lower SCS than that of allele B (p<0.05. Thus, the allele A might play an important role in mastitis resistance in cows. Conclusion: The relationship between the bovine mastitis trait and the polymorphism of TLR4 gene indicated that the bovine TLR4 gene may play an important role in mastitis resistance.

  13. PRC2 regulates RNA polymerase III transcribed non-translated RNA gene transcription through EZH2 and SUZ12 interaction with TFIIIC complex

    Science.gov (United States)

    Liu, Chang; Li, Shuai; Dai, Xiaoyan; Ma, Ji; Wan, Junhu; Jiang, Hao; Wang, Peng; Liu, Zhaoli; Zhang, Hongquan

    2015-01-01

    Polycomb repression complex 2 (PRC2) component EZH2 tri-methylates H3K27 and exerts epigenetic repression on target gene expression. EZH2-mediated epigenetic control of RNA polymerase II (Pol II) transcribed coding gene transcription has been well established. However, little is known about EZH2-mediated epigenetic regulation of RNA polymerase III (Pol III) transcription. Here we present a paradigm that EZH2 is involved in the repression of Pol III transcription via interaction with transcriptional factor complex IIIC (TFIIIC). EZH2 and H3K27me3 co-occupy the promoter of tRNATyr, 5S rRNA and 7SL RNA genes. Depletion of EZH2 or inhibition of EZH2 methyltransferase activity led to upregulation of Pol III target gene transcription. EZH2-mediated repression of Pol III transcribed gene expression requires presence of SUZ12. SUZ12 was able to interact with TFIIIC complex and knockdown of SUZ12 decreased occupancy of EZH2 and H3K27me3 at the promoter of Pol III target genes. Our findings pointed out a previously unidentified role of PRC2 complex in suppressing transcription of Pol III transcribed non-translated RNA genes, putting Pol III on a new layer of epigenetic regulation. PMID:26038315

  14. Quantitative profiling of housekeeping and Epstein-Barr virus gene transcription in Burkitt lymphoma cell lines using an oligonucleotide microarray

    Directory of Open Access Journals (Sweden)

    Niggli Felix K

    2006-06-01

    Full Text Available Abstract Background The Epstein-Barr virus (EBV is associated with lymphoid malignancies, including Burkitt's lymphoma (BL, and can transform human B cells in vitro. EBV-harboring cell lines are widely used to investigate lymphocyte transformation and oncogenesis. Qualitative EBV gene expression has been extensively described, but knowledge of quantitative transcription is lacking. We hypothesized that transcription levels of EBNA1, the gene essential for EBV persistence within an infected cell, are similar in BL cell lines. Results To compare quantitative gene transcription in the BL cell lines Namalwa, Raji, Akata, Jijoye, and P3HR1, we developed an oligonucleotide microarray chip, including 17 housekeeping genes, six latent EBV genes (EBNA1, EBNA2, EBNA3A, EBNA3C, LMP1, LMP2, and four lytic EBV genes (BZLF1, BXLF2, BKRF2, BZLF2, and used the cell line B95.8 as a reference for EBV gene transcription. Quantitative polymerase chain reaction assays were used to validate microarray results. We found that transcription levels of housekeeping genes differed considerably among BL cell lines. Using a selection of housekeeping genes with similar quantitative transcription in the tested cell lines to normalize EBV gene transcription data, we showed that transcription levels of EBNA1 were quite similar in very different BL cell lines, in contrast to transcription levels of other EBV genes. As demonstrated with Akata cells, the chip allowed us to accurately measure EBV gene transcription changes triggered by treatment interventions. Conclusion Our results suggest uniform EBNA1 transcription levels in BL and that microarray profiling can reveal novel insights on quantitative EBV gene transcription and its impact on lymphocyte biology.

  15. Highly conserved regions in Ebola virus RNA dependent RNA polymerase may be act as a universal novel peptide vaccine target: a computational approach.

    Science.gov (United States)

    Oany, Arafat Rahman; Sharmin, Tahmina; Chowdhury, Afrin Sultana; Jyoti, Tahmina Pervin; Hasan, Md Anayet

    2015-12-01

    Ebola virus (EBOV) is such kind of virus which is responsible for 23,825 cases and 9675 deaths worldwide only in 2014 and with an average diseases fatality rate between 25 % and 90 %. Although, medical technology has tried to handle the problems, there is no Food and Drug Administration (FDA)-approved therapeutics or vaccines available for the prevention, post exposure, or treatment of Ebola virus disease (EVD). In the present study, we used the immunoinformatics approach to design a potential epitope-based vaccine against the RNA-dependent RNA polymerase-L of EBOV. BioEdit v7.2.3 sequence alignment editor, Jalview v2 and CLC Sequence Viewer v7.0.2 were used for the initial sequence analysis for securing the conservancy from the sequences. Later the Immune Epitope Database and Analysis Resource (IEDB-AR) was used for the identification of T-cell and B-cellepitopes associated with type I and II major histocompatibility complex molecules analysis. Finally, the population coverage analysis was employed. The core epitope "FRYEFTAPF" was found to be the most potential one, with 100 % conservancy among all the strains of EBOV. It also interacted with both type I and II major histocompatibility complex molecules and is considered as nonallergenic in nature. Finally, with impressive cumulative population coverage of 99.87 % for the both MHC-I and MHC-II class throughout the world population was found for the proposed epitope. To end, the projected peptide gave us a solid stand to propose for vaccine consideration and that might be experimented for its potency in eliciting immunity through humoral and cell mediated immune responses in vitro and in vivo.

  16. A multiplex reverse transcription-nested polymerase chain reaction for detection and differentiation of wild-type and vaccine strains of canine distemper virus

    Directory of Open Access Journals (Sweden)

    Cui Shang-jin

    2010-05-01

    Full Text Available Abstract A multiplex reverse transcription-nested polymerase chain reaction (RT-nPCR method was developed for the detection and differentiation of wild-type and vaccine strains of canine distemper virus (CDV. A pair of primers (P1 and P4 specific for CDV corresponding to the highly conserved region of the CDV genome were used as a common primer pair in the first-round PCR of the nested PCR. Primers P2 specific for CDV wild-type strains, were used as the forward primer together with the common reverse primer P4 in the second round of nested PCR. Primers P3, P5 specific for CDV wild-type strain or vaccine strain, were used as the forward primer together with the common reverse primer P4+P6 in the second round of nested PCR. A fragment of 177 bp was amplified from vaccine strain genomic RNA, and a fragment of 247 bp from wild-type strain genomic RNA in the RT-nPCR, and two fragments of 247 bp and 177 bp were amplified from the mixed samples of vaccine and wild-type strains. No amplification was achieved for uninfected cells, or cells infected with Newcastle disease virus (NDV, canine parvovirus (CPV, canine coronavirus (CCV, rabies virus (RV, or canine adenovirus (CAV. The RT-nPCR method was used to detect 30 field samples suspected of canine distemper from Heilongjiang and Jilin Provinces, and 51 samples in Shandong province. As a result of 30 samples, were found to be wild-type-like, and 5 to be vaccine-strain-like. The RT-nPCR method can be used to effectively detect and differentiate wild-type CDV-infected dogs from dogs vaccinated with CDV vaccine, and thus can be used in clinical detection and epidemiological surveillance.

  17. Phylogenetic analysis of the haemagglutinin gene of current wild-type canine distemper viruses from South Africa: lineage Africa.

    Science.gov (United States)

    Woma, Timothy Y; van Vuuren, Moritz; Bosman, Ana-Mari; Quan, Melvyn; Oosthuizen, Marinda

    2010-07-14

    There are no reports of CDV isolations in southern Africa, and although CDV is said to have geographically distinct lineages, molecular information of African strains has not yet been documented. Viruses isolated in cell cultures were subjected to reverse transcription-polymerase chain reaction (RT-PCR), and the complete H gene was sequenced and phylogenetically analysed with other strains from GenBank. Phylogenetic comparisons of the complete H gene of CDV isolates from different parts of the world (available in GenBank) with wild-type South African isolates revealed nine clades. All South African isolates form a separate African clade of their own and thus are clearly separated from the American, European, Asian, Arctic and vaccine virus clades. It is likely that only the 'African lineage' of CDV may be circulating in South Africa currently, and the viruses isolated from dogs vaccinated against CDV are not the result of reversion to virulence of vaccine strains, but infection with wild-type strains. (c) 2009 Elsevier B.V. All rights reserved.

  18. Pelacakan Virus Bercak Putih pada Udang Vaname (Litopenaeus vannamei di Lombok dengan Real-Time Polymerase Chain Reaction (DETECTION OF WHITE SPOT SYNDROME VIRUS IN LITOPENAEUS VANNAMEI IN LOMBOK ISLAND USING REAL-TIME POLYMERASE CHAIN REACTION

    Directory of Open Access Journals (Sweden)

    Lulu Arafani

    2016-03-01

    Full Text Available White spot syndrome virus (WSSV is one of the most threatening diseases in shrimp and othercrustaceans affecting global shrimp farming. Since firstly detected in Taiwan in 1992, the disease hasspread globally and followed with considerable socio-economic consequences. This research was performedto detect the WSSV infection in shrimp farming in Lombok Island’s (West Nusa Tenggara using real-timepolymerase chain reaction. Samples of vaname (Litopenaeus vannamei were collected from several shrimpfarming in Lombok. Results indicated that the spread of WSSV has reached shrimp farms in Lombok,especially in Lendang Jae, West Lombok. Therefore, a biosurveillance program is strongly recommendedto government to avoid and halt the spread of the disease in East Indonesia region .

  19. Adeno-associated virus (AAV) vectors in cancer gene therapy.

    Science.gov (United States)

    Santiago-Ortiz, Jorge L; Schaffer, David V

    2016-10-28

    Gene delivery vectors based on adeno-associated virus (AAV) have been utilized in a large number of gene therapy clinical trials, which have demonstrated their strong safety profile and increasingly their therapeutic efficacy for treating monogenic diseases. For cancer applications, AAV vectors have been harnessed for delivery of an extensive repertoire of transgenes to preclinical models and, more recently, clinical trials involving certain cancers. This review describes the applications of AAV vectors to cancer models and presents developments in vector engineering and payload design aimed at tailoring AAV vectors for transduction and treatment of cancer cells. We also discuss the current status of AAV clinical development in oncology and future directions for AAV in this field. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Adeno-associated virus (AAV) gene therapy for neurological disease.

    Science.gov (United States)

    Weinberg, Marc S; Samulski, R Jude; McCown, Thomas J

    2013-06-01

    Diseases of the central nervous system (CNS) have provided enormous opportunities for the therapeutic application of viral vector gene transfer. Adeno-associated virus (AAV) has been the vector of choice in recent clinical trials of neurological disease, including Parkinson's and Alzheimer's disease, due to the safety, efficacy, and stability of AAV gene transfer to the CNS. This review highlights the strategies employed for improving direct and peripheral targeting of therapeutic vectors to CNS tissue, and considers the significance of cellular and tissue transduction specificity, transgene regulation, and other variables that influence achievement of successful therapeutic goals. This article is part of the Special Issue entitled 'New Targets and Approaches to the Treatment of Epilepsy'. Copyright © 2012 Elsevier Ltd. All rights reserved.

  1. Radiochemotherapy of hepatocarcinoma via lentivirus-mediated transfer of human sodium iodide symporter gene and herpes simplex virus thymidine kinase gene

    Energy Technology Data Exchange (ETDEWEB)

    Chen Libo, E-mail: libochen888@hotmail.com [Department of Nuclear Medicine, Shanghai Sixth People' s Hospital, Shanghai Jiao Tong University, Shanghai 200233 (China); Guo Guoying [Xinyuan Institute of Medicine and Biotechnology, School of Life Sciences, Zhejiang Sci-Tech University, Hangzhou 310018 (China); Liu Tianjing; Guo Lihe [Division of Biochemistry and Cell Biology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China); Zhu Ruisen [Department of Nuclear Medicine, Shanghai Sixth People' s Hospital, Shanghai Jiao Tong University, Shanghai 200233 (China)

    2011-07-15

    Herpes simplex virus thymidine kinase (HSV-TK) gene/ganciclovir (GCV) system has been widely used as a traditional gene therapy modality, and the sodium/iodide symporter gene (NIS) has been found to be a novel therapeutic gene. Since the therapeutic effects of radioiodine therapy or prodrug chemotherapy on cancers following NIS or HSV-TK gene transfer need to be enhanced, this study was designed to investigate the feasibility of radiochemotherapy for hepatocarcinoma via coexpression of NIS gene and HSV-TK gene. Methods: HepG2 cells were stably transfected with NIS, TK and GFP gene via recombinant lentiviral vector and named HepG2/NTG. Gene expression was examined by reverse transcriptase polymerase chain reaction, fluorescence imaging and iodide uptake. The therapeutic effects were assessed by MTT assay and clonogenic assay. Results: HepG2/NTG cells concentrated {sup 125}I{sup -} up to 76-fold higher than the wild-type cells within 20 min, and the efflux happened with a T{sub 1/2eff} of less than 10 min. The iodide uptake in HepG2/NTG cells was specifically inhibited by sodium perchlorate. Dose-dependent toxicity to HepG2/NTG cells by either GCV or {sup 131}I was revealed by clonogenic assay and MTT assay, respectively. The survival rate of HepG2/NTG cells decreased to 49.7%{+-}2.5%, 43.4%{+-}2.8% and 8.6%{+-}1.2% after exposure to {sup 131}I, GCV and combined therapy, respectively. Conclusion: We demonstrate that radiochemotherapy of hepatocarcinoma via lentiviral-mediated coexpression of NIS gene and HSV-TK gene leads to stronger killing effect than single treatment, and in vivo studies are needed to verify these findings.

  2. Cellular unfolded protein response against viruses used in gene therapy

    Directory of Open Access Journals (Sweden)

    Dwaipayan eSen

    2014-05-01

    Full Text Available Viruses are excellent vehicles for gene therapy due to their natural ability to infect and deliver the cargo to specific tissues with high efficiency. Although such vectors are usually ‘gutted’ and are replication defective, they are subjected to clearance by the host cells by immune recognition and destruction. Unfolded protein response (UPR is a naturally evolved cyto-protective signaling pathway which is triggered due to endoplasmic reticulum (ER stress caused by accumulation of unfolded/misfolded proteins in its lumen. The UPR signaling consists of three signaling pathways, namely PKR-like ER kinase, activating transcription factor 6, and inositol-requiring protein-1. Once activated, UPR triggers the production of ER molecular chaperones and stress response proteins to help reduce the protein load within the ER. This occurs by degradation of the misfolded proteins and ensues in the arrest of protein translation machinery. If the burden of protein load in ER is beyond its processing capacity, UPR can activate pro-apoptotic pathways or autophagy leading to cell death. Viruses are naturally evolved in hijacking the host cellular translation machinery to generate a large amount of proteins. This phenomenon disrupts ER homeostasis and leads to ER stress. Alternatively, in the case of gutted vectors used in gene therapy, the excess load of recombinant vectors administered and encountered by the cell can trigger UPR. Thus, in the context of gene therapy, UPR becomes a major roadblock that can potentially trigger inflammatory responses against the vectors and reduce the efficiency of gene transfer.

  3. Systematic analysis of viral genes responsible for differential virulence between American and Australian West Nile virus strains.

    Science.gov (United States)

    Setoh, Yin Xiang; Prow, Natalie A; Rawle, Daniel J; Tan, Cindy Si En; Edmonds, Judith H; Hall, Roy A; Khromykh, Alexander A

    2015-06-01

    A variant Australian West Nile virus (WNV) strain, WNVNSW2011, emerged in 2011 causing an unprecedented outbreak of encephalitis in horses in south-eastern Australia. However, no human cases associated with this strain have yet been reported. Studies using mouse models for WNV pathogenesis showed that WNVNSW2011 was less virulent than the human-pathogenic American strain of WNV, New York 99 (WNVNY99). To identify viral genes and mutations responsible for the difference in virulence between WNVNSW2011 and WNVNY99 strains, we constructed chimeric viruses with substitution of large genomic regions coding for the structural genes, non-structural genes and untranslated regions, as well as seven individual non-structural gene chimeras, using a modified circular polymerase extension cloning method. Our results showed that the complete non-structural region of WNVNSW2011, when substituted with that of WNVNY99, significantly enhanced viral replication and the ability to suppress type I IFN response in cells, resulting in higher virulence in mice. Analysis of the individual non-structural gene chimeras showed a predominant contribution of WNVNY99 NS3 to increased virus replication and evasion of IFN response in cells, and to virulence in mice. Other WNVNY99 non-structural proteins (NS2A, NS4B and NS5) were shown to contribute to the modulation of IFN response. Thus a combination of non-structural proteins, likely NS2A, NS3, NS4B and NS5, is primarily responsible for the difference in virulence between WNVNSW2011 and WNVNY99 strains, and accumulative mutations within these proteins would likely be required for the Australian WNVNSW2011 strain to become significantly more virulent. © 2015 The Authors.

  4. Replication-Competent Influenza A Virus That Encodes a Split-Green Fluorescent Protein-Tagged PB2 Polymerase Subunit Allows Live-Cell Imaging of the Virus Life Cycle

    Science.gov (United States)

    Avilov, Sergiy V.; Moisy, Dorothée; Munier, Sandie; Schraidt, Oliver

    2012-01-01

    Studies on the intracellular trafficking of influenza virus ribonucleoproteins are currently limited by the lack of a method enabling their visualization during infection in single cells. This is largely due to the difficulty of encoding fluorescent fusion proteins within the viral genome. To circumvent this limitation, we used the split-green fluorescent protein (split-GFP) system (S. Cabantous, T. C. Terwilliger, and G. S. Waldo, Nat. Biotechnol. 23:102–107, 2005) to produce a quasi-wild-type recombinant A/WSN/33/influenza virus which allows expression of individually fluorescent PB2 polymerase subunits in infected cells. The viral PB2 proteins were fused to the 16 C-terminal amino acids of the GFP, whereas the large transcomplementing GFP fragment was supplied by transient or stable expression in cultured cells that were permissive to infection. This system was used to characterize the intranuclear dynamics of PB2 by fluorescence correlation spectroscopy and to visualize the trafficking of viral ribonucleoproteins (vRNPs) by dynamic light microscopy in live infected cells. Following nuclear export, vRNPs showed a transient pericentriolar accumulation and intermittent rapid (∼1 μm/s), directional movements in the cytoplasm, dependent on both microtubules and actin filaments. Our data establish the potential of split-GFP-based recombinant viruses for the tracking of viral proteins during a quasi-wild-type infection. This new virus, or adaptations of it, will be of use in elucidating many aspects of influenza virus host cell interactions as well as in screening for new antiviral compounds. Furthermore, the existence of cell lines stably expressing the complementing GFP fragment will facilitate applications to many other viral and nonviral systems. PMID:22114331

  5. A potentially novel overlapping gene in the genomes of Israeli acute paralysis virus and its relatives

    Directory of Open Access Journals (Sweden)

    Price Nicholas

    2009-09-01

    Full Text Available Abstract The Israeli acute paralysis virus (IAPV is a honeybee-infecting virus that was found to be associated with colony collapse disorder. The IAPV genome contains two genes encoding a structural and a nonstructural polyprotein. We applied a recently developed method for the estimation of selection in overlapping genes to detect purifying selection and, hence, functionality. We provide evolutionary evidence for the existence of a functional overlapping gene, which is translated in the +1 reading frame of the structural polyprotein gene. Conserved orthologs of this putative gene, which we provisionally call pog (predicted overlapping gene, were also found in the genomes of a monophyletic clade of dicistroviruses that includes IAPV, acute bee paralysis virus, Kashmir bee virus, and Solenopsis invicta (red imported fire ant virus 1.

  6. Adeno-associated virus-targeted disruption of the CFTR gene in cloned ferrets

    National Research Council Canada - National Science Library

    Sun, Xingshen; Yan, Ziying; Yi, Yaling; Li, Ziyi; Lei, Diana; Rogers, Christopher S; Chen, Juan; Zhang, Yulong; Welsh, Michael J; Leno, Gregory H; Engelhardt, John F

    2008-01-01

    .... In this study, we describe the production of a CFTR gene-deficient model in the domestic ferret using recombinant adeno-associated virus-mediated gene targeting in fibroblasts, followed by nuclear transfer cloning...

  7. Comparison of the nucleoprotein genes of a chicken and a mink influenza A H 10 virus.

    Science.gov (United States)

    Reinhardt, U; Scholtissek, C

    1988-01-01

    The base sequences of the coding region of the nucleoprotein (NP) genes of two H 10 influenza A viruses, one avian (virus N) and one mink virus, have been determined by primer extension. When the NP genes and the NP sequences derived from the only open reading frame of the two H 10 viruses were compared with those of other human and avian influenza A viruses, it turned out that the mink virus NP was highly related to that of other avian strains, but differed from that of the human strains. Comparison of the NP genes of the mink and avian strains of European origin suggests a direct lineage between them. Since the NP plays a major role in species specificity, it is assumed that an avian influenza virus has directly invaded the mink population.

  8. High rates of virus-induced gene silencing by tobacco rattle virus in Populus.

    Science.gov (United States)

    Shen, Zedan; Sun, Jian; Yao, Jun; Wang, Shaojie; Ding, Mingquan; Zhang, Huilong; Qian, Zeyong; Zhao, Nan; Sa, Gang; Zhao, Rui; Shen, Xin; Polle, Andrea; Chen, Shaoliang

    2015-09-01

    Virus-induced gene silencing (VIGS) has been shown to be an effective tool for investigating gene functions in herbaceous plant species, but has rarely been tested in trees. The establishment of a fast and reliable transformation system is especially important for woody plants, many of which are recalcitrant to transformation. In this study, we established a tobacco rattle virus (TRV)-based VIGS system for two Populus species, Populus euphratica and P. × canescens. Here, TRV constructs carrying a 266 bp or a 558 bp fragment of the phytoene desaturase (PDS) gene were Agrobacterium-infiltrated into leaves of the two poplar species. Agrobacterium-mediated delivery of the shorter insert, TRV2-PePDS266, into the host poplars resulted in expected photobleaching in both tree species, but not the longer insert, PePDS558. The efficiency of VIGS was temperature-dependent, increasing by raising the temperature from 18 to 28 °C. The optimized TRV-VIGS system at 28 °C resulted in a high silencing frequency and efficiency up to 65-73 and 83-94%, respectively, in the two tested poplars. Moreover, syringe inoculation of Agrobacterium in 100 mM acetosyringone induced a more efficient silencing in the two poplar species, compared with other agroinfiltration methods, e.g., direct injection, misting and agrodrench. There were plant species-related differences in the response to VIGS because the photobleaching symptoms were more severe in P. × canescens than in P. euphratica. Furthermore, VIGS-treated P. euphratica exhibited a higher recovery rate (50%) after several weeks of the virus infection, compared with TRV-infected P. × canescens plants (20%). Expression stability of reference genes was screened to assess the relative abundance of PePDS mRNA in VIGS-treated P. euphratica and P. × canescens. PeACT7 was stably expressed in P. euphratica and UBQ-L was selected as the most suitable reference gene for P. × canescens using three different

  9. Evaluation of a rapid analyte measurement platform and real-time reverse-transcriptase polymerase chain reaction assay West Nile virus detection system in mosquito pools.

    Science.gov (United States)

    Burkhalter, Kristen L; Horiuchi, Kalanthe; Biggerstaff, Brad J; Savage, Harry M; Nasci, Roger S

    2014-03-01

    We evaluated the commercially available Rapid Analyte Measurement Platform (RAMP) West Nile virus (WNV) antigen detection test for sensitivity and consistency with real-time reverse transcriptase polymerase chain reaction (RT-PCR) confirmation testing. Panels of samples consisting of WNV-spiked mosquito pools and negative control pools were sent to 20 mosquito abatement districts (MADs) that processed the pools using the RAMP assay. The samples were then sent to the reference laboratories used by the MADs for confirmation by real-time RT-PCR. Positive pools with virus titers of roughly 1-3 log10 PFU/ml had RAMP scores above the RAMP test positive cutoff score of 30 RAMP units, but these virus-positive samples could not be reliably confirmed by real-time RT-PCR testing. Pools with virus titers > or =4 log10 PFU/ml scored > or =50 RAMP units. Real-time RT-PCR results varied among the confirmation laboratories. With few exceptions, pools returning a RAMP score of > or =100 were confirmed with real-time RT-PCR, while pools returning a RAMP score of 50-99 appeared to be at the limit of real-time RT-PCR detection. Therefore, we recommend using a positive cutoff of 50 RAMP units with no real-time RT-PCR confirmation to maximize speed, efficiency, and economy of the RAMP assay. A more conservative approach would be to implement a "gray zone" range of 50-100 RAMP units. Pools scoring within the gray zone could be submitted for real-time RT-PCR confirmation with the understanding that positive pools may not confirm due to the inhibitory effect of the RAMP buffer on the real-time RT-PCR assay. We also conducted a series of experiments using laboratory-prepared mosquito pools spiked with WNV to compare mosquito homogenization buffers, pool sizes, and grinding methods in order to determine how these variables affect the RAMP and real-time RT-PCR assay results.

  10. Development of next-generation respiratory virus vaccines through targeted modifications to viral immunomodulatory genes

    Science.gov (United States)

    Stobart, Christopher C.; Moore, Martin L.

    2016-01-01

    Vaccines represent one of the greatest contributions of the scientific community to global health. Yet, many pathogens remain either unchallenged or inadequately hindered by commercially available vaccines. Respiratory viruses pose distinct and difficult challenges due to their ability to rapidly spread, adapt, and modify the host immune response. Considerable research has been directed to understand the role of respiratory virus immunomodulatory proteins and how they influence the host immune response. We review here efforts to develop next-generation vaccines through targeting these key immunomodulatory genes in influenza virus, coronaviruses, respiratory syncytial virus, measles virus, and mumps virus. PMID:26434947

  11. Host determinant residue lysine 627 lies on the surface of a discrete, folded domain of influenza virus polymerase PB2 subunit.

    Directory of Open Access Journals (Sweden)

    Franck Tarendeau

    Full Text Available Understanding how avian influenza viruses adapt to human hosts is critical for the monitoring and prevention of future pandemics. Host specificity is determined by multiple sites in different viral proteins, and mutation of only a limited number of these sites can lead to inter-species transmission. Several of these sites have been identified in the viral polymerase, the best characterised being position 627 in the PB2 subunit. Efficient viral replication at the relatively low temperature of the human respiratory tract requires lysine 627 rather than the glutamic acid variant found systematically in avian viruses. However, the molecular mechanism by which any of these host specific sites determine host range are unknown, although adaptation to host factors is frequently evoked. We used ESPRIT, a library screening method, to identify a new PB2 domain that contains a high density of putative host specific sites, including residue 627. The X-ray structure of this domain (denoted the 627-domain exhibits a novel fold with the side-chain of Lys627 solvent exposed. The structure of the K627E mutated domain shows no structural differences but the charge reversal disrupts a striking basic patch on the domain surface. Five other recently proposed host determining sites of PB2 are also located on the 627-domain surface. The structure of the complete C-terminal region of PB2 comprising the 627-domain and the previously identified NLS-domain, which binds the host nuclear import factor importin alpha, was also determined. The two domains are found to pack together with a largely hydrophilic interface. These data enable a three-dimensional mapping of approximately half of PB2 sites implicated in cross-species transfer onto a single structural unit. Their surface location is consistent with roles in interactions with other viral proteins or host factors. The identification and structural characterization of these well-defined PB2 domains will help design

  12. Biological characterization and variability of the nucleocapsid protein gene of Groundnut bud necrosis virus isolates infecting pea from India

    Directory of Open Access Journals (Sweden)

    Mohammad AKRAM

    2012-09-01

    Full Text Available A disease of pea characterized by browning in veins, leaves and stems, mostly in growing tips, and brown circular spots on pods, was recorded in four districts of Uttar Pradesh, India. The causal agent of this disease was detected by reverse transcription-polymerase chain reaction (RT-PCR using primers pair HRP 26/HRP 28 and identified as Groundnut bud necrosis virus (GBNV on the basis of nucleocapsid protein (NP gene sequence. Virus isolates from Bareilly (BRY, Kanpur (KNP, Udham Singh Nagar (USN and Shahjahanpur (SJP were designated as GBNV-[Pea_BRY], GBNV-[Pea_KNP], GBNV-[Pea_USN] and GBNV-[Pea_SJP] and their NP genes sequenced. The sequence data of each isolate were deposited at NCBI database (JF281101-JF281104. The complete nucleotide sequence of the NP genes of all the GBNV isolates had a single open reading frame of 831 nucleotides and 276 amino acids. The isolates had among them 2% variability at amino acid level and 2‒3 variability at nucleotide level, but had variability with other GBNV isolates of fabaceous hosts in the range of 0‒6% at amino acid level and 1‒8% at nucleotide level. Though this variation in nucleotide sequences of GBNV isolates from fabaceous hosts is within the limits of species demarcation for tospoviruses, formation of a separate cluster within the GBNV isolates indicates the possibility of distinct variants in GBNV.

  13. [Cloning and expression of 3C protease gene from foot-and-mouth disease virus in insect cell].

    Science.gov (United States)

    Cao, Yimei; Lu, Zengjun; Sun, Pu; Sun, Jiachuan; Liu, Zaixin

    2008-03-01

    The 3C protease from foot-and-mouth disease virus (FMDV 3Cpro) is critical for viral pathogenesis, has vital roles in both processing of the polyprotein precursor and RNA replication, and is a potential anti-viral drug target. In the study, 3C gene of FMDV from serotype Asia I was obtained through Polymerase Chain Reaction (PCR), and subcloned into baculovirus transfer vector pMelBac-B. The recombinant transfer plasmid and linearized baculovirus DNA were co-transfected into sf9 insect cell, and the recombinant baculovirus were screened by plaque cloning and PCR identification. After amplification of recombinant baculovirus on cell passages, the recombinant virus were seeded on sf9 cell with 10 multiplicity of infection (MOI), and cells were harvested 72 hours after infection. The expressing product of 3C gene in insect cells was detected by Sodium Dodecyl Sulphate PolyAcrylamide Gel Electrophoresis (SDS-PAGE) and Western blot. The result demonstrated that the 3C gene was successfully expressed in insect cells. The product was a 23 kDa protein and could be recognized by anti-FMDV serum in western blot. The results provide a basis for research of the assembly of FMDV empty capsids in vitro and the design of antivirus drug.

  14. Sequence analysis of the capsid gene of Aichi viruses detected from Japan, Bangladesh, Thailand, and Vietnam.

    Science.gov (United States)

    Pham, Ngan Thi Kim; Trinh, Quang Duy; Khamrin, Pattara; Nguyen, Tuan Anh; Dey, Shuvra Kanti; Phan, Tung Gia; Hoang, Le Phuc; Maneekarn, Niwat; Okitsu, Shoko; Mizuguchi, Masashi; Ushijima, Hiroshi

    2008-07-01

    Sequence analysis of the capsid gene of Aichi viruses was performed on 12 strains detected in Japan, Bangladesh, Thailand, and Vietnam during 2002-2005. The phylogenetic tree constructed from 17 nucleotide sequences of the capsid gene of the strains studied and reference strains demonstrated that Aichi virus strains clustered into two branches. A classification of Aichi viruses based on the capsid gene was proposed, in which lineage I consists of the Aichi virus strains detected from Japan, Thailand, Vietnam, and Germany, and lineage II includes Bangladeshi strains and a Brazilian strain.

  15. Virus-induced gene silencing of Mlo genes induces powdery mildew resistance in Triticum aestivum.

    Science.gov (United States)

    Várallyay, Eva; Giczey, Gábor; Burgyán, József

    2012-07-01

    Powdery mildew is one of the most important cereal diseases worldwide. Genetic analysis has revealed that mutant alleles of the Mlo gene cause broad-spectrum resistance against this pathogen in barley. In this study, the possibility of inducing broad-spectrum powdery mildew resistance against this pathogen by RNAi of the barley Mlo ortholog in wheat was examined using virus-induced gene silencing (VIGS). A clear correlation was found between resistance and accumulation of Mlo-specific siRNAs, raising the possibility of designing powdery mildew resistance in wheat by RNA silencing using both transgenic and non-transgenic approaches.

  16. Derepression of a novel class of vaccinia virus genes upon DNA replication

    NARCIS (Netherlands)

    Vos, J C; Stunnenberg, H.G.

    1988-01-01

    A novel class of vaccinia virus genes, called intermediate, is expressed immediately post-replication and prior to the onset of late gene transcription. Intermediate transcription is dependent on trans-acting factors which are present in an active state in virus-infected cells prior to the onset of

  17. Microarray-driven validation of reference genes for quantitative real-time polymerase chain reaction in a rat vocal fold model of mucosal injury.

    Science.gov (United States)

    Chang, Zhen; Ling, Changying; Yamashita, Masaru; Welham, Nathan V

    2010-11-15

    Relative quantification by normalization against a stably expressed reference gene is a widely used data analysis method in microarray and quantitative real-time polymerase chain reaction (qRT-PCR) platforms; however, recent evidence suggests that many commonly utilized reference genes are unstable in certain experimental systems and situations. The primary aim of this study, therefore, was to screen and identify stably expressed reference genes in a well-established rat model of vocal fold mucosal injury. We selected and evaluated the expression stability of nine candidate reference genes. Ablim1, Sptbn1, and Wrnip1 were identified as stably expressed in a model-specific microarray dataset and were further validated as suitable reference genes in an independent qRT-PCR experiment using 2(-DeltaCT) and pairwise comparison-based (geNorm) analyses. Parallel analysis of six commonly used reference genes identified Sdha as the only stably expressed candidate in this group. Sdha, Sptbn1, and the geometric mean of Sdha and Sptbn1 each provided accurate normalization of target gene Tgfb1; Gapdh, the least stable candidate gene in our dataset, provided inaccurate normalization and an invalid experimental result. The stable reference genes identified here are suitable for accurate normalization of target gene expression in vocal fold mucosal injury experiments. Copyright 2010 Elsevier Inc. All rights reserved.

  18. Computational fitness landscape for all gene-order permutations of an RNA virus.

    Directory of Open Access Journals (Sweden)

    Kwang-il Lim

    2009-02-01

    Full Text Available How does the growth of a virus depend on the linear arrangement of genes in its genome? Answering this question may enhance our basic understanding of virus evolution and advance applications of viruses as live attenuated vaccines, gene-therapy vectors, or anti-tumor therapeutics. We used a mathematical model for vesicular stomatitis virus (VSV, a prototype RNA virus that encodes five genes (N-P-M-G-L, to simulate the intracellular growth of all 120 possible gene-order variants. Simulated yields of virus infection varied by 6,000-fold and were found to be most sensitive to gene-order permutations that increased levels of the L gene transcript or reduced levels of the N gene transcript, the lowest and highest expressed genes of the wild-type virus, respectively. Effects of gene order on virus growth also depended upon the host-cell environment, reflecting different resources for protein synthesis and different cell susceptibilities to infection. Moreover, by computationally deleting intergenic attenuations, which define a key mechanism of transcriptional regulation in VSV, the variation in growth associated with the 120 gene-order variants was drastically narrowed from 6,000- to 20-fold, and many variants produced higher progeny yields than wild-type. These results suggest that regulation by intergenic attenuation preceded or co-evolved with the fixation of the wild type gene order in the evolution of VSV. In summary, our models have begun to reveal how gene functions, gene regulation, and genomic organization of viruses interact with their host environments to define processes of viral growth and evolution.

  19. A novel recombinant pseudorabies virus expressing parvovirus VP2 gene: Immunogenicity and protective efficacy in swine

    OpenAIRE

    Chen, Yang; Guo, Wanzhu; Xu, Zhiwen; Yan, Qigui; Luo, Yan; Shi, Qian; Chen, Dishi; Zhu, Ling; Wang, Xiaoyu

    2011-01-01

    Abstract Background Porcine parvovirus (PPV) VP2 gene has been successfully expressed in many expression systems resulting in self-assembly of virus-like particles (VLPs) with similar morphology to the native capsid. Here, a pseudorabies virus (PRV) system was adopted to express the PPV VP2 gene. Methods A recombinant PRV SA215/VP2 was obtained by homologous recombination between the vector PRV viral DNA and a transfer plasmid. Then recombinant virus was purified with plaque purification, and...

  20. Integrated analysis of miRNAs and transcriptomes in Aedes albopictus midgut reveals the differential expression profiles of immune-related genes during dengue virus serotype-2 infection.

    Science.gov (United States)

    Liu, Yan-Xia; Li, Fen-Xiang; Liu, Zhuan-Zhuan; Jia, Zhi-Rong; Zhou, Yan-He; Zhang, Hao; Yan, Hui; Zhou, Xian-Qiang; Chen, Xiao-Guang

    2016-06-01

    Mosquito microRNAs (miRNAs) are involved in host-virus interaction, and have been reported to be altered by dengue virus (DENV) infection in Aedes albopictus (Diptera: Culicidae). However, little is known about the molecular mechanisms of Aedes albopictus midgut-the first organ to interact with DENV-involved in its resistance to DENV. Here we used high-throughput sequencing to characterize miRNA and messenger RNA (mRNA) expression patterns in Aedes albopictus midgut in response to dengue virus serotype 2. A total of three miRNAs and 777 mRNAs were identified to be differentially expressed upon DENV infection. For the mRNAs, we identified 198 immune-related genes and 31 of them were differentially expressed. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses also showed that the differentially expressed immune-related genes were involved in immune response. Then the differential expression patterns of six immune-related genes and three miRNAs were confirmed by real-time reverse transcription polymerase chain reaction. Furthermore, seven known miRNA-mRNA interaction pairs were identified by aligning our two datasets. These analyses of miRNA and mRNA transcriptomes provide valuable information for uncovering the DENV response genes and provide a basis for future study of the resistance mechanisms in Aedes albopictus midgut. © 2016 Institute of Zoology, Chinese Academy of Sciences.

  1. Comparison of host cell gene expression in cowpox, monkeypox or vaccinia virus-infected cells reveals virus-specific regulation of immune response genes.

    Science.gov (United States)

    Bourquain, Daniel; Dabrowski, Piotr Wojtek; Nitsche, Andreas

    2013-02-20

    Animal-borne orthopoxviruses, like monkeypox, vaccinia and the closely related cowpox virus, are all capable of causing zoonotic infections in humans, representing a potential threat to human health. The disease caused by each virus differs in terms of symptoms and severity, but little is yet know about the reasons for these varying phenotypes. They may be explained by the unique repertoire of immune and host cell modulating factors encoded by each virus. In this study, we analysed the specific modulation of the host cell's gene expression profile by cowpox, monkeypox and vaccinia virus infection. We aimed to identify mechanisms that are either common to orthopoxvirus infection or specific to certain orthopoxvirus species, allowing a more detailed description of differences in virus-host cell interactions between individual orthopoxviruses. To this end, we analysed changes in host cell gene expression of HeLa cells in response to infection with cowpox, monkeypox and vaccinia virus, using whole-genome gene expression microarrays, and compared these to each other and to non-infected cells. Despite a dominating non-responsiveness of cellular transcription towards orthopoxvirus infection, we could identify several clusters of infection-modulated genes. These clusters are either commonly regulated by orthopoxvirus infection or are uniquely regulated by infection with a specific orthopoxvirus, with major differences being observed in immune response genes. Most noticeable was an induction of genes involved in leukocyte migration and activation in cowpox and monkeypox virus-infected cells, which was not observed following vaccinia virus infection. Despite their close genetic relationship, the expression profiles induced by infection with different orthopoxviruses vary significantly. It may be speculated that these differences at the cellular level contribute to the individual characteristics of cowpox, monkeypox and vaccinia virus infections in certain host species.

  2. Transmission of Influenza A Viruses

    Science.gov (United States)

    Neumann, Gabriele; Kawaoka, Yoshihiro

    2015-01-01

    Influenza A viruses cause respiratory infections that range from asymptomatic to deadly in humans. Widespread outbreaks (pandemics) are attributable to ‘novel’ viruses that possess a viral hemagglutinin (HA) gene to which humans lack immunity. After a pandemic, these novel viruses form stable virus lineages in humans and circulate until they are replaced by other novel viruses. The factors and mechanisms that facilitate virus transmission among hosts and the establishment of novel lineages are not completely understood, but the HA and basic polymerase 2 (PB2) proteins are thought to play essential roles in these processes by enabling avian influenza viruses to infect mammals and replicate efficiently in their new host. Here, we summarize our current knowledge of the contributions of HA, PB2, and other viral components to virus transmission and the formation of new virus lineages. PMID:25812763

  3. Resistance of Cloned 1F5 Chimeric Anti-CD20 Antibody Heavy-Chain Gene to DNA Polymerase due to a Predicted Hairpin Structure

    Directory of Open Access Journals (Sweden)

    Fatemeh Khademi

    2016-11-01

    Full Text Available Background: Formation of secondary structure such as DNA hairpins or loops may influence molecular genetics methods and PCR based approaches necessary for genetic engineering, in addition to gene regulation. Materials and Methods: A polymerase chain reaction with splice overlap extension (SOE-PCR was used to create fully synthetic 1F5 chimeric anti-CD20 heavy- and light-chain genes. The chimeric genes were cloned into the pCR-Blunt II-TOPO vector following by cloning into the pBudCE4.1 expression vector. Prediction of secondary structure was performed with the Vienna RNAfold webserver. PCR and sequencing across the predicted secondary structure of chimeric 1F5 heavy-chain gene was performed with multiple protocols for standard and GC-rich templates. Results: In our attempt to design vectors aimed to generate mouse-human chimeric antibody against CD20 (1F5, we found that the coding sequence of 1F5 chimeric heavy-chain gene constructed by SOE-PCR was resistant to polymerase during both PCR and sequencing reactions. Furthermore, we were also unable to analysis some positive transformants by restriction enzyme digestion. Encountering such difficulties to identify the cloned anti-CD20 chimeric heavy-chain gene, we found that the chimeric heavy-chain sequence is highly GC-rich and predicted to form a stable secondary structure. Conclusion: In conclusion, for the first time, we reported several difficulties with production of therapeutic chimeric 1F5 anti-CD20 antibody due to a predicted hairpin cluster correlates with barriers to PCR, sequencing and possibly restriction analysis. Our findings provide a probable note for researchers experiencing technical difficulties with construction of chimeric anti-CD20 antibody 1F5 gene vectors and also with other genes and molecular biology techniques requiring PCR-based method or restriction enzyme analysis. 

  4. The Diagnosis of Genital Herpes – Beyond Culture: An Evidence-Based Guide for the Utilization of Polymerase Chain Reaction and Herpes Simplex Virus Type-Specific Serology

    Directory of Open Access Journals (Sweden)

    S Ratnam

    2007-01-01

    Full Text Available Accurate identification of persons with genital herpes is necessary for optimal patient management and prevention of transmission. Because of inherent inaccuracies, clinical diagnosis of genital herpes should be confirmed by laboratory testing for the causative agents herpes simplex virus type 1 (HSV-1 and HSV type 2 (HSV-2. Further identification of the HSV type is valuable for counselling on the natural history of infection and risk of transmission. Laboratory methods include antigen detection, culture, polymerase chain reaction (PCR and conventional and type-specific serology (TSS. PCR has, by far, the greater sensitivity and should be the test of choice for symptomatic cases. HSV-2 TSS is indicated for patients with genital lesions in whom antigen detection, culture or PCR fail to detect HSV, and for patients who are asymptomatic but have a history suggestive of genital herpes. HSV-2 TSS is further indicated for patients infected with HIV. HSV-2 TSS along with HSV-1 TSS may be considered, as appropriate, in evaluating infection and/or immune status in couples discordant for genital herpes, women who develop their first clinical episode of genital herpes during pregnancy, asymptomatic pregnant women whose partners have a history of genital herpes or HIV infection, and women contemplating pregnancy or considering sexual partnership with those with a history of genital herpes. The above should be performed in conjunction with counselling of infected persons and their sex partners.

  5. Diagnostic Usefulness of Varicella-Zoster Virus Real-Time Polymerase Chain Reaction Analysis of DNA in Saliva and Plasma Specimens From Patients With Herpes Zoster.

    Science.gov (United States)

    Park, Seong Yeon; Kim, Ji Yeun; Kim, Ji-Ae; Kwon, Ji-Soo; Kim, Sun-Mi; Jeon, Na Young; Kim, Min-Chul; Chong, Yong Pil; Lee, Sang-Oh; Choi, Sang-Ho; Kim, Yang Soo; Woo, Jun Hee; Kim, Sung-Han

    2017-12-27

    We evaluated the diagnostic usefulness of polymerase chain reaction (PCR) analysis for detecting varicella-zoster virus (VZV) infection and reactivation of VZV, using DNA extracted from saliva and plasma specimens obtained from subjects with suspected herpes zoster and from healthy volunteers during stressful and nonstressful conditions. There were 52 patients with a diagnosis of herpes zoster (group 1), 30 with a diagnosis of zoster-mimicking disease (group 2), and 27 healthy volunteers (group 3). Saliva and plasma samples were evaluated for VZV DNA by real-time PCR analysis. Among patients with suspected herpes zoster (ie, patients in groups 1 and 2), the sensitivity of PCR analysis of salivary DNA for detecting VZV (88%; 95% confidence interval [CI], 74%-95%) was significantly higher than that of PCR analysis of plasma DNA (28%; 95% CI, 16%-44%; P .99). VZV DNA was not detected in saliva and plasma samples from group 3 (0%; 95% CI, 0%-14%). Real-time PCR analysis of salivary DNA is more sensitive than that of plasma DNA for detecting VZV among patients with suspected herpes zoster. We found no subclinical reactivation of VZV in group 3 following exposure to common stressful conditions.

  6. Variation in Bluetongue virus real-time reverse transcription polymerase chain reaction assay results in blood samples of sheep, cattle, and alpaca.

    Science.gov (United States)

    Brito, Barbara P; Gardner, Ian A; Hietala, Sharon K; Crossley, Beate M

    2011-07-01

    Bluetongue is a vector-borne viral disease that affects domestic and wild ruminants. The epidemiology of this disease has recently changed, with occurrence in new geographic areas. Various real-time quantitative reverse transcription polymerase chain reaction (real-time qRT-PCR) assays are used to detect Bluetongue virus (BTV); however, the impact of biologic differences between New World camelids and domestic ruminant samples on PCR efficiency, for which the BTV real-time qRT-PCR was initially validated are unknown. New world camelids are known to have important biologic differences in whole blood composition, including hemoglobin concentration, which can alter PCR performance. In the present study, sheep, cattle, and alpaca blood were spiked with BTV serotypes 10, 11, 13, and 17 and analyzed in 10-fold dilutions by real-time qRT-PCR to determine if species affected nucleic acid recovery and assay performance. A separate experiment was performed using spiked alpaca blood subsequently diluted in 10-fold series in sheep blood to assess the influence of alpaca blood on performance efficiency of the BTV real-time qRT-PCR assay. Results showed that BTV-specific nucleic acid detection from alpaca blood was consistently 1-2 logs lower than from sheep and cattle blood, and results were similar for each of the 4 BTV serotypes analyzed.

  7. Detection of replication-defective hepatitis A virus based on the correlation between real-time polymerase chain reaction and ELISA in situ results

    Directory of Open Access Journals (Sweden)

    Alyne Moraes Costa

    2013-02-01

    Full Text Available ELISA in situ can be used to titrate hepatitis A virus (HAV particles and real-time polymerase chain reaction (RT-PCR has been shown to be a fast method to quantify the HAV genome. Precise quantification of viral concentration is necessary to distinguish between infectious and non-infectious particles. The purpose of this study was to compare cell culture and RT-PCR quantification results and determine whether HAV genome quantification can be correlated with infectivity. For this purpose, three stocks of undiluted, five-fold diluted and 10-fold diluted HAV were prepared to inoculate cells in a 96-well plate. Monolayers were then incubated for seven, 10 and 14 days and the correlation between the ELISA in situ and RT-PCR results was evaluated. At 10 days post-incubation, the highest viral load was observed in all stocks of HAV via RT-PCR (10(5 copies/mL (p = 0.0002, while ELISA revealed the highest quantity of particles after 14 days (optical density = 0.24, p < 0.001. At seven days post-infection, there was a significant statistical correlation between the results of the two methods, indicating equivalents titres of particles and HAV genome during this period of infection. The results reported here indicate that the duration of growth of HAV in cell culture must be taken into account to correlate genome quantification with infectivity.

  8. Cyclosporin A associated helicase-like protein facilitates the association of hepatitis C virus RNA polymerase with its cellular cyclophilin B.

    Directory of Open Access Journals (Sweden)

    Kengo Morohashi

    Full Text Available BACKGROUND: Cyclosporin A (CsA is well known as an immunosuppressive drug useful for allogeneic transplantation. It has been reported that CsA inhibits hepatitis C virus (HCV genome replication, which indicates that cellular targets of CsA regulate the viral replication. However, the regulation mechanisms of HCV replication governed by CsA target proteins have not been fully understood. PRINCIPAL FINDINGS: Here we show a chemical biology approach that elucidates a novel mechanism of HCV replication. We developed a phage display screening to investigate compound-peptide interaction and identified a novel cellular target molecule of CsA. This protein, named CsA associated helicase-like protein (CAHL, possessed RNA-dependent ATPase activity that was negated by treatment with CsA. The downregulation of CAHL in the cells resulted in a decrease of HCV genome replication. CAHL formed a complex with HCV-derived RNA polymerase NS5B and host-derived cyclophilin B (CyPB, known as a cellular cofactor for HCV replication, to regulate NS5B-CyPB interaction. CONCLUSIONS: We found a cellular factor, CAHL, as CsA associated helicase-like protein, which would form trimer complex with CyPB and NS5B of HCV. The strategy using a chemical compound and identifying its target molecule by our phage display analysis is useful to reveal a novel mechanism underlying cellular and viral physiology.

  9. Gene Technology for Papaya Ringspot Virus Disease Management

    Science.gov (United States)

    Azad, Md. Abul Kalam; Sidik, Nik Marzuki

    2014-01-01

    Papaya (Carica papaya) is severely damaged by the papaya ringspot virus (PRSV). This review focuses on the development of PRSV resistant transgenic papaya through gene technology. The genetic diversity of PRSV depends upon geographical distribution and the influence of PRSV disease management on a sequence of PRSV isolates. The concept of pathogen-derived resistance has been employed for the development of transgenic papaya, using a coat protein-mediated, RNA-silencing mechanism and replicase gene-mediated transformation for effective PRSV disease management. The development of PRSV-resistant papaya via post-transcriptional gene silencing is a promising technology for PRSV disease management. PRSV-resistant transgenic papaya is environmentally safe and has no harmful effects on human health. Recent studies have revealed that the success of adoption of transgenic papaya depends upon the application, it being a commercially viable product, bio-safety regulatory issues, trade regulations, and the wider social acceptance of the technology. This review discusses the genome and the genetic diversity of PRSV, host range determinants, molecular diagnosis, disease management strategies, the development of transgenic papaya, environmental issues, issues in the adoption of transgenic papaya, and future directions for research. PMID:24757435

  10. Gene technology for papaya ringspot virus disease management.

    Science.gov (United States)

    Azad, Md Abul Kalam; Amin, Latifah; Sidik, Nik Marzuki

    2014-01-01

    Papaya (Carica papaya) is severely damaged by the papaya ringspot virus (PRSV). This review focuses on the development of PRSV resistant transgenic papaya through gene technology. The genetic diversity of PRSV depends upon geographical distribution and the influence of PRSV disease management on a sequence of PRSV isolates. The concept of pathogen-derived resistance has been employed for the development of transgenic papaya, using a coat protein-mediated, RNA-silencing mechanism and replicase gene-mediated transformation for effective PRSV disease management. The development of PRSV-resistant papaya via post-transcriptional gene silencing is a promising technology for PRSV disease management. PRSV-resistant transgenic papaya is environmentally safe and has no harmful effects on human health. Recent studies have revealed that the success of adoption of transgenic papaya depends upon the application, it being a commercially viable product, bio-safety regulatory issues, trade regulations, and the wider social acceptance of the technology. This review discusses the genome and the genetic diversity of PRSV, host range determinants, molecular diagnosis, disease management strategies, the development of transgenic papaya, environmental issues, issues in the adoption of transgenic papaya, and future directions for research.

  11. The p10 gene of Bombyx mori nucleopolyhedrosis virus encodes a ...

    Indian Academy of Sciences (India)

    In baculovirus-based high-level expression of cloned foreign genes, the viral very late gene promoters of polyhedrin (polh) and p10 are extensively exploited. Here we report the cloning and characterization of the p10 gene from a local isolate of Bombyx mori nucleopolyhedrosis virus (BmNPV). The gene harbours a 213-bp ...

  12. dDsk2 regulates H2Bub1 and RNA polymerase II pausing at dHP1c complex target genes.

    Science.gov (United States)

    Kessler, Roman; Tisserand, Johan; Font-Burgada, Joan; Reina, Oscar; Coch, Laura; Attolini, Camille Stephan-Otto; Garcia-Bassets, Ivan; Azorín, Fernando

    2015-04-28

    dDsk2 is a conserved extraproteasomal ubiquitin receptor that targets ubiquitylated proteins for degradation. Here we report that dDsk2 plays a nonproteolytic function in transcription regulation. dDsk2 interacts with the dHP1c complex, localizes at promoters of developmental genes and is required for transcription. Through the ubiquitin-binding domain, dDsk2 interacts with H2Bub1, a modification that occurs at dHP1c complex-binding sites. H2Bub1 is not required for binding of the complex; however, dDsk2 depletion strongly reduces H2Bub1. Co-depletion of the H2Bub1 deubiquitylase dUbp8/Nonstop suppresses this reduction and rescues expression of target genes. RNA polymerase II is strongly paused at promoters of dHP1c complex target genes and dDsk2 depletion disrupts pausing. Altogether, these results suggest that dDsk2 prevents dUbp8/Nonstop-dependent H2Bub1 deubiquitylation at promoters of dHP1c complex target genes and regulates RNA polymerase II pausing. These results expand the catalogue of nonproteolytic functions of ubiquitin receptors to the epigenetic regulation of chromatin modifications.

  13. Epstein-Barr virus candidate genes and multiple sclerosis.

    Science.gov (United States)

    Claire Simon, Kelly; Schmidt, Hollie; Loud, Sara; Ascherio, Alberto

    2015-01-01

    Previous infection with Epstein-Barr virus (EBV) and a history of infectious mononucleosis (IM) have been previously associated with an increased risk of multiple sclerosis (MS). Whether there are common genetic factors that may partially explain these associations has not been thoroughly explored. To investigate whether select polymorphisms in genes associated with IM susceptibility are related to MS risk-a self-reported history of IM or antibody titer against Epstein-Barr virus nuclear antigen 1 (anti-EBNA1). A case-control study including 1213 MS cases and 454 controls enrolled in the Accelerated Cure Project for MS (ACP) Repository. Select polymorphisms in HLA-A, SH2D1A and IL15RA and anti-EBNA1 Ab titers were measured using stored blood samples provided by participants. Generalized linear models were used to assess the associations between select polymorphisms and odds of MS, odds of IM or anti-EBNA1 Ab titers. No significant associations were observed between the selected polymorphisms and odds of MS, odds of IM or anti-EBNA1 Ab titer. It is unlikely that any of the studied polymorphisms contribute to the explaining the association between anti-EBNA1 Ab titer or history of IM and MS. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. Both cis and trans Activities of Foot-and-Mouth Disease Virus 3D Polymerase Are Essential for Viral RNA Replication.

    Science.gov (United States)

    Herod, Morgan R; Ferrer-Orta, Cristina; Loundras, Eleni-Anna; Ward, Joseph C; Verdaguer, Nuria; Rowlands, David J; Stonehouse, Nicola J

    2016-08-01

    The Picornaviridae is a large family of positive-sense RNA viruses that contains numerous human and animal pathogens, including foot-and-mouth disease virus (FMDV). The picornavirus replication complex comprises a coordinated network of protein-protein and protein-RNA interactions involving multiple viral and host-cellular factors. Many of the proteins within the complex possess multiple roles in viral RNA replication, some of which can be provided in trans (i.e., via expression from a separate RNA molecule), while others are required in cis (i.e., expressed from the template RNA molecule). In vitro studies have suggested that multiple copies of the RNA-dependent RNA polymerase (RdRp) 3D are involved in the viral replication complex. However, it is not clear whether all these molecules are catalytically active or what other function(s) they provide. In this study, we aimed to distinguish between catalytically active 3D molecules and those that build a replication complex. We report a novel nonenzymatic cis-acting function of 3D that is essential for viral-genome replication. Using an FMDV replicon in complementation experiments, our data demonstrate that this cis-acting role of 3D is distinct from the catalytic activity, which is predominantly trans acting. Immunofluorescence studies suggest that both cis- and trans-acting 3D molecules localize to the same cellular compartment. However, our genetic and structural data suggest that 3D interacts in cis with RNA stem-loops that are essential for viral RNA replication. This study identifies a previously undescribed aspect of picornavirus replication complex structure-function and an important methodology for probing such interactions further. Foot-and-mouth disease virus (FMDV) is an important animal pathogen responsible for foot-and-mouth disease. The disease is endemic in many parts of the world with outbreaks within livestock resulting in major economic losses. Propagation of the viral genome occurs within

  15. Protection from rabies by a vaccinia virus recombinant containing the rabies virus glycoprotein gene.

    OpenAIRE

    Wiktor, T. J.; Macfarlan, R I; Reagan, K J; Dietzschold, B; Curtis, P. J.; Wunner, W. H.; Kieny, M P; Lathe, R; Lecocq, J P; Mackett, M.

    1984-01-01

    Inoculation of rabbits and mice with a vaccinia-rabies glycoprotein recombinant (V-RG) virus resulted in rapid induction of high concentrations of rabies virus-neutralizing antibodies and protection from severe intracerebral challenge with several strains of rabies virus. Protection from virus challenge also was achieved against the rabies-related Duvenhage virus but not against the Mokola virus. Effective immunization by V-RG depended on the expression of a rabies glycoprotein that registere...

  16. Protocol: using virus-induced gene silencing to study the arbuscular mycorrhizal symbiosis in Pisum sativum

    DEFF Research Database (Denmark)

    Grønlund, Mette; Olsen, Anne; Johansen, Elisabeth

    2010-01-01

    Virus-induced gene silencing (VIGS) is an alternative reverse genetics tool for silencing of genes in some plants, which are difficult to transform. The pea early-browning virus (PEBV) has been developed as a VIGS vector and used in pea for functional analysis of several genes. However......, the available PEBV-VIGS protocols are inadequate for studying genes involved in the symbiosis with arbuscular mycorrhizal fungi (AMF). Here we describe a PEBV-VIGS protocol suitable for reverse genetics studies in pea of genes involved in the symbiosis with AMF and show its effectiveness in silencing genes...

  17. The BFRF1 Gene of Epstein-Barr Virus Encodes a Novel Protein

    Science.gov (United States)

    Farina, Antonella; Santarelli, Roberta; Gonnella, Roberta; Bei, Roberto; Muraro, Raffaella; Cardinali, Giorgia; Uccini, Stefania; Ragona, Giuseppe; Frati, Luigi; Faggioni, Alberto; Angeloni, Antonio

    2000-01-01

    Computer analysis of the Epstein-Barr virus (EBV) genome indicates there are ∼100 open reading frames (ORFs). Thus far about 30 EBV genes divided into the categories latent and lytic have been identified. The BamHI F region of EBV is abundantly transcribed during lytic replication. This region is highly conserved among herpesviruses, thus suggesting that some common function could be retained in the ORFs encompassed within this viral fragment. To identify putative novel proteins and possible new markers for viral replication, we focused our attention on the first rightward ORF in the BamHI F region (BFRF1). Histidine and glutathione S-transferase-tagged BFRF1 fusion proteins were synthesized to produce a mouse monoclonal antibody (MAb). Analysis of human sera revealed a high seroprevalence of antibodies to BFRF1 in patients affected by nasopharyngeal carcinoma or Burkitt's lymphoma, whereas no humoral response to BFRF1 could be detected among healthy donors. An anti-BFRF1 MAb recognizes a doublet migrating at 37 to 38 kDa in cells extracts from EBV-infected cell lines following lytic cycle activation and in an EBV-negative cell line (DG75) transfected with a plasmid expressing the BFRF1 gene. Northern blot analysis allowed the detection of a major transcript of 3.7 kb highly expressed in EBV-positive lytic cycle-induced cell lines. Treatment with inhibitors of viral DNA polymerase, such as phosphonoacetic acid and acyclovir, reduced but did not abolish the transcription of BFRF1, thus indicating that BFRF1 can be classified as an early gene. Cell fractionation experiments, as well as immunolocalization by immunofluorescence microscopy, immunohistochemistry, and immunoelectron microscopy, showed that BFRF1 is localized on the plasma membrane and nuclear compartments of the cells and is a structural component of the viral particle. Identification of BFRF1 provides a new marker with which to monitor EBV infection and might help us better understand the biology of

  18. Selection of reference genes for quantitative real-time reverse transcription-polymerase chain reaction in concanavalin A-induced hepatitis model.

    Science.gov (United States)

    Shi, Guojun; Zhang, Zhijian; Feng, Dechun; Xu, Yan; Lu, Yan; Wang, Jiqiu; Jiang, Jingjing; Zhang, Zhiguo; Li, Xiaoying; Ning, Guang

    2010-06-01

    Quantitative real-time reverse transcription-polymerase chain reaction (Q-PCR) has become an indispensable technique for accurate determination of gene expression in various samples. In mice, intravenous injection of concanavalin A (ConA) leads to acute hepatitis and liver injury. Functional studies based on this model have provided insights for understanding the mechanisms of liver injury. However, no data have been reported to validate reference genes during the progression of ConA-induced hepatitis (CIH). In this study, IkappaBalpha and C/EBPbeta messenger RNA (mRNA) levels were examined using Q-PCR with ACTB as the reference gene after ConA injection. However, we got inconsistent results with previous reports determining IkappaBalpha and C/EBPbeta mRNA expression levels. The results indicate the necessity for stability analysis of candidate reference genes in the CIH model. geNorm, NormFinder, and BestKeeper software analysis indicates that ACTB is the most unstable gene during CIH progression among the 10 reference genes tested, whereas RPLP0 or HPRT1 is the most stable one. This study demonstrates that some of the commonly used reference genes are inadequate for normalization of Q-PCR data due to their expression instability. Furthermore, this study validates HPRT1 and RPLP0 as appropriate reference genes for Q-PCR analysis in the CIH model. Copyright 2010 Elsevier Inc. All rights reserved.

  19. Simian virus 40 vectors for pulmonary gene therapy

    Directory of Open Access Journals (Sweden)

    Oppenheim Ariella

    2007-10-01

    Full Text Available Abstract Background Sepsis remains the leading cause of death in critically ill patients. One of the primary organs affected by sepsis is the lung, presenting as the Acute Respiratory Distress Syndrome (ARDS. Organ damage in sepsis involves an alteration in gene expression, making gene transfer a potential therapeutic modality. This work examines the feasibility of applying simian virus 40 (SV40 vectors for pulmonary gene therapy. Methods Sepsis-induced ARDS was established by cecal ligation double puncture (2CLP. SV40 vectors carrying the luciferase reporter gene (SV/luc were administered intratracheally immediately after sepsis induction. Sham operated (SO as well as 2CLP rats given intratracheal PBS or adenovirus expressing luciferase served as controls. Luc transduction was evaluated by in vivo light detection, immunoassay and luciferase mRNA detection by RT-PCR in tissue harvested from septic rats. Vector abundance and distribution into alveolar cells was evaluated using immunostaining for the SV40 VP1 capsid protein as well as by double staining for VP1 and for the surfactant protein C (proSP-C. Immunostaining for T-lymphocytes was used to evaluate the cellular immune response induced by the vector. Results Luc expression measured by in vivo light detection correlated with immunoassay from lung tissue harvested from the same rats. Moreover, our results showed vector presence in type II alveolar cells. The vector did not induce significant cellular immune response. Conclusion In the present study we have demonstrated efficient uptake and expression of an SV40 vector in the lungs of animals with sepsis-induced ARDS. These vectors appear to be capable of in vivo transduction of alveolar type II cells and may thus become a future therapeutic tool.

  20. Origin and Evolution of RNA-Dependent RNA Polymerase.

    Science.gov (United States)

    de Farias, Savio T; Dos Santos Junior, Ariosvaldo P; Rêgo, Thais G; José, Marco V

    2017-01-01

    RNA-dependent RNA polymerases (RdRp) are very ancient enzymes and are essential for all viruses with RNA genomes. We reconstruct the origin and evolution of this polymerase since the initial stages of the origin of life. The origin of the RdRp was traced back from tRNA ancestors. At the origin of the RdRp the most ancient part of the protein is the cofactor-binding site that had the capacity of binding to simple molecules as magnesium, calcium, and ribonucleotides. Our results suggest that RdRp originated from junctions of proto-tRNAs that worked as the first genes at the emergence of the primitive translation system, where the RNA was the informational molecule. The initial domain, worked as a building block for the emergence of the fingers and thumb domains. From the ancestral RdRp, we could establish the evolutionary stages of viral evolution from a rooted ancestor to modern viruses. It was observed that the selective pressure under the RdRp was the organization and functioning of the genome, where RNA double-stranded and RNA single-stranded virus formed a separate group. We propose an evolutionary route to the polymerases and the results suggest an ancient scenario for the origin of RNA viruses.

  1. Reference Gene Selection for qPCR Analysis in Tomato-Bipartite Begomovirus Interaction and Validation in Additional Tomato-Virus Pathosystems.

    Directory of Open Access Journals (Sweden)

    Ana L M Lacerda

    Full Text Available Quantitative Polymerase Chain Reaction (qPCR is currently the most sensitive technique used for absolute and relative quantification of a target gene transcript, requiring the use of appropriated reference genes for data normalization. To accurately estimate the relative expression of target tomato (Solanum lycopersicum L. genes responsive to several virus species in reverse transcription qPCR analysis, the identification of reliable reference genes is mandatory. In the present study, ten reference genes were analyzed across a set of eight samples: two tomato contrasting genotypes ('Santa Clara', susceptible, and its near-isogenic line 'LAM 157', resistant; subjected to two treatments (inoculation with Tomato chlorotic mottle virus (ToCMoV and its mock-inoculated control and in two distinct times after inoculation (early and late. Reference genes stability was estimated by three statistical programs (geNorm, NormFinder and BestKeeper. To validate the results over broader experimental conditions, a set of ten samples, corresponding to additional three tomato-virus pathosystems that included tospovirus, crinivirus and tymovirus + tobamovirus, was analyzed together with the tomato-ToCMoV pathosystem dataset, using the same algorithms. Taking into account the combined analyses of the ranking order outputs from the three algorithms, TIP41 and EF1 were identified as the most stable genes for tomato-ToCMoV pathosystem, and TIP41 and EXP for the four pathosystems together, and selected to be used as reference in the forthcoming expression qPCR analysis of target genes in experimental conditions involving the aforementioned tomato-virus pathosystems.

  2. A multiple reverse transcription-polymerase chain reaction assay for simultaneous detection and differentiation of latent viruses and apscarviroids in apple trees

    Science.gov (United States)

    Apple chlorotic leaf spot virus (ACLSV), Apple stem grooving virus (ASGV), and Apple stem pitting virus (ASPV) are three latent viruses frequently occurring in apple trees worldwide. In field orchards, these viruses are frequently found in a mixed infection with viroids in the genus Apscarviroid, in...

  3. The Cockayne syndrome group A gene encodes a WD repeat protein that interacts with CSB protein and a subunit of RNA polymerase II TFIIH.

    Science.gov (United States)

    Henning, K A; Li, L; Iyer, N; McDaniel, L D; Reagan, M S; Legerski, R; Schultz, R A; Stefanini, M; Lehmann, A R; Mayne, L V; Friedberg, E C

    1995-08-25

    The hereditary disease Cockayne syndrome (CS) is characterized by a complex clinical phenotype. CS cells are abnormally sensitive to ultraviolet radiation and are defective in the repair of transcriptionally active genes. The cloned CSB gene encodes a member of a protein family that includes the yeast Snf2 protein, a component of the transcriptional regulator Swi/Snf. We report the cloning of the CSA cDNA, which can encode a WD repeat protein. Mutations in the cDNA have been identified in CS-A cell lines. CSA protein interacts with CSB protein and with p44 protein, a subunit of the human RNA polymerase II transcription factor IIH. These observations suggest that the products of the CSA and CSB genes are involved in transcription.

  4. Multiplex reverse transcription-polymerase chain reaction combined with on-chip electrophoresis as a rapid screening tool for candidate gene sets

    DEFF Research Database (Denmark)

    Wittig, Rainer; Salowsky, Rüdiger; Blaich, Stephanie

    2005-01-01

    Combining multiplex reverse transcription-polymerase chain reaction (mRT-PCR) with microfluidic amplicon analysis, we developed an assay for the rapid and reliable semiquantitative expression screening of 11 candidate genes for drug resistance in human malignant melanoma. The functionality...... of this approach was demonstrated by low interexperimental variations of amplicon quantities after endpoint analysis. When applied to RNA samples derived from drug-sensitive and -resistant melanoma cell lines, mRT-PCR delivered results qualitatively concordant with data obtained from Northern blot and array...... analyses. The screening of additional melanoma cell lines resulted in distinct expression patterns for ten candidate genes. Our approach reveals a rapid and easy-to-handle alternative for candidate gene set evaluation from limited amounts of RNA....

  5. Disruption of TLR3 signaling due to cleavage of TRIF by the hepatitis A virus protease-polymerase processing intermediate, 3CD.

    Directory of Open Access Journals (Sweden)

    Lin Qu

    2011-09-01

    Full Text Available Toll-like receptor 3 (TLR3 and cytosolic RIG-I-like helicases (RIG-I and MDA5 sense viral RNAs and activate innate immune signaling pathways that induce expression of interferon (IFN through specific adaptor proteins, TIR domain-containing adaptor inducing interferon-β (TRIF, and mitochondrial antiviral signaling protein (MAVS, respectively. Previously, we demonstrated that hepatitis A virus (HAV, a unique hepatotropic human picornavirus, disrupts RIG-I/MDA5 signaling by targeting MAVS for cleavage by 3ABC, a precursor of the sole HAV protease, 3C(pro, that is derived by auto-processing of the P3 (3ABCD segment of the viral polyprotein. Here, we show that HAV also disrupts TLR3 signaling, inhibiting poly(I:C-stimulated dimerization of IFN regulatory factor 3 (IRF-3, IRF-3 translocation to the nucleus, and IFN-β promoter activation, by targeting TRIF for degradation by a distinct 3ABCD processing intermediate, the 3CD protease-polymerase precursor. TRIF is proteolytically cleaved by 3CD, but not by the mature 3C(pro protease or the 3ABC precursor that degrades MAVS. 3CD-mediated degradation of TRIF depends on both the cysteine protease activity of 3C(pro and downstream 3D(pol sequence, but not 3D(pol polymerase activity. Cleavage occurs at two non-canonical 3C(pro recognition sequences in TRIF, and involves a hierarchical process in which primary cleavage at Gln-554 is a prerequisite for scission at Gln-190. The results of mutational studies indicate that 3D(pol sequence modulates the substrate specificity of the upstream 3C(pro protease when fused to it in cis in 3CD, allowing 3CD to target cleavage sites not normally recognized by 3C(pro. HAV thus disrupts both RIG-I/MDA5 and TLR3 signaling pathways through cleavage of essential adaptor proteins by two distinct protease precursors derived from the common 3ABCD polyprotein processing intermediate.

  6. Recombinant Hendra viruses expressing a reporter gene retain pathogenicity in ferrets.

    Science.gov (United States)

    Marsh, Glenn A; Virtue, Elena R; Smith, Ina; Todd, Shawn; Arkinstall, Rachel; Frazer, Leah; Monaghan, Paul; Smith, Greg A; Broder, Christopher C; Middleton, Deborah; Wang, Lin-Fa

    2013-03-25

    Hendra virus (HeV) is an Australian bat-borne zoonotic paramyxovirus that repeatedly spills-over to horses causing fatal disease. Human cases have all been associated with close contact with infected horses. A full-length antigenome clone of HeV was assembled, a reporter gene (GFP or luciferase) inserted between the P and M genes and transfected to 293T cells to generate infectious reporter gene-encoding recombinant viruses. These viruses were then assessed in vitro for expression of the reporter genes. The GFP expressing recombinant HeV was used to challenge ferrets to assess the virulence and tissue distribution by monitoring GFP expression in infected cells. Three recombinant HeV constructs were successfully cloned and rescued; a wild-type virus, a GFP-expressing virus and a firefly luciferase-expressing virus. In vitro characterisation demonstrated expression of the reporter genes, with levels proportional to the initial inoculum levels. Challenge of ferrets with the GFP virus demonstrated maintenance of the fatal phenotype with disease progressing to death consistent with that observed previously with the parental wild-type isolate of HeV. GFP expression could be observed in infected tissues collected from animals at euthanasia. Here, we report on the first successful rescue of recombinant HeV, including wild-type virus and viruses expressing two different reporter genes encoded as an additional gene cassette inserted between the P and M genes. We further demonstrate that the GFP virus retained the ability to cause fatal disease in a well-characterized ferret model of henipavirus infection despite the genome being an extra 1290 nucleotides in length.

  7. Programmed ribosomal frameshift alters expression of west nile virus genes and facilitates virus replication in birds and mosquitoes.

    Directory of Open Access Journals (Sweden)

    Ezequiel Balmori Melian

    2014-11-01

    Full Text Available West Nile virus (WNV is a human pathogen of significant medical importance with close to 40,000 cases of encephalitis and more than 1,600 deaths reported in the US alone since its first emergence in New York in 1999. Previous studies identified a motif in the beginning of non-structural gene NS2A of encephalitic flaviviruses including WNV which induces programmed -1 ribosomal frameshift (PRF resulting in production of an additional NS protein NS1'. We have previously demonstrated that mutant WNV with abolished PRF was attenuated in mice. Here we have extended our previous observations by showing that PRF does not appear to have a significant role in virus replication, virion formation, and viral spread in several cell lines in vitro. However, we have also shown that PRF induces an over production of structural proteins over non-structural proteins in virus-infected cells and that mutation abolishing PRF is present in ∼11% of the wild type virus population. In vivo experiments in house sparrows using wild type and PRF mutant of New York 99 strain of WNV viruses showed some attenuation for the PRF mutant virus. Moreover, PRF mutant of Kunjin strain of WNV showed significant decrease compared to wild type virus infection in dissemination of the virus from the midgut through the haemocoel, and ultimately the capacity of infected mosquitoes to transmit virus. Thus our results demonstrate an important role for PRF in regulating expression of viral genes and consequently virus replication in avian and mosquito hosts.

  8. Reference gene selection for quantitative reverse transcription-polymerase chain reaction normalization during in vitro adventitious rooting in Eucalyptus globulus Labill

    Directory of Open Access Journals (Sweden)

    Pasquali Giancarlo

    2010-09-01

    Full Text Available Abstract Background Eucalyptus globulus and its hybrids are very important for the cellulose and paper industry mainly due to their low lignin content and frost resistance. However, rooting of cuttings of this species is recalcitrant and exogenous auxin application is often necessary for good root development. To date one of the most accurate methods available for gene expression analysis is quantitative reverse transcription-polymerase chain reaction (qPCR; however, reliable use of this technique requires reference genes for normalization. There is no single reference gene that can be regarded as universal for all experiments and biological materials. Thus, the identification of reliable reference genes must be done for every species and experimental approach. The present study aimed at identifying suitable control genes for normalization of gene expression associated with adventitious rooting in E. globulus microcuttings. Results By the use of two distinct algorithms, geNorm and NormFinder, we have assessed gene expression stability of eleven candidate reference genes in E. globulus: 18S, ACT2, EF2, EUC12, H2B, IDH, SAND, TIP41, TUA, UBI and 33380. The candidate reference genes were evaluated in microccuttings rooted in vitro, in presence or absence of auxin, along six time-points spanning the process of adventitious rooting. Overall, the stability profiles of these genes determined with each one of the algorithms were very similar. Slight differences were observed in the most stable pair of genes indicated by each program: IDH and SAND for geNorm, and H2B and TUA for NormFinder. Both programs indentified UBI and 18S as the most variable genes. To validate these results and select the most suitable reference genes, the expression profile of the ARGONAUTE1 gene was evaluated in relation to the most stable candidate genes indicated by each algorithm. Conclusion Our study showed that expression stability varied between putative reference genes

  9. Reference gene selection for quantitative reverse transcription-polymerase chain reaction normalization during in vitro adventitious rooting in Eucalyptus globulus Labill.

    Science.gov (United States)

    de Almeida, Márcia R; Ruedell, Carolina M; Ricachenevsky, Felipe K; Sperotto, Raul A; Pasquali, Giancarlo; Fett-Neto, Arthur G

    2010-09-20

    Eucalyptus globulus and its hybrids are very important for the cellulose and paper industry mainly due to their low lignin content and frost resistance. However, rooting of cuttings of this species is recalcitrant and exogenous auxin application is often necessary for good root development. To date one of the most accurate methods available for gene expression analysis is quantitative reverse transcription-polymerase chain reaction (qPCR); however, reliable use of this technique requires reference genes for normalization. There is no single reference gene that can be regarded as universal for all experiments and biological materials. Thus, the identification of reliable reference genes must be done for every species and experimental approach. The present study aimed at identifying suitable control genes for normalization of gene expression associated with adventitious rooting in E. globulus microcuttings. By the use of two distinct algorithms, geNorm and NormFinder, we have assessed gene expression stability of eleven candidate reference genes in E. globulus: 18S, ACT2, EF2, EUC12, H2B, IDH, SAND, TIP41, TUA, UBI and 33380. The candidate reference genes were evaluated in microccuttings rooted in vitro, in presence or absence of auxin, along six time-points spanning the process of adventitious rooting. Overall, the stability profiles of these genes determined with each one of the algorithms were very similar. Slight differences were observed in the most stable pair of genes indicated by each program: IDH and SAND for geNorm, and H2B and TUA for NormFinder. Both programs identified UBI and 18S as the most variable genes. To validate these results and select the most suitable reference genes, the expression profile of the ARGONAUTE1 gene was evaluated in relation to the most stable candidate genes indicated by each algorithm. Our study showed that expression stability varied between putative reference genes tested in E. globulus. Based on the AGO1 relative expression

  10. INFUENCE OF GENE POLIMORPHISM ON THE COURSE OF EPSTEIN-BARR VIRUS INFECTION

    Directory of Open Access Journals (Sweden)

    Sorokina O.G.

    2017-06-01

    Full Text Available Introduction. Currently, infectious diseases occupy a dominant place in human pathology. The relevance of the Epstein-Barr virus infection (VEB is due to a high degree of infection of the population around the world, as well as specific antibodies to this virus, detected in almost 95% of the adult population. Material and methods. We have examined 96 patients with chronic VEB infection, the main clinical manifestations of which were various immunopathological and immunodeficiency states, as well as 10 patients who had undergone a history of VEB without any complaints at the moment. The comparison group consisted of 10 clinically healthy people who had no record of infectious mononucleosis. Polymorphism of the genes was determined using the RFLP method (polymorphism of the length of restriction fragments and the real-time PCR method using the Corbett Research Rotor-Gene-3000 and the DNA-detecting DT-96 amplifier. To detect the polymorphisms under study, amplification of certain sections of the corresponding genes was carried out. To determine the allelic variation of the IL28B gene, a commercial DNA-technology test system was used. SNP 39743165T> G (rs8099917 and SNP 39738787C> T (rs 2979860 of the IL-28B gene were used to detect point mutations using polymerase chain reaction and polymorphism of restriction fragment lengths. As a material for the study, DNA obtained from leukocytes was used with commercial reagents to extract DNA from the clinical material "Cytolysin" by AmpliSens (Russia. Statistical processing of the results of the study was carried out in accordance with the recommendations for statistical processing of biomedical data. The statistical software package STATISTICA 10.0 was used. Results and discussion. A group of patients with a record of VEB who do not currently have any complaints, and also in the comparison group for the IL-28B gene, found the CC genotype at the locus rs12979860, and the TT genotype at the locus rs8099917

  11. A high throughput barley stripe mosaic virus vector for virus induced gene silencing in monocots and dicots.

    Directory of Open Access Journals (Sweden)

    Cheng Yuan

    Full Text Available Barley stripe mosaic virus (BSMV is a single-stranded RNA virus with three genome components designated alpha, beta, and gamma. BSMV vectors have previously been shown to be efficient virus induced gene silencing (VIGS vehicles in barley and wheat and have provided important information about host genes functioning during pathogenesis as well as various aspects of genes functioning in development. To permit more effective use of BSMV VIGS for functional genomics experiments, we have developed an Agrobacterium delivery system for BSMV and have coupled this with a ligation independent cloning (LIC strategy to mediate efficient cloning of host genes. Infiltrated Nicotiana benthamiana leaves provided excellent sources of virus for secondary BSMV infections and VIGS in cereals. The Agro/LIC BSMV VIGS vectors were able to function in high efficiency down regulation of phytoene desaturase (PDS, magnesium chelatase subunit H (ChlH, and plastid transketolase (TK gene silencing in N. benthamiana and in the monocots, wheat, barley, and the model grass, Brachypodium distachyon. Suppression of an Arabidopsis orthologue cloned from wheat (TaPMR5 also interfered with wheat powdery mildew (Blumeria graminis f. sp. tritici infections in a manner similar to that of the A. thaliana PMR5 loss-of-function allele. These results imply that the PMR5 gene has maintained similar functions across monocot and dicot families. Our BSMV VIGS system provides substantial advantages in expense, cloning efficiency, ease of manipulation and ability to apply VIGS for high throughput genomics studies.

  12. Characterization of a Novel Megabirnavirus from Sclerotinia sclerotiorum Reveals Horizontal Gene Transfer from Single-Stranded RNA Virus to Double-Stranded RNA Virus

    Science.gov (United States)

    Wang, Minghong; Wang, Yong; Sun, Xiangzhong; Cheng, Jiasen; Fu, Yanping; Liu, Huiquan; Jiang, Daohong; Ghabrial, Said A.

    2015-01-01

    ABSTRACT Mycoviruses have been detected in all major groups of filamentous fungi, and their study represents an important branch of virology. Here, we characterized a novel double-stranded RNA (dsRNA) mycovirus, Sclerotinia sclerotiorum megabirnavirus 1 (SsMBV1), in an apparently hypovirulent strain (SX466) of Sclerotinia sclerotiorum. Two similarly sized dsRNA segments (L1- and L2-dsRNA), the genome of SsMBV1, are packaged in rigid spherical particles purified from strain SX466. The full-length cDNA sequence of L1-dsRNA/SsMBV1 comprises two large open reading frames (ORF1 and ORF2), which encode a putative coat protein and an RNA-dependent RNA polymerase (RdRp), respectively. Phylogenetic analysis of the RdRp domain clearly indicates that SsMBV1 is related to Rosellinia necatrix megabirnavirus 1 (RnMBV1). L2-dsRNA/SsMBV1 comprises two nonoverlapping ORFs (ORFA and ORFB) encoding two hypothetical proteins with unknown functions. The 5′-terminal regions of L1- and L2-dsRNA/SsMBV1 share strictly conserved sequences and form stable stem-loop structures. Although L2-dsRNA/SsMBV1 is dispensable for replication, genome packaging, and pathogenicity of SsMBV1, it enhances transcript accumulation of L1-dsRNA/SsMBV1 and stability of virus-like particles (VLPs). Interestingly, a conserved papain-like protease domain similar to a multifunctional protein (p29) of Cryphonectria hypovirus 1 was detected in the ORFA-encoded protein of L2-dsRNA/SsMBV1. Phylogenetic analysis based on the protease domain suggests that horizontal gene transfer may have occurred from a single-stranded RNA (ssRNA) virus (hypovirus) to a dsRNA virus, SsMBV1. Our results reveal that SsMBV1 has a slight impact on the fundamental biological characteristics of its host regardless of the presence or absence of L2-dsRNA/SsMBV1. IMPORTANCE Mycoviruses are widespread in all major fungal groups, and they possess diverse genomes of mostly ssRNA and dsRNA and, recently, circular ssDNA. Here, we have characterized

  13. Characterization of a Novel Megabirnavirus from Sclerotinia sclerotiorum Reveals Horizontal Gene Transfer from Single-Stranded RNA Virus to Double-Stranded RNA Virus.

    Science.gov (United States)

    Wang, Minghong; Wang, Yong; Sun, Xiangzhong; Cheng, Jiasen; Fu, Yanping; Liu, Huiquan; Jiang, Daohong; Ghabrial, Said A; Xie, Jiatao

    2015-08-01

    Mycoviruses have been detected in all major groups of filamentous fungi, and their study represents an important branch of virology. Here, we characterized a novel double-stranded RNA (dsRNA) mycovirus, Sclerotinia sclerotiorum megabirnavirus 1 (SsMBV1), in an apparently hypovirulent strain (SX466) of Sclerotinia sclerotiorum. Two similarly sized dsRNA segments (L1- and L2-dsRNA), the genome of SsMBV1, are packaged in rigid spherical particles purified from strain SX466. The full-length cDNA sequence of L1-dsRNA/SsMBV1 comprises two large open reading frames (ORF1 and ORF2), which encode a putative coat protein and an RNA-dependent RNA polymerase (RdRp), respectively. Phylogenetic analysis of the RdRp domain clearly indicates that SsMBV1 is related to Rosellinia necatrix megabirnavirus 1 (RnMBV1). L2-dsRNA/SsMBV1 comprises two nonoverlapping ORFs (ORFA and ORFB) encoding two hypothetical proteins with unknown functions. The 5'-terminal regions of L1- and L2-dsRNA/SsMBV1 share strictly conserved sequences and form stable stem-loop structures. Although L2-dsRNA/SsMBV1 is dispensable for replication, genome packaging, and pathogenicity of SsMBV1, it enhances transcript accumulation of L1-dsRNA/SsMBV1 and stability of virus-like particles (VLPs). Interestingly, a conserved papain-like protease domain similar to a multifunctional protein (p29) of Cryphonectria hypovirus 1 was detected in the ORFA-encoded protein of L2-dsRNA/SsMBV1. Phylogenetic analysis based on the protease domain suggests that horizontal gene transfer may have occurred from a single-stranded RNA (ssRNA) virus (hypovirus) to a dsRNA virus, SsMBV1. Our results reveal that SsMBV1 has a slight impact on the fundamental biological characteristics of its host regardless of the presence or absence of L2-dsRNA/SsMBV1. Mycoviruses are widespread in all major fungal groups, and they possess diverse genomes of mostly ssRNA and dsRNA and, recently, circular ssDNA. Here, we have characterized a novel dsRNA virus

  14. The use of a one-step real-time reverse transcription polymerase chain reaction (rRT-PCR) for the surveillance of viral hemorrhagic septicemia virus (VHSV) in Minnesota.

    Science.gov (United States)

    Phelps, Nicholas B D; Patnayak, Devi P; Jiang, Yin; Goyal, Sagar M

    2012-12-01

    Viral hemorrhagic septicemia virus (VHSV) is a highly contagious and pathogenic virus of fish. The virus infects more than 70 fish species worldwide, in both fresh and salt water. A new viral strain (VHSV-IVb) has proven both virulent and persistent, spreading throughout the Great Lakes of North America and to inland water bodies in the region. To better understand the geographic distribution of the virus, we used a modified real-time reverse transcription polymerase chain reaction (rRT-PCR) assay for high-throughput testing of fish for VHSV. The assay was shown to be twice as sensitive as the gold standard, virus isolation, and did not cross react with other viruses found in fish. In addition, the diagnostic turnaround time was reduced from 28 to 30 d for virus isolation to 2-4 d for rRT-PCR. To demonstrate the usefulness of the rRT-PCR assay, 115 high-priority water bodies in Minnesota were tested by both methods from April 2010 to June 2011. All survey sites tested negative for VHSV by both methods. The survey results have informed fisheries managers on the absence of VHSV in Minnesota and have better prepared them for the eventual arrival of the disease. In addition, the results demonstrate the value of this rRT-PCR as a surveillance tool to rapidly identify an outbreak so that it can be controlled in a timely manner.

  15. Development and application of a multiplex reverse-transcription polymerase chain reaction assay for screening a global collection of Citrus tristeza virus isolates.

    Science.gov (United States)

    Roy, Avijit; Ananthakrishnan, G; Hartung, John S; Brlansky, R H

    2010-10-01

    The emerging diversity of Citrus tristeza virus (CTV) genotypes has complicated detection and diagnostic measures and prompted the search for new differentiation methods. To simplify the identification and differentiation of CTV genotypes, a multiplex reverse-transcription polymerase chain reaction (RT-PCR) technique for the screening of CTV isolates was developed. Variable regions within the open reading frame (ORF)-1a of diverse CTV genotypes were identified to develop first a simplex (S) and then a hexaplex (H) RT-PCR. CTV isolates have been grouped previously into five genotypes (namely, T3, T30, T36, VT, and B165) based on the nucleotide sequence comparisons and phylogenetic analyses. Nucleotide sequences from GenBank were used to design species and genotype-specific primers (GSPs). The GSPs were initially used for reliable detection of all CTV genotypes using S-RT-PCR. Furthermore, detection of all five recognized CTV genotypes was established using the H-RT-PCR. Six amplicons, one generic to all CTV isolates and one for each of the five recognized genotypes, were identified on the basis of their size and were confirmed by sequence analysis. In all, 175 CTV isolates from 29 citrus-growing countries were successfully analyzed by S- and H-RT-PCR. Of these, 97 isolates contained T36 genotypes, 95 contained T3 genotypes, 76 contained T30 genotypes, 71 contained VT genotypes, and 24 contained B165 genotype isolates. In total, 126 isolates contained mixed infections of 2 to 5 of the known CTV genotypes. Two of the CTV isolates could not be assigned to a known genotype. H-RT-PCR provides a sensitive, specific, reliable, and rapid way to screen for CTV genotypes compared with other methods for CTV genotype detection. Efficient identification of CTV genotypes will facilitate a better understanding of CTV isolates, including the possible interaction of different genotypes in causing or preventing diseases. The methods described can also be used in virus-free citrus

  16. Ribose 2'-O methylation of the vesicular stomatitis virus mRNA cap precedes and facilitates subsequent guanine-N-7 methylation by the large polymerase protein.

    Science.gov (United States)

    Rahmeh, Amal A; Li, Jianrong; Kranzusch, Philip J; Whelan, Sean P J

    2009-11-01

    During conventional mRNA cap formation, two separate methyltransferases sequentially modify the cap structure, first at the guanine-N-7 (G-N-7) position and subsequently at the ribose 2'-O position. For vesicular stomatitis virus (VSV), a prototype of the nonsegmented negative-strand RNA viruses, the two methylase activities share a binding site for the methyl donor S-adenosyl-l-methionine and are inhibited by individual amino acid substitutions within the C-terminal domain of the large (L) polymerase protein. This led to the suggestion that a single methylase domain functions for both 2'-O and G-N-7 methylations. Here we report a trans-methylation assay that recapitulates both ribose 2'-O and G-N-7 modifications by using purified recombinant L and in vitro-synthesized RNA. Using this assay, we demonstrate that VSV L typically modifies the 2'-O position of the cap prior to the G-N-7 position and that G-N-7 methylation is diminished by pre-2'-O methylation of the substrate RNA. Amino acid substitutions in the C terminus of L that prevent all cap methylation in recombinant VSV (rVSV) partially retain the ability to G-N-7 methylate a pre-2'-O-methylated RNA, therefore uncoupling the effect of substitutions in the C terminus of the L protein on the two methylations. In addition, we show that the 2'-O and G-N-7 methylase activities act specifically on RNA substrates that contain the conserved elements of a VSV mRNA start at the 5' terminus. This study provides new mechanistic insights into the mRNA cap methylase activities of VSV L, demonstrates that 2'-O methylation precedes and facilitates subsequent G-N-7 methylation, and reveals an RNA sequence and length requirement for the two methylase activities. We propose a model of regulation of the activity of the C terminus of L protein in 2'-O and G-N-7 methylation of the cap structure.

  17. Study of Epstein-Barr virus expression in Burkitt′s lymphoma by polymerase chain reaction and in situ hybridization: A study in Iran

    Directory of Open Access Journals (Sweden)

    Parviz Deyhimi

    2014-01-01

    Full Text Available Background: The association of Epstein-Barr virus (EBV with Burkitt′s lymphoma (BL is variable in different geographic regions. In developing countries, the association of EBV with BL is regarded to be of an endemic-type in equatorial Africa ( > 95% and sporadic-type in the developed countries (15-30%. The purpose of this study is to assess the frequency of EBV infection in BL, in Iran. The study also aims to compare Ribonucleic acid (RNA in situ hybridization (RISH, the standard diagnostic method, with the polymerase chain reaction (PCR-based method for diagnosing BL. Materials and Methods: In this epidemiological study, the paraffinized specimens of 18 cases of BL were selected. Next, the ISH of EBV-encoded RNA (EBER-RISH and PCR assays that were based on Epstein Barr Nuclear Antigen 2 ( EBNA2 amplification were used. The EBV strain was determined by PCR. The data were analyzed using the SPSS10 software and by performing Pearson correlation coefficient formula at a significant level of 0.05. Results: EBV RNA was detected in 50% of the BL specimens. Type 1 and 2 accounted for 70 and 30% of the cases, respectively. Regarding RISH as the standard method for EBV diagnosis, the PCR assays showed a sensitivity and specificity of 100 and 88.9%, respectively. Conclusion: According to the obtained findings, the frequency of EBV in BL was 50% and PCR and RISH showed high concordance and sensitivity in EBV detection. Therefore, PCR can be used as a faster method for EBV detection in high-risk geographical regions.

  18. Incidence of and factors associated with false positives in laboratory diagnosis of norovirus infection by amplification of the RNA-dependent RNA polymerase gene.

    Directory of Open Access Journals (Sweden)

    Fang-Ru Lin

    Full Text Available BACKGROUND: Conventional reverse transcription-polymerase chain reaction (RT-PCR amplification of the RNA-dependent RNA polymerase (RdRp gene remains a used method for the rapid detection of norovirus (NV in clinical laboratories. The incidence of and factors associated with false positives in this assay have not been previously evaluated. METHODS/PRINCIPAL FINDINGS: After an NV outbreak caused by the GII.4 Sydney strain in 2012, we reanalysed 250 stool samples positive for NV by RdRp gene detection. True positives were confirmed in 154 (61.6% samples by successful amplification and sequencing confirmation of the viral protein 1 gene. Of the remaining 96 samples that underwent RT-PCR for the RdRp gene, 34 samples yielded PCR products of the expected length. However, the sequences of the amplicons belonged to the human genome, with 91-97% matched nucleotide sequences, indicating false positives. Multivariate analysis of the clinical features of the patients identified a positive stool culture for bacteria (adjusted odds ratio [aOR] 9.07, 95% adjusted confidence interval [aCI] 2.17-37.92, P = .003 and the use of parenteral antibiotics (aOR 5.55, 95% aCI 1.21-24.73, P = .027 as significant and independent factors associated with false positives. CONCLUSION: Conventional RT-PCR targeting the RdRp gene of NV can lead to false positives in patients with bacterial enterocolitis by incidental amplification of DNA from a human source.

  19. Reference gene identification for reverse transcription-quantitative polymerase chain reaction analysis in an ischemic wound-healing model.

    Science.gov (United States)

    Ruedrich, Elizabeth D; Henzel, Mary K; Hausman, Bryan S; Bogie, Kath M

    2013-12-01

    Reference genes are often used in RT-quantitative PCR (qPCR) analysis to normalize gene expression levels to a gene that is expressed stably across study groups. They ultimately serve as a control in RT-qPCR analysis, producing more accurate interpretation of results. Whereas many reference genes have been used in various wound-healing studies, the most stable reference gene for ischemic wound-healing analysis has yet to be identified. The goal of this study was to determine systematically the most stable reference gene for studying gene expression in a rat ischemic wound-healing model using RT-qPCR. Twelve commonly used reference genes were analyzed using RT-qPCR and geNorm data analysis to determine stability across normal and ischemic skin tissue. It was ultimately determined that Ubiquitin C (UBC) and β-2 Microglobulin (B2M) are the most stably conserved reference genes across normal and ischemic skin tissue. UBC and B2M represent reliable reference genes for RT-qPCR studies in the rat ischemic wound model and are unaffected by sustained tissue ischemia. The geometric mean of these two stable genes provides an accurate normalization factor. These results provide insight on dependence of reference-gene stability on experimental parameters and the importance of such reference-gene investigations.

  20. Identification and comparative analysis of the chloroplast α-subunit gene of DNA-dependent RNA polymerase from seven Euglena species

    Science.gov (United States)

    Sheveleva, Elena V.; Giordani, Nicole V.; Hallick, Richard B.

    2002-01-01

    When the sequence of the Euglena gracilis chloroplast genome was reported in 1993 the α-subunit gene (rpoA) of RNA polymerase appeared to be missing, based on a comparison of all putative reading frames to the then known rpoA loci. Since there has been a large increase in known rpoA sequences, the question of a Euglena chloroplast rpoA gene was re-examined. A previously described unknown reading frame of 161 codons was found to be part of an rpoA gene split by a single group III intron. This rpoA gene, which is highly variable from species to species, was then isolated and characterized in five other euglenoid species, Euglena anabaena, Euglena granulata, Euglena myxocylindracea, Euglena stellata and Euglena viridis, and in the Astasia longa plastid genome. All seven Euglena rpoA genes have either one or three group III introns. The rpoA gene products in Euglena spp. appear to be the most variable in this gene family when compared to the rpoA gene in other species of bacteria, algae and plants. Additionally, Euglena rpoA proteins lack a C-terminal domain required for interaction with some regulatory proteins, a feature shared only with some chlorophyte green algae. The E.gracilis rpoA gene is the distal cistron of a multigene cluster that includes genes for carbohydrate biosynthesis, photosynthetic electron transport, an antenna complex and ribosomal proteins. This study provides new insights into the transcription system of euglenoid plastids, the organization of the plastid genome, group III intron evolution and euglenoid phylogeny. PMID:11861918

  1. Different responses in the expression of alginases, alginate polymerase and acetylation genes during alginate production by Azotobacter vinelandii under oxygen-controlled conditions.

    Science.gov (United States)

    Díaz-Barrera, Alvaro; Maturana, Nataly; Pacheco-Leyva, Ivette; Martínez, Irene; Altamirano, Claudia

    2017-07-01

    Alginate production and gene expression of genes involved in alginate biosynthesis were evaluated in continuous cultures under dissolved oxygen tension (DOT) controlled conditions. Chemostat at 8% DOT showed an increase in the specific oxygen uptake rate [Formula: see text] from 10.9 to 45.3 mmol g -1  h -1 by changes in the dilution rate (D) from 0.06 to 0.10 h -1 , whereas under 1% DOT the [Formula: see text] was not affected. Alginate molecular weight was not affected by DOT. However, chemostat at 1% DOT showed a downregulation up to 20-fold in genes encoding both the alginate polymerase (alg8, alg44), alginate acetylases (algV, algI) and alginate lyase AlgL. alyA1 and algE7 lyases gene expressions presented an opposite behavior by changing the DOT, suggesting that A. vinelandii can use specific depolymerases depending on the oxygen level. Overall, the DOT level have a differential effect on genes involved in alginate synthesis, thus a gene expression equilibrium determines the production of alginates of similar molecular weight under DOT controlled.

  2. Identification, cloning, and expression analysis of three putative Lymantria dispar nuclear polyhedrosis virus immediate early genes

    Science.gov (United States)

    James M. Slavicek; Nancy Hayes-Plazolles

    1991-01-01

    Viral immediate early gene products are usually regulatory proteins that control expression of other viral genes at the transcriptional level or are proteins that are part of the viral DNA replication complex. The identification and functional characterization of the immediate early gene products of Lymantria dispar nuclear polyhedrosis virus (LdNPV...

  3. A plant virus evolved by acquiring multiple nonconserved genes to extend its host range.

    Science.gov (United States)

    Tatineni, Satyanarayana; Robertson, Cecile J; Garnsey, Stephen M; Dawson, William O

    2011-10-18

    Viruses have evolved as combinations of genes whose products interact with cellular components to produce progeny virus throughout the plants. Some viral genes, particularly those that are involved in replication and assembly, tend to be relatively conserved, whereas other genes that have evolved for interactions with the specific host for movement and to counter host-defense systems tend to be less conserved. Closteroviridae encode 1-5 nonconserved ORFs. Citrus tristeza virus (CTV), a Closterovirus, possesses nonconserved p33, p18, and p13 genes that are expendable for systemic infection of the two laboratory hosts, Citrus macrophylla and Mexican lime. In this study, we show that the extended host range of CTV requires these nonconserved genes. The p33 gene was required to systemically infect sour orange and lemon trees, whereas either the p33 or the p18 gene was sufficient for systemic infection of grapefruit trees and the p33 or the p13 gene was sufficient for systemic infection of calamondin plants. Thus, these three genes are required for systemic infection of the full host range of CTV, but different genes were specific for different hosts. Remarkably, either of two genes was sufficient for infection of some citrus hybrids. These findings suggest that CTV acquired multiple nonconserved genes (p33, p18, and p13) and, as a result, gained the ability to interact with multiple hosts, thus extending its host range during the course of evolution. These results greatly extend the complexity of known virus-plant interactions.

  4. Identification and characterization of the ecdysteroid UDPglucosyltransferase gene of the Lymantria dispar multinucleocapsid nuclear polyhedrosis virus

    Science.gov (United States)

    Christopher I. Riegel; Carita Lanner-Herrera; James M. Slavicek

    1994-01-01

    We have located, cloned, sequenced and characterized the ecdysteroid UDP-glucosyltransferase gene (egt) gene from the baculovirus Lymantria dispar multinucleocapsid nuclear polyhedrosis virus,(LdMNPV), which is specific for the gypsy moth (L. dispar). The egt gene from the related baculovirus Autographa californica...

  5. Transcriptional Organization and In Vivo Role of the Escherichia coli rsd Gene, Encoding the Regulator of RNA Polymerase Sigma D

    OpenAIRE

    Jishage, Miki; Ishihama, Akira

    1999-01-01

    The regulator of sigma D (Rsd) was identified as an RNA polymerase ς70-associated protein in stationary-phase Escherichia coli with the inhibitory activity of ς70-dependent transcription in vitro (M. Jishage and A. Ishihama, Proc. Natl. Acad. Sci. USA 95:4953–4958, 1998). Primer extension analysis of rsd mRNA indicated the presence of two promoters, ςS-dependent P1 and ς70-dependent P2 with the gearbox sequence. To get insight into the in vivo role of Rsd, the expressi...

  6. Comparison of the fast track diagnostics respiratory 21 and Seegene Allplex multiplex polymerase chain reaction assays for the detection of respiratory viruses.

    Science.gov (United States)

    Barratt, Kevin; Anderson, Trevor P; Fahey, Jennifer A; Jennings, Lance C; Werno, Anja M; Murdoch, David R

    2017-04-01

    Real-time multiplex PCR assays are increasingly used for respiratory virus detection, and offer automated analysis in a closed tube system, but they have the disadvantage of low-throughput due to multiplexing limitations. In this study, the established fast-track respiratory 21 assay (FTD) (fast-track diagnostics, Junglinster Luxembourg) was compared to the new Seegene Allplex assay (Seegene) (Seegene Inc. Seoul, Korea) which offers greater multiplexing as multiple targets can be detected in each fluorescence channel. The Seegene Allplex assay is quicker to perform than previous Seegene respiratory multiplex assays. The assays were evaluated using 199 mostly upper respiratory tract samples. A respiratory pathogen was found in 127/199 (63.8%) of samples by the FTD assay and 123/199 (61.8%) using the Seegene assay. Kappa agreement was between 0.87 and 1 for all targets except human bocavirus and adenovirus. Although the performance of the assays were similar, the Seegene assay had the advantage of simultaneous detection of two gene targets for each of the common Influenza A subtypes, improved throughput of 30 samples per run and automated result analysis. The FTD assay could only test 17 samples per run but validation for use on several different real-time thermal cyclers made it easier to integrate into an existing laboratory system. Both assays were cost effective compared to in-house multiplex PCR respiratory virus screening.

  7. Optimized cDNA libraries for virus-induced gene silencing (VIGS using tobacco rattle virus

    Directory of Open Access Journals (Sweden)

    Page Jonathan E

    2008-01-01

    Full Text Available Abstract Background Virus-induced gene silencing (VIGS has emerged as a method for performing rapid loss-of-function experiments in plants. Despite its expanding use, the effect of host gene insert length and other properties on silencing efficiency have not been systematically tested. In this study, we probed the optimal properties of cDNA fragments of the phytoene desaturase (PDS gene for efficient VIGS in Nicotiana benthamiana using tobacco rattle virus (TRV. Results NbPDS inserts of between 192 bp and 1304 bp led to efficient silencing as determined by analysis of leaf chlorophyll a levels. The region of the NbPDS cDNA used for silencing had a small effect on silencing efficiency with 5' and 3' located inserts performing more poorly than those from the middle. Silencing efficiency was reduced by the inclusion of a 24 bp poly(A or poly(G homopolymeric region. We developed a method for constructing cDNA libraries for use as a source of VIGS-ready constructs. Library construction involved the synthesis of cDNA on a solid phase support, digestion with RsaI to yield short cDNA fragments lacking poly(A tails and suppression subtractive hybridization to enrich for differentially expressed transcripts. We constructed two cDNA libraries from methyl-jasmonate treated N. benthamiana roots and obtained 2948 ESTs. Thirty percent of the cDNA inserts were 401–500 bp in length and 99.5% lacked poly(A tails. To test the efficiency of constructs derived from the VIGS-cDNA libraries, we silenced the nicotine biosynthetic enzyme, putrescine N-methyltransferase (PMT, with ten different VIGS-NbPMT constructs ranging from 122 bp to 517 bp. Leaf nicotine levels were reduced by more than 90% in all plants infected with the NbPMT constructs. Conclusion Based on the silencing of NbPDS and NbPMT, we suggest the following design guidelines for constructs in TRV vectors: (1 Insert lengths should be in the range of ~200 bp to ~1300 bp, (2 they should be positioned in

  8. Pathogenicity of Alternanthera mosaic virus is affected by determinants in RNA-dependent RNA polymerase and by reduced efficacy of silencing suppression in a movement-competent TGB1.

    Science.gov (United States)

    Lim, Hyoun-Sub; Vaira, Anna Maria; Reinsel, Michael D; Bae, Hanhong; Bailey, Bryan A; Domier, Leslie L; Hammond, John

    2010-01-01

    Four biologically active cDNA clones were derived from the Alternanthera mosaic virus (AltMV; genus Potexvirus) isolate, AltMV-SP, which differ in symptoms in infected Nicotiana benthamiana plants. Two clones induced necrosis and plant death; a mixture of all four clones induced milder symptoms than AltMV-SP. Replication of all clones was enhanced by a minimum of fourfold at 15 degrees C. A mixture of clones 4-7 (severe) and 3-1 (mild) was indistinguishable from AltMV-SP, but the ratio of 4-7 to 3-1 differed at 25 and 15 degrees C. RNA copy numbers of mixed infections were always below those of 4-7 alone. Determinants of symptom severity were identified in both Pol and TGB1; the mildest (4-1) and most severe (3-7) clones differed at three residues in the 'core' Pol domain [R(1110)P, K(1121)R, R(1255)K] and one [S(1535)P] in the C-terminal Pol domain of RNA-dependent RNA polymerase, and one in TGB1 [P(88)L]. Pol [P(1110),R(1121),K(1255)]+TGB1(L(88))] always induced systemic necrosis at 15 degrees C. Gene exchanges of Pol and TGB1 each affected replication and symptom expression, with TGB1(P(88)) significantly reducing silencing suppression. The difference in silencing suppression between TGB1(P(88)) and TGB1(L(88)) was confirmed by an agroinfiltration assay. Further, co-expression of TGB1(P(88)) and TGB1(L(88)) resulted in interference in the suppression of silencing by TGB1(L(88)). Yeast two-hybrid analysis confirmed that TGB1(P(88)) and TGB1(L(88)) interact. These results identify a TGB1 residue that significantly affects replication and silencing suppression, but maintains full movement functions.

  9. Mutations in the S gene region of hepatitis B virus genotype D in ...

    Indian Academy of Sciences (India)

    The gene region of the hepatitis B virus (HBV) is responsible for the expression of surface antigens and includes the 'a'-determinant region. Thus, mutation(s) in this region would afford HBV variants a distinct survival advantage, permitting the mutant virus to escape from the immune system. The aim of this study was to ...

  10. Identification and phylogeny of a protein kinase gene of white spot syndrome virus

    NARCIS (Netherlands)

    Hulten, van M.C.W.; Vlak, J.M.

    2001-01-01

    White spot syndrome virus (WSSV) is a virus infecting shrimp and other crustaceans, which is unclassified taxonomically. A 2193 bp long open reading frame, encoding a putative protein kinase (PK), was found on a 8.4 kb EcoRI fragment of WSSV proximal to the gene for the major envelope protein

  11. Horizontal gene transfer events reshape the global landscape of arm race between viruses and homo sapiens

    Science.gov (United States)

    Chen, Dong-Sheng; Wu, Yi-Quan; Zhang, Wei; Jiang, San-Jie; Chen, Shan-Ze

    2016-01-01

    Horizontal gene transfer (HGT) drives the evolution of recipient organism particularly if it provides a novel function which enhances the fitness or its adaption to the environment. Virus-host co-evolution is attractive for studying co-evolutionary processes, since viruses strictly replicate inside of the host cells and thus their evolution is inexorably tangled with host biology. HGT, as a mechanism of co-evolution between human and viruses, has been widely documented, however, the roles HGT play during the interaction between human and viruses are still in their infancy. In this study, we performed a comprehensive analysis on the genes horizontally transferred between viruses and their corresponding human hosts. Our study suggests that the HGT genes in human are predominantly enriched in immune related GO terms while viral HGT genes are tend to be encoded by viruses which promote the invasion of immune system of hosts. Based on our results, it gives us a hint about the evolution trajectory of HGT events. Overall, our study suggests that the HGT between human and viruses are highly relevant to immune interaction and probably reshaped the arm race between hosts and viruses. PMID:27270140

  12. Oncolysis of canine tumor cells by myxoma virus lacking the serp2 gene.

    Science.gov (United States)

    Urbasic, Ashlee S; Hynes, Stacy; Somrak, Amy; Contakos, Stacey; Rahman, Masmudur M; Liu, Jia; MacNeill, Amy L

    2012-08-01

    To determine the oncolytic efficacy of an attenuated form of myxoma virus lacking the serp2 gene in canine tumor cells. Primary cells were isolated from tumors that were surgically removed from dogs and from connective tissue obtained from the cadaver of a dog. Cells of various established cell lines from tumors and nontumorous tissues were obtained. Experiments were performed with cells in monolayer culture. Cell cultures were inoculated with wild-type myxoma viruses or myxoma viruses lacking the serp2 gene, and measures of cytopathic effects, viral growth kinetics, and cell death and apoptosis were determined. Myxoma viruses replicated in cells of many of the primary and established canine tumor cell lines. Canine tumor cells in which expression of activated protein kinase B was upregulated were more permissive to myxoma virus infection than were cells in which expression of activated protein kinase B was not upregulated. Myxoma viruses lacking the serp2 gene caused more cytopathic effects in canine tumor cells because of apoptosis than did wild-type myxoma viruses. Results of the present study indicated myxoma viruses lacking the serp2 gene may be useful for treatment of cancer in dogs. Impact for Human Medicine-Results of the present study may be useful for development of novel oncolytic treatments for tumors in humans.

  13. Tobacco Rattle Virus-Based Silencing of Enoyl-CoA Reductase Gene and Its Role in Resistance Against Cotton Wilt Disease.

    Science.gov (United States)

    Mustafa, Roma; Hamza, Muhammad; Kamal, Hira; Mansoor, Shahid; Scheffler, Jodi; Amin, Imran

    2017-07-01

    A Tobacco rattle virus (TRV)-based virus-induced gene silencing assay was employed as a reverse genetic approach to study gene function in cotton (Gossypium hirsutum). This approach was used to investigate the function of the Enoyl-CoA reductase (GhECR) gene in pathogen defense. Amino acid sequence alignment of Arabidopsis ECR with homologous sequence from G. hirsutum, G. arboreum, G. herbaceum and G. barbadense showed that ECRs are highly conserved among these species. TRV-based silencing of GhECR gene in G. hirsutum induced a cell death/necrotic lesion-like phenotype. Reverse transcription polymerase chain reaction (RT-PCR) and real-time quantitative PCR showed reduced GhECR mRNA levels in TRV inoculated plants. Three isolates of Verticillium dahliae (V. dahliae) and Fusarium oxysporum f. sp. vasinfectum (FOV) were used to infect GhECR-silenced plants. Out of 6 races of 2 pathogens, down regulation of GhECR gene resulted in reduced resistance. This is the first report showing that cotton GhECR gene is involved in resistance to different strains of V. dahliae and FOV.

  14. Oxylipin biosynthesis genes positively regulate programmed cell death during compatible infections with the synergistic pair potato virus X-potato virus Y and Tomato spotted wilt virus.

    Science.gov (United States)

    García-Marcos, Alberto; Pacheco, Remedios; Manzano, Aranzazu; Aguilar, Emmanuel; Tenllado, Francisco

    2013-05-01

    One of the most severe symptoms caused by compatible plant-virus interactions is systemic necrosis, which shares common attributes with the hypersensitive response to incompatible pathogens. Although several studies have identified viral symptom determinants responsible for systemic necrosis, mechanistic models of how they contribute to necrosis in infected plants remain scarce. Here, we examined the involvement of different branches of the oxylipin biosynthesis pathway in the systemic necrosis response caused either by the synergistic interaction of Potato virus X with Potato virus Y (PVX-PVY) or by Tomato spotted wilt virus (TSWV) in Nicotiana benthamiana. Silencing either 9-lipoxygenase (LOX), 13-LOX, or α-dioxygenase-1 (α-DOX-1) attenuated the programmed cell death (PCD)-associated symptoms caused by infection with either PVX-PVY or TSWV. In contrast, silencing of the jasmonic acid perception gene, COI1 (Coronatine insensitive 1), expedited cell death during infection with compatible viruses. This correlated with an enhanced expression of oxylipin biosynthesis genes and dioxygenase activity in PVX-PVY-infected plants. Moreover, the Arabidopsis thaliana double lox1 α-dox-1 mutant became less susceptible to TSWV infection. We conclude that oxylipin metabolism is a critical component that positively regulates the process of PCD during compatible plant-virus interactions but does not play a role in restraining virus accumulation in planta.

  15. Validation of reference genes for gene expression studies in virus-infected Nicotiana benthamiana using quantitative real-time PCR.

    Directory of Open Access Journals (Sweden)

    Deshui Liu

    Full Text Available Nicotiana benthamiana is the most widely-used experimental host in plant virology. The recent release of the draft genome sequence for N. benthamiana consolidates its role as a model for plant-pathogen interactions. Quantitative real-time PCR (qPCR is commonly employed for quantitative gene expression analysis. For valid qPCR analysis, accurate normalisation of gene expression against an appropriate internal control is required. Yet there has been little systematic investigation of reference gene stability in N. benthamiana under conditions of viral infections. In this study, the expression profiles of 16 commonly used housekeeping genes (GAPDH, 18S, EF1α, SAMD, L23, UK, PP2A, APR, UBI3, SAND, ACT, TUB, GBP, F-BOX, PPR and TIP41 were determined in N. benthamiana and those with acceptable expression levels were further selected for transcript stability analysis by qPCR of complementary DNA prepared from N. benthamiana leaf tissue infected with one of five RNA plant viruses (Tobacco necrosis virus A, Beet black scorch virus, Beet necrotic yellow vein virus, Barley stripe mosaic virus and Potato virus X. Gene stability was analysed in parallel by three commonly-used dedicated algorithms: geNorm, NormFinder and BestKeeper. Statistical analysis revealed that the PP2A, F-BOX and L23 genes were the most stable overall, and that the combination of these three genes was sufficient for accurate normalisation. In addition, the suitability of PP2A, F-BOX and L23 as reference genes was illustrated by expression-level analysis of AGO2 and RdR6 in virus-infected N. benthamiana leaves. This is the first study to systematically examine and evaluate the stability of different reference genes in N. benthamiana. Our results not only provide researchers studying these viruses a shortlist of potential housekeeping genes to use as normalisers for qPCR experiments, but should also guide the selection of appropriate reference genes for gene expression studies of N

  16. Reverse transcription quantitative real-time polymerase chain reaction reference genes in the spared nerve injury model of neuropathic pain: validation and literature search.

    Science.gov (United States)

    Piller, Nicolas; Decosterd, Isabelle; Suter, Marc R

    2013-07-10

    The reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a widely used, highly sensitive laboratory technique to rapidly and easily detect, identify and quantify gene expression. Reliable RT-qPCR data necessitates accurate normalization with validated control genes (reference genes) whose expression is constant in all studied conditions. This stability has to be demonstrated.We performed a literature search for studies using quantitative or semi-quantitative PCR in the rat spared nerve injury (SNI) model of neuropathic pain to verify whether any reference genes had previously been validated. We then analyzed the stability over time of 7 commonly used reference genes in the nervous system - specifically in the spinal cord dorsal horn and the dorsal root ganglion (DRG). These were: Actin beta (Actb), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal proteins 18S (18S), L13a (RPL13a) and L29 (RPL29), hypoxanthine phosphoribosyltransferase 1 (HPRT1) and hydroxymethylbilane synthase (HMBS). We compared the candidate genes and established a stability ranking using the geNorm algorithm. Finally, we assessed the number of reference genes necessary for accurate normalization in this neuropathic pain model. We found GAPDH, HMBS, Actb, HPRT1 and 18S cited as reference genes in literature on studies using the SNI model. Only HPRT1 and 18S had been once previously demonstrated as stable in RT-qPCR arrays. All the genes tested in this study, using the geNorm algorithm, presented gene stability values (M-value) acceptable enough for them to qualify as potential reference genes in both DRG and spinal cord. Using the coefficient of variation, 18S failed the 50% cut-off with a value of 61% in the DRG. The two most stable genes in the dorsal horn were RPL29 and RPL13a; in the DRG they were HPRT1 and Actb. Using a 0.15 cut-off for pairwise variations we found that any pair of stable reference gene was sufficient for the normalization process

  17. Multi-gene detection and identification of mosquito-borne RNA viruses using an oligonucleotide microarray.

    Directory of Open Access Journals (Sweden)

    Nathan D Grubaugh

    Full Text Available BACKGROUND: Arthropod-borne viruses are important emerging pathogens world-wide. Viruses transmitted by mosquitoes, such as dengue, yellow fever, and Japanese encephalitis viruses, infect hundreds of millions of people and animals each year. Global surveillance of these viruses in mosquito vectors using molecular based assays is critical for prevention and control of the associated diseases. Here, we report an oligonucleotide DNA microarray design, termed ArboChip5.1, for multi-gene detection and identification of mosquito-borne RNA viruses from the genera Flavivirus (family Flaviviridae, Alphavirus (Togaviridae, Orthobunyavirus (Bunyaviridae, and Phlebovirus (Bunyaviridae. METHODOLOGY/PRINCIPAL FINDINGS: The assay utilizes targeted PCR amplification of three genes from each virus genus for electrochemical detection on a portable, field-tested microarray platform. Fifty-two viruses propagated in cell-culture were used to evaluate the specificity of the PCR primer sets and the ArboChip5.1 microarray capture probes. The microarray detected all of the tested viruses and differentiated between many closely related viruses such as members of the dengue, Japanese encephalitis, and Semliki Forest virus clades. Laboratory infected mosquitoes were used to simulate field samples and to determine the limits of detection. Additionally, we identified dengue virus type 3, Japanese encephalitis virus, Tembusu virus, Culex flavivirus, and a Quang Binh-like virus from mosquitoes collected in Thailand in 2011 and 2012. CONCLUSIONS/SIGNIFICANCE: We demonstrated that the described assay can be utilized in a comprehensive field surveillance program by the broad-range amplification and specific identification of arboviruses from infected mosquitoes. Furthermore, the microarray platform can be deployed in the field and viral RNA extraction to data analysis can occur in as little as 12 h. The information derived from the ArboChip5.1 microarray can help to establish

  18. A novel recombinant pseudorabies virus expressing parvovirus VP2 gene: Immunogenicity and protective efficacy in swine.

    Science.gov (United States)

    Chen, Yang; Guo, Wanzhu; Xu, Zhiwen; Yan, Qigui; Luo, Yan; Shi, Qian; Chen, Dishi; Zhu, Ling; Wang, Xiaoyu

    2011-06-16

    Porcine parvovirus (PPV) VP2 gene has been successfully expressed in many expression systems resulting in self-assembly of virus-like particles (VLPs) with similar morphology to the native capsid. Here, a pseudorabies virus (PRV) system was adopted to express the PPV VP2 gene. A recombinant PRV SA215/VP2 was obtained by homologous recombination between the vector PRV viral DNA and a transfer plasmid. Then recombinant virus was purified with plaque purification, and its identity confirmed by PCR amplification, Western blot and indirect immunofluorescence (IFA) analyses. Electronic microscopy of PRV SA215/VP2 confirmed self-assembly of both pseudorabies virus and VLPs from VP2 protein. Immunization of piglets with recombinant virus elicited PRV-specific and PPV-specific humoral immune responses and provided complete protection against a lethal dose of PRV challenges. Gilts immunized with recombinant viruses induced PPV-specific antibodies, and significantly reduced the mortality rate of (1 of 28) following virulent PPV challenge compared with the control (7 of 31). Furthermore, PPV virus DNA was not detected in the fetuses of recombinant virus immunized gilts. In this study, a recombinant PRV SA215/VP2 virus expressing PPV VP2 protein was constructed using PRV SA215 vector. The safety, immunogenicity, and protective efficacy of the recombinant virus were demonstrated in piglets and primiparous gilts. This recombinant PRV SA215/VP2 represents a suitable candidate for the development of a bivalent vaccine against both PRV and PPV infection.

  19. Determination of suitable housekeeping genes for normalisation of quantitative real time PCR analysis of cells infected with human immunodeficiency virus and herpes viruses

    Directory of Open Access Journals (Sweden)

    Wilkinson John

    2007-12-01

    Full Text Available Abstract The choice of an appropriate housekeeping gene for normalisation purposes has now become an essential requirement when designing QPCR experiments. This is of particular importance when using QPCR to measure viral and cellular gene transcription levels in the context of viral infections as viruses can significantly interfere with host cell pathways, the components of which traditional housekeeping genes often encode. In this study we have determined the reliability of 10 housekeeping genes in context of four heavily studied viral infections; human immunodeficiency virus type 1, herpes simplex virus type 1, cytomegalovirus and varicella zoster virus infections using a variety of cell types and virus strains. This provides researchers of these viruses with a shortlist of potential housekeeping genes to use as normalisers for QPCR experiments.

  20. Gene delivery systems: Bridging the gap between recombinant viruses and artificial vectors.

    Science.gov (United States)

    Navarro; Oudrhiri; Fabrega; Lehn

    1998-03-02

    Although most research in the field of somatic gene therapy has investigated the use of recombinant viruses for transferring genes into somatic target cells, various methods for nonviral gene delivery have also been proposed. Both types of gene delivery systems have advantages and drawbacks. Schematically, viral vectors are particularly efficient for gene delivery, whereas nonviral systems are free of the difficulties associated with the use of recombinant viruses but need to be further optimized to reach their full potential. In order to bridge the gap between viral vectors and synthetic reagents, we discuss here some specific features of the viral vector systems of today that could advantageously be taken into account for the design of improved nonviral gene delivery systems. Indeed, although nonviral systems differ fundamentally from viral systems, one possible approach towards enhanced artificial reagents aims at developing 'artificial viruses' that mimic the highly efficient processes of viral infection.

  1. Ebola Virus GP Gene Polyadenylation Versus RNA Editing.

    Science.gov (United States)

    Volchkova, Valentina A; Vorac, Jaroslav; Repiquet-Paire, Laurie; Lawrence, Philip; Volchkov, Viktor E

    2015-10-01

    Synthesis of Ebola virus (EBOV) surface glycoprotein (GP) is dependent on transcriptional RNA editing. Northern blot analysis of EBOV-infected cells using GP-gene-specific probes reveals that, in addition to full-length GP messenger RNAs (mRNAs), a shorter RNA is also synthesized, representing >40% of the total amount of GP mRNA. Sequence analysis demonstrates that this RNA is a truncated version of the full-length GP mRNA that is polyadenylated at the editing site and thus lacks a stop codon. An absence of detectable levels of protein synthesis in cellulo is consistent with the existence of tight regulation of the translation of such mRNA. However, nonstop GP mRNA was shown to be only slightly less stable than the same mRNA containing a stop codon, against the general belief in nonstop decay mechanisms aimed at detecting and destroying mRNAs lacking a stop codon. In conclusion, we demonstrate that the editing site indeed serves as a cryptic transcription termination/polyadenylation site, which rarely also functions to edit GP mRNA for expression of surface GP. This new data suggest that the downregulation of surface GP expression is even more dramatic than previously thought, reinforcing the importance of the GP gene editing site for EBOV replication and pathogenicity. © The Author 2015. 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.

  2. Overcoming the cystic fibrosis sputum barrier to leading adeno-associated virus gene therapy vectors

    National Research Council Canada - National Science Library

    Schuster, Benjamin S; Kim, Anthony J; Kays, Joshua C; Kanzawa, Mia M; Guggino, William B; Boyle, Michael P; Rowe, Steven M; Muzyczka, Nicholas; Suk, Jung Soo; Hanes, Justin

    2014-01-01

    .... We investigated whether CF sputum acts as a barrier to leading adeno-associated virus (AAV) gene vectors, including AAV2, the only serotype tested in CF clinical trials, and AAV1, a leading candidate for future trials...

  3. Widespread Horizontal Gene Transfer from Circular Single-stranded DNA Viruses to Eukaryotic Genomes

    Directory of Open Access Journals (Sweden)

    Xie Jiatao

    2011-09-01

    Full Text Available Abstract Background In addition to vertical transmission, organisms can also acquire genes from other distantly related species or from their extra-chromosomal elements (plasmids and viruses via horizontal gene transfer (HGT. It has been suggested that phages represent substantial forces in prokaryotic evolution. In eukaryotes, retroviruses, which can integrate into host genome as an obligate step in their replication strategy, comprise approximately 8% of the human genome. Unlike retroviruses, few members of other virus families are known to transfer genes to host genomes. Results Here we performed a systematic search for sequences related to circular single-stranded DNA (ssDNA viruses in publicly available eukaryotic genome databases followed by comprehensive phylogenetic analysis. We conclude that the replication initiation protein (Rep-related sequences of geminiviruses, nanoviruses and circoviruses have been frequently transferred to a broad range of eukaryotic species, including plants, fungi, animals and protists. Some of the transferred viral genes were conserved and expressed, suggesting that these genes have been coopted to assume cellular functions in the host genomes. We also identified geminivirus-like and parvovirus-like transposable elements in genomes of fungi and lower animals, respectively, and thereby provide direct evidence that eukaryotic transposons could derive from ssDNA viruses. Conclusions Our discovery extends the host range of circular ssDNA viruses and sheds light on the origin and evolution of these viruses. It also suggests that ssDNA viruses act as an unforeseen source of genetic innovation in their hosts.

  4. Adeno-associated virus-mediated gene transfer.

    Science.gov (United States)

    Srivastava, Arun

    2008-09-01

    Although the remarkable versatility and efficacy of recombinant adeno-associated virus 2 (AAV2) vectors in transducing a wide variety of cells and tissues in vitro, and in numerous pre-clinical animal models of human diseases in vivo, have been well established, the published literature is replete with controversies with regard to the efficacy of AAV2 vectors in hematopoietic stem cell (HSC) transduction. A number of factors have contributed to these controversies, the molecular bases of which have begun to come to light in recent years. With the availability of several novel serotypes (AAV1 through AAV12), rational design of AAV capsid mutants, and strategies (self-complementary vector genomes, hematopoietic cell-specific promoters), it is indeed becoming feasible to achieve efficient transduction of HSC by AAV vectors. Using a murine serial bone marrow transplantation model in vivo, we have recently documented stable integration of the proviral AAV genome into mouse chromosomes, which does not lead to any overt hematological abnormalities. Thus, a better understanding of the AAV-HSC interactions, and the availability of a vast repertoire of novel serotype and capsid mutant vectors, are likely to have significant implications in the use of AAV vectors in high-efficiency transduction of HSCs as well as in gene therapy applications involving the hematopoietic system. (c) 2008 Wiley-Liss, Inc.

  5. Expression of human poly (ADP-ribose) polymerase 1 in Saccharomyces cerevisiae: Effect on survival, homologous recombination and identification of genes involved in intracellular localization

    Energy Technology Data Exchange (ETDEWEB)

    La Ferla, Marco; Mercatanti, Alberto; Rocchi, Giulia; Lodovichi, Samuele; Cervelli, Tiziana; Pignata, Luca [Yeast Genetics and Genomics, Institute of Clinical Physiology, National Council of Research (CNR), via Moruzzi 1, 56122 Pisa (Italy); Caligo, Maria Adelaide [Section of Genetic Oncology, University Hospital and University of Pisa, via Roma 57, 56125 Pisa (Italy); Galli, Alvaro, E-mail: alvaro.galli@ifc.cnr.it [Yeast Genetics and Genomics, Institute of Clinical Physiology, National Council of Research (CNR), via Moruzzi 1, 56122 Pisa (Italy)

    2015-04-15

    Highlights: • The human poly (ADP-ribose) polymerase 1 (PARP-1) gene affects growth and UV-induced homologous recombination in yeast. • PARP-1 chemical inhibition impacts yeast growth and UV-induced recombination. • A genome-wide screen identifies 99 yeast genes that suppress the growth defect inferred by PARP-1. • Bioinformatics analysis identifies 41 human orthologues that may have a role in PARP-1 intracellular localization. • The findings suggest that PARP-1 nuclear localization may affect the response to PARP inhibitors in cancer therapy. - Abstract: The poly (ADP-ribose) polymerase 1 (PARP-1) actively participates in a series of functions within the cell that include: mitosis, intracellular signaling, cell cycle regulation, transcription and DNA damage repair. Therefore, inhibition of PARP1 has a great potential for use in cancer therapy. As resistance to PARP inhibitors is starting to be observed in patients, thus the function of PARP-1 needs to be studied in depth in order to find new therapeutic targets. To gain more information on the PARP-1 activity, we expressed PARP-1 in yeast and investigated its effect on cell growth and UV induced homologous recombination. To identify candidate genes affecting PARP-1 activity and cellular localization, we also developed a yeast genome wide genetic screen. We found that PARP-1 strongly inhibited yeast growth, but when yeast was exposed to the PARP-1 inhibitor 6(5-H) phenantridinone (PHE), it recovered from the growth suppression. Moreover, we showed that PARP-1 produced PAR products in yeast and we demonstrated that PARP-1 reduced UV-induced homologous recombination. By genome wide screening, we identified 99 mutants that suppressed PARP-1 growth inhibition. Orthologues of human genes were found for 41 of these yeast genes. We determined whether the PARP-1 protein level was altered in strains which are deleted for the transcription regulator GAL3, the histone H1 gene HHO1, the HUL4 gene, the

  6. Temperature-Sensitive Mutants in the Influenza A Virus RNA Polymerase: Alterations in the PA Linker Reduce Nuclear Targeting of the PB1-PA Dimer and Result in Viral Attenuation.

    Science.gov (United States)

    Da Costa, Bruno; Sausset, Alix; Munier, Sandie; Ghounaris, Alexandre; Naffakh, Nadia; Le Goffic, Ronan; Delmas, Bernard

    2015-06-01

    The influenza virus RNA-dependent RNA polymerase catalyzes genome replication and transcription within the cell nucleus. Efficient nuclear import and assembly of the polymerase subunits PB1, PB2, and PA are critical steps in the virus life cycle. We investigated the structure and function of the PA linker (residues 197 to 256), located between its N-terminal endonuclease domain and its C-terminal structured domain that binds PB1, the polymerase core. Circular dichroism experiments revealed that the PA linker by itself is structurally disordered. A large series of PA linker mutants exhibited a temperature-sensitive (ts) phenotype (reduced viral growth at 39.5°C versus 37°C/33°C), suggesting an alteration of folding kinetic parameters. The ts phenotype was associated with a reduced efficiency of replication/transcription of a pseudoviral reporter RNA in a minireplicon assay. Using a fluorescent-tagged PB1, we observed that ts and lethal PA mutants did not efficiently recruit PB1 to reach the nucleus at 39.5°C. A protein complementation assay using PA mutants, PB1, and β-importin IPO5 tagged with fragments of the Gaussia princeps luciferase showed that increasing the temperature negatively modulated the PA-PB1 and the PA-PB1-IPO5 interactions or complex stability. The selection of revertant viruses allowed the identification of different types of compensatory mutations located in one or the other of the three polymerase subunits. Two ts mutants were shown to be attenuated and able to induce antibodies in mice. Taken together, our results identify a PA domain critical for PB1-PA nuclear import and that is a "hot spot" to engineer ts mutants that could be used to design novel attenuated vaccines. By targeting a discrete domain of the PA polymerase subunit of influenza virus, we were able to identify a series of 9 amino acid positions that are appropriate to engineer temperature-sensitive (ts) mutants. This is the first time that a large number of ts mutations were

  7. Identification of Suitable Reference Genes for Normalization of Real-Time Quantitative Polymerase Chain Reaction in an Intestinal Graft-Versus-Host Disease Mouse Model.

    Science.gov (United States)

    Li, X; Qiao, J; Yang, N; Mi, H; Chu, P; Xia, Y; Yao, H; Liu, Y; Qi, K; Yan, Z; Zeng, L; Xu, K

    2015-01-01

    With the development of real-time quantitative polymerase chain reaction (RT-qPCR) and intensive research on acute graft-versus-host disease (GVHD), selecting the best reference gene for normalization of RT-qPCR analysis in a GVHD model becomes more and more important. In this study, we aimed to identify suitable reference genes for mRNA studies in an intestinal GVHD mouse model after bone marrow transplantation (BMT). BALB/c recipients received 7.5 Gy total body irradiation (TBI) followed by injection of 5 × 10(6) bone marrow cells, without infusion of spleen cells for BMT, with infusion of 5 × 10(5) or 2.5 × 10(6) spleen cells for mild or moderate GVHD, respectively. Healthy mice were chosen as normal control subjects. Duodenum, jejunum, ileum, colon, and small intestine were collected at days 7, 14, 21, and 28 after transplantation. Transcription levels of 9 candidate genes, B2M, SDHA, HPRT, ACTB, GAPDH, HMBS, TBP, YWHAZ, and RPLP0, in each tissue were measured with the use of RT-qPCR. Combined data from these tissues in each group were defined as all samples. The expression stability of these genes was analyzed with the use of Genorm, Normfinder, Bestkeeper, and ΔCt. Our results showed that in all samples, ACTB and HMBS displayed the highest and lowest expression levels, respectively. Genorm identified HRPT and SDHA as the most stable reference genes, whereas Normfinder and ΔCt method showed HPRT as the most stably expressed gene. Bestkeeper ranked YWHAZ and HPRT as the top 2 most suitable genes. In conclusion, HPRT was recommended as the most suitable reference gene after comprehensive ranking, suggesting that it could be used as an internal control for mRNA studies in intestinal GVHD after BMT. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. holE, the gene coding for the theta subunit of DNA polymerase III of Escherichia coli: characterization of a holE mutant and comparison with a dnaQ (epsilon-subunit) mutant.

    OpenAIRE

    Slater, S C; Lifsics, M R; O'Donnell, M; Maurer, R

    1994-01-01

    DNA polymerase III holoenzyme is a multiprotein complex responsible for the bulk of chromosomal replication in Escherichia coli and Salmonella typhimurium. The catalytic core of the holoenzyme is an alpha epsilon theta heterotrimer that incorporates both a polymerase subunit (alpha; dnaE) and a proofreading subunit (epsilon; dnaQ). The role of theta is unknown. Here, we describe a null mutation of holE, the gene for theta. A strain carrying this mutation was fully viable and displayed no muta...

  9. Real time reverse transcription (RRT)-polymerase chain reaction (PCR) methods for detection of pandemic (H1N1) 2009 influenza virus and European swine influenza A virus infections in pigs.

    Science.gov (United States)

    Slomka, Marek J; Densham, Anstice L E; Coward, Vivien J; Essen, Steve; Brookes, Sharon M; Irvine, Richard M; Spackman, Erica; Ridgeon, Jonathan; Gardner, Rebecca; Hanna, Amanda; Suarez, David L; Brown, Ian H

    2010-09-01

    There is a requirement to detect and differentiate pandemic (H1N1) 2009 (H1N1v) and established swine influenza A viruses (SIVs) by real time reverse transcription (RRT) PCR methods. First, modify an existing matrix (M) gene RRT PCR for sensitive generic detection of H1N1v and other European SIVs. Second, design an H1 RRT PCR to specifically detect H1N1v infections. RRT PCR assays were used to test laboratory isolates of SIV (n = 51; 37 European and 14 North American), H1N1v (n = 5) and avian influenza virus (AIV; n = 43). Diagnostic sensitivity and specificity were calculated for swabs (n = 133) and tissues (n = 116) collected from field cases and pigs infected experimentally with SIVs and H1N1v. The "perfect match" M gene RRT PCR was the most sensitive variant of this test for detection of established European SIVs and H1N1v. H1 RRT PCR specifically detected H1N1v but not European SIVs. Validation with clinical specimens included comparison with virus isolation (VI) as a "gold standard", while field infection with H1N1v in swine was independently confirmed by sequencing H1N1v amplified by conventional RT PCR. "Perfect match" M gene RRT PCR had 100% sensitivity and 95.2% specificity for swabs, 93.6% and 98.6% for tissues. H1 RRT PCR demonstrated sensitivity and specificity of 100% and 99.1%, respectively, for the swabs, and 100% and 100% for the tissues. Two RRT PCRs for the purposes of (i) generic detection of SIV and H1N1v infection in European pigs, and for (ii) specific detection of H1N1v (pandemic influenza) infection were validated.

  10. Polymerase chain reaction genotyping of Epstein-Barr virus in scraping samples of the tongue lateral border in HIV-1 seropositive patients

    Directory of Open Access Journals (Sweden)

    TF Robaina

    2008-06-01

    Full Text Available The Epstein-Barr virus (EBV is the etiological agent of oral hairy leukoplakia (OHL, an oral lesion with important diagnostic and prognostic value in acquired immunodeficiency disease syndrome. The two EBV genotypes, EBV-1 and EBV-2, can be distinguished by divergent gene sequences encoding the EBNA-2, 3A, 3B, and 3C proteins. The purpose of this study was to identify the EBV genotype prevalent in 53 samples of scrapings from the lateral border of the tongue of HIV-1 seropositive patients, with and without OHL, and to correlate the genotypes with presence of clinical or subclinical OHL with the clinic data collected. EBV-1 and EBV-2 were identified through PCR and Nested-PCR based on sequence differences of the EBNA-2 gene. EBV-1 was identified in the 31 samples (15 without OHL, 7 with clinical OHL and 9 with subclinical OHL, EBV-2 in 12 samples (10 without OHL, 1 with clinical and 1 subclinical OHL, and a mixed infection in 10 samples (2 without OHL, 3 with clinical and 5 with subclinical OHL. The presence of EBV-1 was higher in women, but a significant statistical result relating one the EBV genotypes to the development of OHL was not found. We conclude that the oral epithelium in HIV-1 seropositive patients can be infected by EBV-1, EBV-2 or by a mixed viral population.

  11. Association between chronic hepatitis B virus infection and HLA-DP gene polymorphisms in the Turkish population.

    Science.gov (United States)

    Akgöllü, Ersin; Bilgin, Ramazan; Akkız, Hikmet; Ülger, Yakup; Kaya, Berrin Yalınbaş; Karaoğullarından, Ümit; Arslan, Yusuf Kemal

    2017-03-15

    Hepatitis B virus (HBV) affects approximately 360 million people worldwide. 10-15% of patients with chronic HBV develop liver cirrhosis (LC), liver failure and hepatocellular carcinoma (HCC). Chronic HBV infection or HBV clearance is influenced by both viral and host factors. In genome-wide association studies (GWAS), the human leukocyte antigen (HLA) gene polymorphisms rs3077 and rs9277535 were identified to be associated with chronic hepatitis B. HLA genes have been linked to immune response to infectious agents. Genetic variants in HLA genes influence HLA mRNA expression which might also affect antigen presentation. We evaluated the association between HLA gene polymorphisms and the risk for persistent HBV infection. In the current study, HLA gene polymorphisms were investigated in a case-control study of 294 chronic HBV patients and 234 persons with HBV natural clearance by using a real-time polymerase chain reaction (RT-PCR). The results showed that rs9277535 allele frequency is associated with HBV infection in the Turkish subjects examined (P=0.048). However, no association was found for rs3077. Additionally, the AG haplotype block showed a protective effect against the risk of persistent HBV infection (for the rs3077A/rs9277535G, OR=0.52; 95% 0.34-0.80, P=0.003). Our results, for the first time, demonstrate that HLA-DPB1 gene rs9277535A allele has a major effect on the risk of persistent HBV infection. We suggest that further independent studies are necessary to clarify the association of these polymorphisms with persistence or natural clearance of HBV infection in Caucasian populations. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Artificial virus as trump-card to resolve exigencies in targeted gene delivery.

    Science.gov (United States)

    Ajithkumar, K C; Pramod, K

    2017-05-28

    Viruses are potent pathogens that can effectively deliver the genetic material to susceptible host cells. This capability is beneficially utilized for successfully deliver the genetic material. However, the therapeutic use of viruses for gene delivery is controversial because, the potentially replicable genomes recombine or integrates with the cell DNA resulted with immunogenicity, ranging from inflammation to death. Thus, the need for potentially effective nonviral gene therapy vehicles arises and the non-viral vectors, protein only particles and virus like particles (VLP) can be engineered which contain all the required functional modules in single chain molecules. These are resembles to viruses called artificial virus. The artificial virus eliminates the disadvantages of the viral vectors but retain the beneficial effects of the viruses. Need for further functionalization can be avoided by this approach because independent elements such as cell ligands, membrane active peptides, etc can be incorporated in to the protein. The protein-DNA complexes appeared as tight and monodisperse spherical nanoparticles of nearly 80 nm size resemble bacterial inclusion bodies. The nucleic acids act as compacting, molecular glue that affects the conformation of the protein units, altering the α- helix structure, minimizing their aggregation tendency and promoting receptor mediated cell uptake and proper intracellular trafficking to the cell nucleus. Such tunable system mimics the activities of infected viruses and used for the safe and effective delivery of drugs and genetic material in gene therapy. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  13. Isolation and characterization of the genes for two small RNAs of herpesvirus papio and their comparison with Epstein-Barr virus-encoded EBER RNAs.

    Science.gov (United States)

    Howe, J G; Shu, M D

    1988-08-01

    Genes for the Epstein-Barr virus-encoded RNAs (EBERs), two low-molecular-weight RNAs encoded by the human gammaherpesvirus Epstein-Barr virus (EBV), hybridize to two small RNAs in a baboon cell line that contains a similar virus, herpesvirus papio (HVP). The genes for the HVP RNAs (HVP-1 and HVP-2) are located together in the small unique region at the left end of the viral genome and are transcribed by RNA polymerase III in a rightward direction, similar to the EBERs. There is significant similarity between EBER1 and HVP-1 RNA, except for an insert of 22 nucleotides which increases the length of HVP-1 RNA to 190 nucleotides. There is less similarity between the sequences of EBER2 and HVP-2 RNA, but both have a length of about 170 nucleotides. The predicted secondary structure of each HVP RNA is remarkably similar to that of the respective EBER, implying that the secondary structures are important for function. Upstream from the initiation sites of all four RNA genes are several highly conserved sequences which may function in the regulation of transcription. The HVP RNAs, together with the EBERs, are highly abundant in transformed cells and are efficiently bound by the cellular La protein.

  14. Association of TNF-Alpha gene polymorphisms and susceptibility to hepatitis B virus infection in Egyptians.

    Science.gov (United States)

    Talaat, Roba M; Abdelkhalek, Mohamed S; El-Maadawy, Eman A; Abdel-Mageed, Wael S; El-Shenawy, Soha Z; Osman, Mohamed A

    2017-11-01

    Tumor necrosis factor alpha (TNF-α) is one of the important cytokine in generating an immune response against hepatitis B virus (HBV). Genetic polymorphisms might influence gene transcription, leading to disturbance in cytokine production. We hypothesized that single nucleotide polymorphism (SNPs) in TNF-α gene could affect the pathogenesis of HBV. To test this hypothesis, we investigated the role of TNF-α polymorphism [-863C/A (rs1800630), -308G/A (rs1800629), -376G/A (rs1800750), -857C/T (rs1799724) and +489G/A (rs1800610)] in the susceptibility to chronic hepatitis B (CHB) infection. Polymorphisms of the TNF-α (-863C/A (rs1800630), -308G/A) were analyzed by Polymerase chain reaction sequence specific primer (PCR-SSP) while TNF-α (-376G/A, -857C/T and +489G/A) by PCR-restriction fragment length polymorphism (PCR-RFLP) in 104 patients with CHB and 104 healthy controls. The plasma level of TNF-α was measured using Enzyme-linked immunosorbent assay (ELISA). The study showed a significant increase in the frequency of -863CC, -376GA, -857CC, -857TT and +489GA genotypes and -863C, -376A, -857C, and +489A alleles in CHB patients compared to controls. In addition, CAGCG haplotype had a highest frequency in CHB patients. A strong Linkage Disequilibrium (LD) between TNF-α -863C/A (rs1800630) and -376G/A (D' = 0.7888, r 2  = 0.0200); -308G/A and -857C/T (D' = 0.9213, r 2  = 0.1770); -308G/A and +489G/A (D' = 0.9088, r 2  = 0.1576) was demonstrated. CHB patients had significantly lower levels of TNF-α compared to controls. In conclusion, our preliminary results suggest that -863C/A (rs1800630), -308G/A, -376G/A, and +489G/A of the TNF-α gene may play a role in HBV susceptibility in Egyptians. The significant reduction in TNF-α in CHB patient was independent of any particular genotype/haplotype in TNF-α. Copyright © 2017 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

  15. Glycoprotein is enough for sindbis virus-derived DNA vector to express heterogenous genes

    Directory of Open Access Journals (Sweden)

    Fu Juanjuan

    2011-07-01

    Full Text Available Abstract To investigate the necessity and potential application of structural genes for expressing heterogenous genes from Sindbis virus-derived vector, the DNA-based expression vector pVaXJ was constructed by placing the recombinant genome of sindbis-like virus XJ-160 under the control of the human cytomegalovirus (CMV promoter of the plasmid pVAX1, in which viral structural genes were replaced by a polylinker cassette to allow for insertion of heterologous genes. The defect helper plasmids pVaE or pVaC were developed by cloning the gene of glycoprotein E3E26KE1 or capsid protein of XJ-160 virus into pVAX1, respectively. The report gene cassette pVaXJ-EGFP or pV-Gluc expressing enhanced green fluorescence protein (EGFP or Gaussia luciferase (G.luc were constructed by cloning EGFP or G.luc gene into pVaXJ. EGFP or G.luc was expressed in the BHK-21 cells co-transfected with report gene cassettes and pVaE at levels that were comparable to those produced by report gene cassettes, pVaC and pVaE and were much higher than the levels produced by report gene cassette and pVaC, suggesting that glycoprotein is enough for Sindbis virus-derived DNA vector to express heterogenous genes in host cells. The method of gene expression from Sindbis virus-based DNA vector only co-transfected with envelop E gene increase the conveniency and the utility of alphavirus-based vector systems in general.

  16. Differential transactivation by orphan nuclear receptor NOR1 and its fusion gene product EWS/NOR1: possible involvement of poly(ADP-ribose) polymerase I, PARP-1.

    Science.gov (United States)

    Ohkura, Naganari; Nagamura, Yuko; Tsukada, Toshihiko

    2008-10-15

    In extraskeletal myxoid chondrosarcoma, a chromosomal translocation creates a gene fusion between EWS and an orphan nuclear receptor, NOR1. The resulting fusion protein EWS/NOR1 has been believed to lead to malignant transformation by functioning as a transactivator for NOR1-target genes. By comparing the gene expression profiles of NOR1- and EWS/NOR1-overexpressing cells, we found that they largely shared up-regulated genes, but no significant correlation was observed with respect to the transactivation levels of each gene. In addition, the proteins associated with NOR1 and EWS/NOR1 were mostly the same in these cells. The results suggest that these proteins differentially transactivate overlapping target genes through a similar transcriptional machinery. To clarify the mechanisms underlying the transcriptional divergence between NOR1 and EWS/NOR1, we searched for alternatively associated proteins, and identified poly(ADP-ribose) polymerase I (PARP-1) as an NOR1-specific binding protein. Consistent with its binding properties, PARP-1 acted as a transcriptional repressor of NOR1, but not EWS/NOR1, in a luciferase reporter assay employing PARP-1(-/-) fibroblasts. Interestingly, suppressive activity of PARP-1 was observed in a DNA response element-specific manner, and in a subtype-specific manner toward the NR4A family (Nur77, Nurr1, and NOR1), suggesting that PARP-1 plays a role in the diversity of transcriptional regulation mediated by the NR4A family in normal cells. Altogether, our findings suggest that NOR1 and EWS/NOR1 regulate overlapping target genes differently by utilizing associated proteins, including PARP-1; and that EWS/NOR1 may acquire oncogenic activities by avoiding (or gaining) transcription factor-specific modulation by the associated proteins. (c) 2008 Wiley-Liss, Inc.

  17. 40 CFR 174.513 - Potato Leaf Roll Virus Resistance Gene (also known as orf1/orf2 gene); exemption from the...

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Potato Leaf Roll Virus</