Broadly-Reactive Neutralizing and Non-neutralizing Antibodies Directed against the H7 Influenza Virus Hemagglutinin Reveal Divergent Mechanisms of Protection.

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Gene S Tan

2016-04-01

Full Text Available In the early spring of 2013, Chinese health authorities reported several cases of H7N9 influenza virus infections in humans. Since then the virus has established itself at the human-animal interface in Eastern China and continues to cause several hundred infections annually. In order to characterize the antibody response to the H7N9 virus we generated several mouse monoclonal antibodies against the hemagglutinin of the A/Shanghai/1/13 (H7N9 virus. Of particular note are two monoclonal antibodies, 1B2 and 1H5, that show broad reactivity to divergent H7 hemagglutinins. Monoclonal antibody 1B2 binds to viruses of the Eurasian and North American H7 lineages and monoclonal antibody 1H5 reacts broadly to virus isolates of the Eurasian lineage. Interestingly, 1B2 shows broad hemagglutination inhibiting and neutralizing activity, while 1H5 fails to inhibit hemagglutination and demonstrates no neutralizing activity in vitro. However, both monoclonal antibodies were highly protective in an in vivo passive transfer challenge model in mice, even at low doses. Experiments using mutant antibodies that lack the ability for Fc/Fc-receptor and Fc/complement interactions suggest that the protection provided by mAb 1H5 is, at least in part, mediated by the Fc-fragment of the mAb. These findings highlight that a protective response to a pathogen may not only be due to neutralizing antibodies, but can also be the result of highly efficacious non-neutralizing antibodies not readily detected by classical in vitro neutralization or hemagglutination inhibition assays. This is of interest because H7 influenza virus vaccines induce only low hemagglutination inhibiting antibody titers while eliciting robust antibody titers as measured by ELISA. Our data suggest that these binding but non-neutralizing antibodies contribute to protection in vivo.

Crystallization and preliminary crystallographic analysis of the measles virus hemagglutinin in complex with the CD46 receptor

International Nuclear Information System (INIS)

Santiago, César; Gutiérrez-Rodríguez, Angel; Tucker, Paul A.; Stehle, Thilo; Casasnovas, José M.

2009-01-01

A complex of the measles virus hemagglutinin and the CD46 receptor representing the initial step of the cell infection has been crystallized. The measles virus (MV) hemagglutinin (MV-H) mediates the attachment of MV particles to cell-surface receptors for entry into host cells. MV uses two receptors for attachment to host cells: the complement-control protein CD46 and the signalling lymphocyte activation molecule (SLAM). The MV-H glycoprotein from an Edmonston MV variant and the MV-binding fragment of the CD46 receptor were overproduced in mammalian cells and used to crystallize an MV-H–CD46 complex. Well diffracting crystals containing two complexes in the asymmetric unit were obtained and the structure of the complex was solved by the molecular-replacement method

Examining the hemagglutinin subtype diversity among wild duck-origin influenza A viruses using ethanol-fixed cloacal swabs and a novel RT-PCR method.

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Wang, Ruixue; Soll, Lindsey; Dugan, Vivien; Runstadler, Jonathan; Happ, George; Slemons, Richard D; Taubenberger, Jeffery K

2008-05-25

This study presents an interconnected approach for circumventing two inherent limitations associated with studies defining the natural history of influenza A viruses in wild birds. The first limiting factor is the ability to maintain a cold chain from specimen collection to the laboratory when study sites are in more remote locations. The second limiting factor is the ability to identify all influenza A virus HA subtypes present in an original sample. We report a novel method for molecular subtyping of avian influenza A virus hemagglutinin genes using degenerate primers designed to amplify all known hemagglutinin subtypes. It was shown previously that templates larger than 200 bp were not consistently amplifiable from ethanol-fixed cloacal swabs. For this study, new primer sets were designed within these constraints. This method was used to perform subtyping RT-PCR on 191 influenza RNA-positive ethanol-fixed cloacal swabs obtained from 880 wild ducks in central Alaska in 2005. Seven different co-circulating hemagglutinin subtypes were identified in this study set, including H1, H3, H4, H5, H6, H8, and H12. In addition, 16% of original cloacal samples showed evidence of mixed infection, with samples yielding from two-to-five different hemagglutinin subtypes. This study further demonstrates the complex ecobiology of avian influenza A viruses in wild birds.

Protection of chickens against H5N1 highly pathogenic avian influenza virus infection by live vaccination with infectious laryngotracheitis virus recombinants expressing H5 hemagglutinin and N1 neuraminidase.

Science.gov (United States)

Pavlova, Sophia P; Veits, Jutta; Keil, Günther M; Mettenleiter, Thomas C; Fuchs, Walter

2009-01-29

Attenuated vaccine strains of the alphaherpesvirus causing infectious laryngotracheitis of chickens (ILTV, gallid herpesvirus 1) can be used for mass application. Previously, we showed that live virus vaccination with recombinant ILTV expressing hemagglutinin of highly pathogenic avian influenza viruses (HPAIV) protected chickens against ILT and fowl plague caused by HPAIV carrying the corresponding hemagglutinin subtypes [Lüschow D, Werner O, Mettenleiter TC, Fuchs W. Protection of chickens from lethal avian influenza A virus infection by live-virus vaccination with infectious laryngotracheitis virus recombinants expressing the hemagglutinin (H5) gene. Vaccine 2001;19(30):4249-59; Veits J, Lüschow D, Kindermann K, Werner O, Teifke JP, Mettenleiter TC, et al. Deletion of the non-essential UL0 gene of infectious laryngotracheitis (ILT) virus leads to attenuation in chickens, and UL0 mutants expressing influenza virus haemagglutinin (H7) protect against ILT and fowl plague. J Gen Virol 2003;84(12):3343-52]. However, protection against H5N1 HPAIV was not satisfactory. Therefore, a newly designed dUTPase-negative ILTV vector was used for rapid insertion of the H5-hemagglutinin, or N1-neuraminidase genes of a recent H5N1 HPAIV isolate. Compared to our previous constructs, protein expression was considerably enhanced by insertion of synthetic introns downstream of the human cytomegalovirus immediate-early promoter within the 5'-nontranslated region of the transgenes. Deletion of the viral dUTPase gene did not affect in vitro replication of the ILTV recombinants, but led to sufficient attenuation in vivo. After a single ocular immunization, all chickens developed H5- or N1-specific serum antibodies. Nevertheless, animals immunized with N1-ILTV died after subsequent H5N1 HPAIV challenge, although survival times were prolonged compared to non-vaccinated controls. In contrast, all chickens vaccinated with either H5-ILTV alone, or H5- and N1-ILTV simultaneously, survived

Conserved epitope on influenza-virus hemagglutinin head defined by a vaccine-induced antibody

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Raymond, Donald D.; Bajic, Goran; Ferdman, Jack; Suphaphiphat, Pirada; Settembre, Ethan C.; Moody, M. Anthony; Schmidt, Aaron G.; Harrison, Stephen C. (Duke-MED); (CH-Boston); (Seqirus)

2017-12-18

Antigenic variation requires frequent revision of annual influenza vaccines. Next-generation vaccine design strategies aim to elicit a broader immunity by directing the human immune response toward conserved sites on the principal viral surface protein, the hemagglutinin (HA). We describe a group of antibodies that recognize a hitherto unappreciated, conserved site on the HA of H1 subtype influenza viruses. Mutations in that site, which required a change in the H1 component of the 2017 vaccine, had not previously “taken over” among circulating H1 viruses. Our results encourage vaccine design strategies that resurface a protein to focus the immune response on a specific region.

The Use of Recombinant Hemagglutinine Protein of Rinderpest Virus in Enzyme Immunoassay

OpenAIRE

BULUT, Hakan; BOLAT, Yusuf

2003-01-01

In this study, Rinderpest virus (RPV) recombinant hemagglutinine protein (rH) fused with protein A region of Staphylococcus aureus was expressed in Escherichia coli and purified by IgG affinity chromatography. rH protein was also used to establish enzyme immunoassay. Therefore, to prevent IgG binding to the protein A the wells coated with the rH proteins were blocked by human serum. Afterwards, RPV antigens were added to the wells to evaluate this assay. To this end, serum from mice immunized...

Recombinant Parainfluenza Virus 5 Expressing Hemagglutinin of Influenza A Virus H5N1 Protected Mice against Lethal Highly Pathogenic Avian Influenza Virus H5N1 Challenge

Science.gov (United States)

Li, Zhuo; Mooney, Alaina J.; Gabbard, Jon D.; Gao, Xiudan; Xu, Pei; Place, Ryan J.; Hogan, Robert J.; Tompkins, S. Mark

2013-01-01

A safe and effective vaccine is the best way to prevent large-scale highly pathogenic avian influenza virus (HPAI) H5N1 outbreaks in the human population. The current FDA-approved H5N1 vaccine has serious limitations. A more efficacious H5N1 vaccine is urgently needed. Parainfluenza virus 5 (PIV5), a paramyxovirus, is not known to cause any illness in humans. PIV5 is an attractive vaccine vector. In our studies, a single dose of a live recombinant PIV5 expressing a hemagglutinin (HA) gene of H5N1 (rPIV5-H5) from the H5N1 subtype provided sterilizing immunity against lethal doses of HPAI H5N1 infection in mice. Furthermore, we have examined the effect of insertion of H5N1 HA at different locations within the PIV5 genome on the efficacy of a PIV5-based vaccine. Interestingly, insertion of H5N1 HA between the leader sequence, the de facto promoter of PIV5, and the first viral gene, nucleoprotein (NP), did not lead to a viable virus. Insertion of H5N1 HA between NP and the next gene, V/phosphorprotein (V/P), led to a virus that was defective in growth. We have found that insertion of H5N1 HA at the junction between the small hydrophobic (SH) gene and the hemagglutinin-neuraminidase (HN) gene gave the best immunity against HPAI H5N1 challenge: a dose as low as 1,000 PFU was sufficient to protect against lethal HPAI H5N1 challenge in mice. The work suggests that recombinant PIV5 expressing H5N1 HA has great potential as an HPAI H5N1 vaccine. PMID:23077314

Accurate Measurement of the Effects of All Amino-Acid Mutations on Influenza Hemagglutinin.

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Doud, Michael B; Bloom, Jesse D

2016-06-03

Influenza genes evolve mostly via point mutations, and so knowing the effect of every amino-acid mutation provides information about evolutionary paths available to the virus. We and others have combined high-throughput mutagenesis with deep sequencing to estimate the effects of large numbers of mutations to influenza genes. However, these measurements have suffered from substantial experimental noise due to a variety of technical problems, the most prominent of which is bottlenecking during the generation of mutant viruses from plasmids. Here we describe advances that ameliorate these problems, enabling us to measure with greatly improved accuracy and reproducibility the effects of all amino-acid mutations to an H1 influenza hemagglutinin on viral replication in cell culture. The largest improvements come from using a helper virus to reduce bottlenecks when generating viruses from plasmids. Our measurements confirm at much higher resolution the results of previous studies suggesting that antigenic sites on the globular head of hemagglutinin are highly tolerant of mutations. We also show that other regions of hemagglutinin-including the stalk epitopes targeted by broadly neutralizing antibodies-have a much lower inherent capacity to tolerate point mutations. The ability to accurately measure the effects of all influenza mutations should enhance efforts to understand and predict viral evolution.

Biosynthesis of measles virus hemagglutinin in persistently infected cells

International Nuclear Information System (INIS)

Bellini, W.J.; Silver, G.D.; McFarlin, D.E.

1983-01-01

The synthesis of the hemagglutinin (HA) glycoprotein of measles virus was investigated in a persistently infected cell line using a monoclonal anti-HA. The synthesis of the HA protein was shown to be associated with the rough endoplasmic reticulum. The unglycosylated (HA 0 ) apoprotein is synthesized as a 65.000 dalton peptide and is inserted into the rough endoplasmic reticulum as a transmembrane protein with approximately 2 to 3000 daltons of the peptide exposed to the cytoplasmic membrane surface. Primary glycosylation of the HA protein was found to occur through the lipid-linked carrier, dolichol-phosphate, as determined by inhibition of glycosylation by tunicamycin. Glycosylation, however, was not a prerequisite for membrane insertion. Endo-β-N-acetyl-Glucosaminidase H digestion of the fully glycosylated HA protein indicated that both simple and complex oligosaccharides are present on the surface glycoprotein. (Author)

The molecular determinants of antibody recognition and antigenic drift in the H3 hemagglutinin of swine influenza A virus

Science.gov (United States)

Influenza A virus (IAV) of the H3 subtype is an important pathogen that affects both humans and swine. The main intervention strategy for preventing infection is vaccination to induce neutralizing antibodies against the surface glycoprotein hemagglutinin (HA). However, due to antigenic drift, vaccin...

The Roles of Hemagglutinin Phe-95 in Receptor Binding and Pathogenicity of Influenza B Virus

Science.gov (United States)

Ni, Fengyun; Mbawuike, Innocent Nnadi; Kondrashkina, Elena; Wang, Qinghua

2014-01-01

Diverged ~4,000 years ago, influenza B virus has several important differences from influenza A virus, including lower receptor-binding affinity and highly restricted host range. Based on our prior structural studies, we hypothesized that a single-residue difference in the receptor-binding site of hemagglutinin (HA), Phe-95 in influenza B virus versus Tyr-98 in influenza A/H1~H15, is possibly a key determinant for the low receptor-binding affinity. Here we demonstrate that the mutation Phe95→Tyr in influenza B virus HA restores all three hydrogen bonds made by Tyr-98 in influenza A/H3 HA and has the potential to enhance receptor binding. However, the full realization of this potential is influenced by the local environment into which the mutation is introduced. The binding and replication of the recombinant viruses correlate well with the receptor-binding capabilities of HA. These results are discussed in relation to the roles of Phe-95 in receptor binding and pathogenicity of influenza B virus. PMID:24503069

Genetic Characterization of the Hemagglutinin Genes of Wild-Type Measles Virus Circulating in China, 1993–2009

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Zhu, Zhen; Liu, Chunyu; Mao, Naiying; Ji, Yixin; Wang, Huiling; Jiang, Xiaohong; Li, Chongshan; Tang, Wei; Feng, Daxing; Wang, Changyin; Zheng, Lei; Lei, Yue; Ling, Hua; Zhao, Chunfang; Ma, Yan; He, Jilan; Wang, Yan; Li, Ping; Guan, Ronghui; Zhou, Shujie; Zhou, Jianhui; Wang, Shuang; Zhang, Hong; Zheng, Huanying; Liu, Leng; Ma, Hemuti; Guan, Jing; Lu, Peishan; Feng, Yan; Zhang, Yanjun; Zhou, Shunde; Xiong, Ying; Ba, Zhuoma; Chen, Hui; Yang, Xiuhui; Bo, Fang; Ma, Yujie; Liang, Yong; Lei, Yake; Gu, Suyi; Liu, Wei; Chen, Meng; Featherstone, David; Jee, Youngmee; Bellini, William J.; Rota, Paul A.; Xu, Wenbo

2013-01-01

Background China experienced several large measles outbreaks in the past two decades, and a series of enhanced control measures were implemented to achieve the goal of measles elimination. Molecular epidemiologic surveillance of wild-type measles viruses (MeV) provides valuable information about the viral transmission patterns. Since 1993, virologic surveillnace has confirmed that a single endemic genotype H1 viruses have been predominantly circulating in China. A component of molecular surveillance is to monitor the genetic characteristics of the hemagglutinin (H) gene of MeV, the major target for virus neutralizing antibodies. Principal Findings Analysis of the sequences of the complete H gene from 56 representative wild-type MeV strains circulating in China during 1993–2009 showed that the H gene sequences were clustered into 2 groups, cluster 1 and cluster 2. Cluster1 strains were the most frequently detected cluster and had a widespread distribution in China after 2000. The predicted amino acid sequences of the H protein were relatively conserved at most of the functionally significant amino acid positions. However, most of the genotype H1 cluster1 viruses had an amino acid substitution (Ser240Asn), which removed a predicted N-linked glycosylation site. In addition, the substitution of Pro397Leu in the hemagglutinin noose epitope (HNE) was identified in 23 of 56 strains. The evolutionary rate of the H gene of the genotype H1 viruses was estimated to be approximately 0.76×10−3 substitutions per site per year, and the ratio of dN to dS (dN/dS) was measles in China. PMID:24073194

Single Dose of Consensus Hemagglutinin-Based Virus-Like Particles Vaccine Protects Chickens against Divergent H5 Subtype Influenza Viruses

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Peipei Wu

2017-11-01

Full Text Available The H5 subtype highly pathogenic avian influenza (HPAI virus is one of the greatest threats to global poultry industry. To develop broadly protective H5 subunit vaccine, a recombinant consensus HA sequence (rHA was constructed and expressed in virus-like particles (rHA VLPs in the baculovirus-insect cell system. The efficacy of the rHA VLPs vaccine with or without immunopotentiator (CVCVA5 was assessed in chickens. Compared to the commercial Re6 or Re6-CVCVA5 vaccines, single dose immunization of chickens with rHA VLPs or rHA-CVCVA5 vaccines induced higher levels of serum hemagglutinin inhibition titers and neutralization titers, mucosal antibodies, IFN-γ and IL-4 cytokines in sera, and cytotoxic T lymphocyte responses. The rHA VLPs vaccine was superior to the commercial Re6 vaccine in conferring cross-protection against different clades of H5 subtype viruses. This study reports that H5 subtype consensus HA VLP single dose vaccination provides broad protection against HPAI virus in chickens.

Hemagglutinin-specific neutralization of subacute sclerosing panencephalitis viruses.

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Miguel Ángel Muñoz-Alía

Full Text Available Subacute sclerosing panencephalitis (SSPE is a progressive, lethal complication of measles caused by particular mutants of measles virus (MeV that persist in the brain despite high levels of neutralizing antibodies. We addressed the hypothesis that antigenic drift is involved in the pathogenetic mechanism of SSPE by analyzing antigenic alterations in the MeV envelope hemagglutinin protein (MeV-H found in patients with SSPE in relation to major circulating MeV genotypes. To this aim, we obtained cDNA for the MeV-H gene from tissue taken at brain autopsy from 3 deceased persons with SSPE who had short (3-4 months, SMa79, average (3.5 years, SMa84, and long (18 years, SMa94 disease courses. Recombinant MeVs with a substituted MeV-H gene were generated by a reverse genetic system. Virus neutralization assays with a panel of anti-MeV-H murine monoclonal antibodies (mAbs or vaccine-immunized mouse anti-MeV-H polyclonal sera were performed to determine the antigenic relatedness. Functional and receptor-binding analysis of the SSPE MeV-H showed activity in a SLAM/nectin-4-dependent manner. Similar to our panel of wild-type viruses, our SSPE viruses showed an altered antigenic profile. Genotypes A, G3, and F (SSPE case SMa79 were the exception, with an intact antigenic structure. Genotypes D7 and F (SSPE SMa79 showed enhanced neutralization by mAbs targeting antigenic site IIa. Genotypes H1 and the recently reported D4.2 were the most antigenically altered genotypes. Epitope mapping of neutralizing mAbs BH015 and BH130 reveal a new antigenic site on MeV-H, which we designated Φ for its intermediate position between previously defined antigenic sites Ia and Ib. We conclude that SSPE-causing viruses show similar antigenic properties to currently circulating MeV genotypes. The absence of a direct correlation between antigenic changes and predisposition of a certain genotype to cause SSPE does not lend support to the proposed antigenic drift as a

Protection against Multiple Subtypes of Influenza Viruses by Virus-Like Particle Vaccines Based on a Hemagglutinin Conserved Epitope

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Shaoheng Chen

2015-01-01

Full Text Available We selected the conserved sequence in the stalk region of influenza virus hemagglutinin (HA trimmer, the long alpha helix (LAH, as the vaccine candidate sequence, and inserted it into the major immunodominant region (MIR of hepatitis B virus core protein (HBc, and, by using the E. coli expression system, we prepared a recombinant protein vaccine LAH-HBc in the form of virus-like particles (VLP. Intranasal immunization of mice with this LAH-HBc VLP plus cholera toxin B subunit with 0.2% of cholera toxin (CTB* adjuvant could effectively elicit humoral and cellular immune responses and protect mice against a lethal challenge of homologous influenza viruses (A/Puerto Rico/8/1934 (PR8 (H1N1. In addition, passage of the immune sera containing specific antibodies to naïve mice rendered them resistant against a lethal homologous challenge. Immunization with LAH-HBc VLP vaccine plus CTB* adjuvant could also fully protect mice against a lethal challenge of the 2009 pandemic H1N1 influenza virus or the avian H9N2 virus and could partially protect mice against a lethal challenge of the avian H5N1 influenza virus. This study demonstrated that the LAH-HBc VLP vaccine based on a conserved sequence of the HA trimmer stalk region is a promising candidate vaccine for developing a universal influenza vaccine against multiple influenza viruses infections.

Stochastic acidification, activation of hemagglutinin and escape of influenza viruses from an endosome

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Lagache, Thibault; Sieben, Christian; Meyer, Tim; Herrmann, Andreas; Holcman, David

2017-06-01

Influenza viruses enter the cell inside an endosome. During the endosomal journey, acidification triggers a conformational change of the virus spike protein hemagglutinin (HA) that results in escape of the viral genome from the endosome into the cytoplasm. It is still unclear how the interplay between acidification and HA conformation changes affects the kinetics of the viral endosomal escape. We develop here a stochastic model to estimate the change of conformation of HAs inside the endosome nanodomain. Using a Markov process, we model the arrival of protons to HA binding sites and compute the kinetics of their accumulation. We compute the Mean First Passage Time (MFPT) of the number of HA bound sites to a threshold, which is used to estimate the HA activation rate for a given pH concentration. The present analysis reveals that HA proton binding sites possess a high chemical barrier, ensuring a stability of the spike protein at sub-acidic pH. We predict that activating more than 3 adjacent HAs is necessary to trigger endosomal fusion and this configuration prevents premature release of viruses from early endosomes

Computational Identification of Antigenicity-Associated Sites in the Hemagglutinin Protein of A/H1N1 Seasonal Influenza Virus.

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Xiaowei Ren

Full Text Available The antigenic variability of influenza viruses has always made influenza vaccine development challenging. The punctuated nature of antigenic drift of influenza virus suggests that a relatively small number of genetic changes or combinations of genetic changes may drive changes in antigenic phenotype. The present study aimed to identify antigenicity-associated sites in the hemagglutinin protein of A/H1N1 seasonal influenza virus using computational approaches. Random Forest Regression (RFR and Support Vector Regression based on Recursive Feature Elimination (SVR-RFE were applied to H1N1 seasonal influenza viruses and used to analyze the associations between amino acid changes in the HA1 polypeptide and antigenic variation based on hemagglutination-inhibition (HI assay data. Twenty-three and twenty antigenicity-associated sites were identified by RFR and SVR-RFE, respectively, by considering the joint effects of amino acid residues on antigenic drift. Our proposed approaches were further validated with the H3N2 dataset. The prediction models developed in this study can quantitatively predict antigenic differences with high prediction accuracy based only on HA1 sequences. Application of the study results can increase understanding of H1N1 seasonal influenza virus antigenic evolution and accelerate the selection of vaccine strains.

Neuraminidase and hemagglutinin matching patterns of a highly pathogenic avian and two pandemic H1N1 influenza A viruses.

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Yonghui Zhang

Full Text Available BACKGROUND: Influenza A virus displays strong reassortment characteristics, which enable it to achieve adaptation in human infection. Surveying the reassortment and virulence of novel viruses is important in the prevention and control of an influenza pandemic. Meanwhile, studying the mechanism of reassortment may accelerate the development of anti-influenza strategies. METHODOLOGY/PRINCIPAL FINDINGS: The hemagglutinin (HA and neuraminidase (NA matching patterns of two pandemic H1N1 viruses (the 1918 and current 2009 strains and a highly pathogenic avian influenza A virus (H5N1 were studied using a pseudotyped particle (pp system. Our data showed that four of the six chimeric HA/NA combinations could produce infectious pps, and that some of the chimeric pps had greater infectivity than did their ancestors, raising the possibility of reassortment among these viruses. The NA of H5N1 (A/Anhui/1/2005 could hardly reassort with the HAs of the two H1N1 viruses. Many biological characteristics of HA and NA, including infectivity, hemagglutinating ability, and NA activity, are dependent on their matching pattern. CONCLUSIONS/SIGNIFICANCE: Our data suggest the existence of an interaction between HA and NA, and the HA NA matching pattern is critical for valid viral reassortment.

Positive Selection on Hemagglutinin and Neuraminidase Genes of H1N1 Influenza Viruses

LENUS (Irish Health Repository)

Li, Wenfu

2011-04-21

Abstract Background Since its emergence in March 2009, the pandemic 2009 H1N1 influenza A virus has posed a serious threat to public health. To trace the evolutionary path of these new pathogens, we performed a selection-pressure analysis of a large number of hemagglutinin (HA) and neuraminidase (NA) gene sequences of H1N1 influenza viruses from different hosts. Results Phylogenetic analysis revealed that both HA and NA genes have evolved into five distinct clusters, with further analyses indicating that the pandemic 2009 strains have experienced the strongest positive selection. We also found evidence of strong selection acting on the seasonal human H1N1 isolates. However, swine viruses from North America and Eurasia were under weak positive selection, while there was no significant evidence of positive selection acting on the avian isolates. A site-by-site analysis revealed that the positively selected sites were located in both of the cleaved products of HA (HA1 and HA2), as well as NA. In addition, the pandemic 2009 strains were subject to differential selection pressures compared to seasonal human, North American swine and Eurasian swine H1N1 viruses. Conclusions Most of these positively and\\/or differentially selected sites were situated in the B-cell and\\/or T-cell antigenic regions, suggesting that selection at these sites might be responsible for the antigenic variation of the viruses. Moreover, some sites were also associated with glycosylation and receptor-binding ability. Thus, selection at these positions might have helped the pandemic 2009 H1N1 viruses to adapt to the new hosts after they were introduced from pigs to humans. Positive selection on position 274 of NA protein, associated with drug resistance, might account for the prevalence of drug-resistant variants of seasonal human H1N1 influenza viruses, but there was no evidence that positive selection was responsible for the spread of the drug resistance of the pandemic H1N1 strains.

Influence of the water molecules near surface of viral protein on virus activation process

Energy Technology Data Exchange (ETDEWEB)

O, Shepelenko S; S, Salnikov A; V, Rak S; P, Goncharova E; B, Ryzhikov A, E-mail: shep@vector.nsc.r, E-mail: shep@ngs.r [Federal State Research Institution State Research Center of Virology and Biotechnology VECTOR of the Federal Service for Surveillance in Consumer Rights Protection and Human Well-being (FSRI SRC VB VECTOR) Koltsovo, Novosibirsk Region (Russian Federation)

2009-06-01

The infection of a cell with influenza virus comprises the stages of receptor binding to the cell membrane, endocytosis of virus particle, and fusion of the virus envelope and cell endosome membrane, which is determined by the conformational changes in hemagglutinin, a virus envelope protein, caused by pH decrease within the endosome. The pH value that induces conformation rearrangements of hemagglutinin molecule considerably varies for different influenza virus strains, first and foremost, due to the differences in amino acid structure of the corresponding proteins. The main goal of this study was to construct a model making it possible to assess the critical pH value characterizing the fusogenic activity of influenza virus hemagglutinin from the data on hemagglutinin structure and experimental verification of this model. Under this model, we assume that when the electrostatic force between interacting hemagglutinin molecules in the virus envelop exceeds a certain value, the hemagglutinin HA1 subunits are arranged so that they form a cavity sufficient for penetration of water molecules. This event leads to an irreversible hydration of the inner fragments of hemagglutinin molecule in a trimer and to the completion of conformational changes. The geometry of electrostatic field in hemagglutinin trimer was calculated taking into account the polarization effects near the interface of two dielectrics, aqueous medium and protein macromolecule. The critical pH values for the conformational changes in hemagglutinin were measured by the erythrocyte hemolysis induced by influenza virus particles when decreasing pH. The critical pH value conditionally separating the pH range into the regions with and without the conformational changes was calculated for several influenza virus H1N1 and H3N2 strains based on the data on the amino acid structure of the corresponding hemagglutinin molecules. Comparison of the theoretical and experimental values of critical pH values for

Immunization with influenza virus hemagglutinin globular region containing the receptor-binding pocket.

Science.gov (United States)

Jeon, Sung Ho; Arnon, Ruth

2002-01-01

The globular region of hemagglutinin (residues 91-261) membrane glycoprotein of influenza virus that encompasses the binding zone to the oligosaccharide receptor of target cells has been cloned by reverse transcriptase-polymerase chain reaction (RT-PCR). This protein segment (denoted HA91-261 peptide) induced significant immune response in mice. The serum antibodies and lung homogenates from the immunized mice cross-reacted with native virus particles. The cellular immunity was manifested by proliferative splenocyte responses and cytokine release indicating T helper type 1 activity. The plasmid DNA containing this segment (denoted pHA91-261) provoked, in addition, a significant cytotoxic T lymphocyte (CTL) response, whereas the HA91-261 protein fragment led to no such response. Both the DNA and the protein fragment of HA91-261 induced significant protection against viral challenge, although the immune response they induce might be along different pathways. Interestingly, the combined DNA priming-protein boosting immunization regimen did not induce protection against viral challenges even though it led to significant humoral immune responses similar to that induced by the peptide vaccine.

Structure, Receptor Binding, and Antigenicity of Influenza Virus Hemagglutinins from the 1957 H2N2 Pandemic

Energy Technology Data Exchange (ETDEWEB)

Xu, Rui; McBride, Ryan; Paulson, James C.; Basler, Christopher F.; Wilson, Ian A. (Sinai); (Scripps)

2010-03-04

The hemagglutinin (HA) envelope protein of influenza viruses mediates essential viral functions, including receptor binding and membrane fusion, and is the major viral antigen for antibody neutralization. The 1957 H2N2 subtype (Asian flu) was one of the three great influenza pandemics of the last century and caused 1 million deaths globally from 1957 to 1968. Three crystal structures of 1957 H2 HAs have been determined at 1.60 to 1.75 {angstrom} resolutions to investigate the structural basis for their antigenicity and evolution from avian to human binding specificity that contributed to its introduction into the human population. These structures, which represent the highest resolutions yet recorded for a complete ectodomain of a glycosylated viral surface antigen, along with the results of glycan microarray binding analysis, suggest that a hydrophobicity switch at residue 226 and elongation of receptor-binding sites were both critical for avian H2 HA to acquire human receptor specificity. H2 influenza viruses continue to circulate in birds and pigs and, therefore, remain a substantial threat for transmission to humans. The H2 HA structure also reveals a highly conserved epitope that could be harnessed in the design of a broader and more universal influenza A virus vaccine.

An induced pocket for the binding of potent fusion inhibitor CL-385319 with H5N1 influenza virus hemagglutinin.

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Runming Li

Full Text Available The influenza glycoprotein hemagglutinin (HA plays crucial roles in the early stage of virus infection, including receptor binding and membrane fusion. Therefore, HA is a potential target for developing anti-influenza drugs. Recently, we characterized a novel inhibitor of highly pathogenic H5N1 influenza virus, CL-385319, which specifically inhibits HA-mediated viral entry. Studies presented here identified the critical binding residues for CL-385319, which clustered in the stem region of the HA trimer by site-directed mutagenesis. Extensive computational simulations, including molecular docking, molecular dynamics simulations, molecular mechanics generalized Born surface area (MM_GBSA calculations, charge density and Laplacian calculations, have been carried out to uncover the detailed molecular mechanism that underlies the binding of CL-385319 to H5N1 influenza virus HA. It was found that the recognition and binding of CL-385319 to HA proceeds by a process of "induced fit" whereby the binding pocket is formed during their interaction. Occupation of this pocket by CL-385319 stabilizes the neutral pH structure of hemagglutinin, thus inhibiting the conformational rearrangements required for membrane fusion. This "induced fit" pocket may be a target for structure-based design of more potent influenza fusion inhibitors.

Accurate Measurement of the Effects of All Amino-Acid Mutations on Influenza Hemagglutinin

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Michael B. Doud

2016-06-01

Full Text Available Influenza genes evolve mostly via point mutations, and so knowing the effect of every amino-acid mutation provides information about evolutionary paths available to the virus. We and others have combined high-throughput mutagenesis with deep sequencing to estimate the effects of large numbers of mutations to influenza genes. However, these measurements have suffered from substantial experimental noise due to a variety of technical problems, the most prominent of which is bottlenecking during the generation of mutant viruses from plasmids. Here we describe advances that ameliorate these problems, enabling us to measure with greatly improved accuracy and reproducibility the effects of all amino-acid mutations to an H1 influenza hemagglutinin on viral replication in cell culture. The largest improvements come from using a helper virus to reduce bottlenecks when generating viruses from plasmids. Our measurements confirm at much higher resolution the results of previous studies suggesting that antigenic sites on the globular head of hemagglutinin are highly tolerant of mutations. We also show that other regions of hemagglutinin—including the stalk epitopes targeted by broadly neutralizing antibodies—have a much lower inherent capacity to tolerate point mutations. The ability to accurately measure the effects of all influenza mutations should enhance efforts to understand and predict viral evolution.

Modified vaccinia virus Ankara expressing the hemagglutinin of pandemic (H1N1) 2009 virus induces cross-protective immunity against Eurasian 'avian-like' H1N1 swine viruses in mice.

Science.gov (United States)

Castrucci, Maria R; Facchini, Marzia; Di Mario, Giuseppina; Garulli, Bruno; Sciaraffia, Ester; Meola, Monica; Fabiani, Concetta; De Marco, Maria A; Cordioli, Paolo; Siccardi, Antonio; Kawaoka, Yoshihiro; Donatelli, Isabella

2014-05-01

To examine cross-reactivity between hemagglutinin (HA) derived from A/California/7/09 (CA/09) virus and that derived from representative Eurasian "avian-like" (EA) H1N1 swine viruses isolated in Italy between 1999 and 2008 during virological surveillance in pigs. Modified vaccinia virus Ankara (MVA) expressing the HA gene of CA/09 virus (MVA-HA-CA/09) was used as a vaccine to investigate cross-protective immunity against H1N1 swine viruses in mice. Two classical swine H1N1 (CS) viruses and four representative EA-like H1N1 swine viruses previously isolated during outbreaks of respiratory disease in pigs on farms in Northern Italy were used in this study. Female C57BL/6 mice were vaccinated with MVA/HA/CA/09 and then challenged intranasally with H1N1 swine viruses. Cross-reactive antibody responses were determined by hemagglutination- inhibition (HI) and virus microneutralizing (MN) assays of sera from MVA-vaccinated mice. The extent of protective immunity against infection with H1N1 swine viruses was determined by measuring lung viral load on days 2 and 4 post-challenge. Systemic immunization of mice with CA/09-derived HA, vectored by MVA, elicited cross-protective immunity against recent EA-like swine viruses. This immune protection was related to the levels of cross-reactive HI antibodies in the sera of the immunized mice and was dependent on the similarity of the antigenic site Sa of H1 HAs. Our findings suggest that the herd immunity elicited in humans by the pandemic (H1N1) 2009 virus could limit the transmission of recent EA-like swine HA genes into the influenza A virus gene pool in humans. © 2013 The Authors Influenza and Other Respiratory Viruses Published by John Wiley & Sons Ltd.

Development and characterization of neutralizing monoclonal antibodies against canine distemper virus hemagglutinin protein.

Science.gov (United States)

Bi, Zhenwei; Xia, Xingxia; Wang, Yongshan; Mei, Yongjie

2015-04-01

Canine distemper virus (CDV) causes a serious multisystemic disease in dogs and other carnivora. Hemagglutinin (H) protein-specific antibodies are mainly responsible for protective immunity against CDV infection. In the present study, six neutralizing MAbs to the H protein of CDV were newly obtained and characterized by immunizing BALB/c mice with a recent Chinese field isolate. Competitive binding inhibition assay revealed that they recognized four distinct antigenic regions of the H protein. Immunofluorescence assay and western blotting showed that all MAbs recognize the conformational rather than the linear epitopes of the H protein. Furthermore, in immunofluorescence and virus neutralization assays, two of the MAbs were found to react only with the recent Chinese field isolate and not with older CDV strains, including vaccine strain Onderstepoort, indicating there are neutralization-related antigenic variations between the recent Chinese field isolate and the older CDV strains examined in this study. The newly established MAbs are useful for differentiating the expanding CDV strains and could be used in immunotherapy and immunodiagnosis against infection with CDV. © 2015 The Societies and Wiley Publishing Asia Pty Ltd.

Molecular Evolution and Characterization of Hemagglutinin (H in Peste des Petits Ruminants Virus.

Directory of Open Access Journals (Sweden)

Zhongxiang Liang

Full Text Available Peste des Petits Ruminants (PPR is an acute, highly contagious, and febrile viral disease that affects both domestic and wild small ruminants. The disease has become a major obstacle to the development of sustainable Agriculture. Hemagglutinin (H, the envelope glycoprotein of Peste des Petits Ruminants Virus (PPRV, plays a crucial role in regulating viral adsorption and entry, thus determining pathogenicity, and release of newly produced viral particles. In order to accurately understand the epidemic of the disease and the interactions between the virus and host, we launch the work. Here, we examined H gene from all four lineages of the PPRV to investigate evolutionary and epidemiologic dynamics of PPRV by the Bayesian method. In addition, we predicted positive selection sites due to selective pressures. Finally, we studied the interaction between H protein and SLAM receptor based on homology model of the complex. Phylogenetic analysis suggested that H gene can also be used to investigate evolutionary and epidemiologic dynamics of PPRV. Positive selection analysis identified four positive selection sites in H gene, in which only one common site (aa246 was detected by two methods, suggesting strong operation structural and/or functional constraint of changes on the H protein. This target site may be of interest for future mutagenesis studies. The results of homology modeling showed PPRVHv-shSLAM binding interface and MVH-maSLAM binding interface were consistent, wherein the groove in the B4 blade and B5 of the head domain of PPRVHv bound to the AGFCC' β-sheets of the membrane-distal ectodomain of shSLAM. The binding regions could provide insight on the nature of the protein for epitope vaccine design, novel drug discovery, and rational drug design against PPRV.

Canine distemper virus neutralization activity is low in human serum and it is sensitive to an amino acid substitution in the hemagglutinin protein

International Nuclear Information System (INIS)

Zhang, Xinsheng; Wallace, Olivia L.; Domi, Arban; Wright, Kevin J.; Driscoll, Jonathan; Anzala, Omu; Sanders, Eduard J.; Kamali, Anatoli; Karita, Etienne; Allen, Susan; Fast, Pat; Gilmour, Jill; Price, Matt A.; Parks, Christopher L.

2015-01-01

Serum was analyzed from 146 healthy adult volunteers in eastern Africa to evaluate measles virus (MV) and canine distemper virus (CDV) neutralizing antibody (nAb) prevalence and potency. MV plaque reduction neutralization test (PRNT) results indicated that all sera were positive for MV nAbs. Furthermore, the 50% neutralizing dose (ND50) for the majority of sera corresponded to antibody titers induced by MV vaccination. CDV nAbs titers were low and generally were detected in sera with high MV nAb titers. A mutant CDV was generated that was less sensitive to neutralization by human serum. The mutant virus genome had 10 nucleotide substitutions, which coded for single amino acid substitutions in the fusion (F) and hemagglutinin (H) glycoproteins and two substitutions in the large polymerase (L) protein. The H substitution occurred in a conserved region involved in receptor interactions among morbilliviruses, implying that this region is a target for cross-reactive neutralizing antibodies. - Highlights: • Screened 146 serum samples for measles virus (MV) and canine distemper virus (CDV) neutralizing antibody (nAb). • MV nAb is prevalent in the sera. • CDV neutralizing activity is generally low or absent and when detected it is present in sera with high MV nAb titers. • A neutralization-resistant CDV mutant was isolated using human serum selection. • A mutation was identified in the receptor-binding region of CDV hemagglutinin protein that confers the neutralization resistance

Canine distemper virus neutralization activity is low in human serum and it is sensitive to an amino acid substitution in the hemagglutinin protein

Energy Technology Data Exchange (ETDEWEB)

Zhang, Xinsheng, E-mail: xzhang@iavi.org [AIDS Vaccine Design and Development Laboratory, International AIDS Vaccine Initiative (IAVI), Brooklyn, NY (United States); Molecular and Cellular Biology Program, State University of New York, Brooklyn, NY (United States); Wallace, Olivia L.; Domi, Arban; Wright, Kevin J.; Driscoll, Jonathan [AIDS Vaccine Design and Development Laboratory, International AIDS Vaccine Initiative (IAVI), Brooklyn, NY (United States); Anzala, Omu [Kenya AIDS Vaccine Initiative (KAVI)-Institute of Clinical Research, Nairobi (Kenya); Sanders, Eduard J. [Centre for Geographic Medicine Research, Kenya Medical Research Institute (KEMRI), Kilifi, Kenya & Centre for Clinical Vaccinology and Tropical Medicine, University of Oxford, Headington (United Kingdom); Kamali, Anatoli [MRC/UVRI Uganda Virus Research Unit on AIDS, Masaka and Entebbe (Uganda); Karita, Etienne [Projet San Francisco, Kigali (Rwanda); Allen, Susan [Department of Pathology and Laboratory Medicine, Emory University, Atlanta, GA (United States); Fast, Pat [Department of Medical Affairs, International AIDS Vaccine Initiative, NY, NY (United States); Gilmour, Jill [Human Immunology Laboratory, International AIDS Vaccine Initiative, London (United Kingdom); Price, Matt A. [Department of Medical Affairs, International AIDS Vaccine Initiative, NY, NY (United States); Department of Epidemiology and Biostatistics, University of California at San Francisco, San Francisco, CA (United States); Parks, Christopher L. [AIDS Vaccine Design and Development Laboratory, International AIDS Vaccine Initiative (IAVI), Brooklyn, NY (United States); Molecular and Cellular Biology Program, State University of New York, Brooklyn, NY (United States)

2015-08-15

Serum was analyzed from 146 healthy adult volunteers in eastern Africa to evaluate measles virus (MV) and canine distemper virus (CDV) neutralizing antibody (nAb) prevalence and potency. MV plaque reduction neutralization test (PRNT) results indicated that all sera were positive for MV nAbs. Furthermore, the 50% neutralizing dose (ND50) for the majority of sera corresponded to antibody titers induced by MV vaccination. CDV nAbs titers were low and generally were detected in sera with high MV nAb titers. A mutant CDV was generated that was less sensitive to neutralization by human serum. The mutant virus genome had 10 nucleotide substitutions, which coded for single amino acid substitutions in the fusion (F) and hemagglutinin (H) glycoproteins and two substitutions in the large polymerase (L) protein. The H substitution occurred in a conserved region involved in receptor interactions among morbilliviruses, implying that this region is a target for cross-reactive neutralizing antibodies. - Highlights: • Screened 146 serum samples for measles virus (MV) and canine distemper virus (CDV) neutralizing antibody (nAb). • MV nAb is prevalent in the sera. • CDV neutralizing activity is generally low or absent and when detected it is present in sera with high MV nAb titers. • A neutralization-resistant CDV mutant was isolated using human serum selection. • A mutation was identified in the receptor-binding region of CDV hemagglutinin protein that confers the neutralization resistance.

Inhibition of influenza virus infection and hemagglutinin cleavage by the protease inhibitor HAI-2

Energy Technology Data Exchange (ETDEWEB)

Hamilton, Brian S.; Chung, Changik; Cyphers, Soreen Y.; Rinaldi, Vera D.; Marcano, Valerie C.; Whittaker, Gary R., E-mail: grw7@cornell.edu

2014-07-25

Highlights: • Biochemical and cell biological analysis of HAI-2 as an inhibitor of influenza HA cleavage activation. • Biochemical and cell biological analysis of HAI-2 as an inhibitor of influenza virus infection. • Comparative analysis of HAI-2 for vesicular stomatitis virus and human parainfluenza virus type-1. • Analysis of the activity of HAI-2 in a mouse model of influenza. - Abstract: Influenza virus remains a significant concern to public health, with the continued potential for a high fatality pandemic. Vaccination and antiviral therapeutics are effective measures to circumvent influenza virus infection, however, multiple strains have emerged that are resistant to the antiviral therapeutics currently on the market. With this considered, investigation of alternative antiviral therapeutics is being conducted. One such approach is to inhibit cleavage activation of the influenza virus hemagglutinin (HA), which is an essential step in the viral replication cycle that permits viral-endosome fusion. Therefore, targeting trypsin-like, host proteases responsible for HA cleavage in vivo may prove to be an effective therapeutic. Hepatocyte growth factor activator inhibitor 2 (HAI-2) is naturally expressed in the respiratory tract and is a potent inhibitor of trypsin-like serine proteases, some of which have been determined to cleave HA. In this study, we demonstrate that HAI-2 is an effective inhibitor of cleavage of HA from the human-adapted H1 and H3 subtypes. HAI-2 inhibited influenza virus H1N1 infection in cell culture, and HAI-2 administration showed protection in a mouse model of influenza. HAI-2 has the potential to be an effective, alternative antiviral therapeutic for influenza.

Inhibition of influenza virus infection and hemagglutinin cleavage by the protease inhibitor HAI-2

International Nuclear Information System (INIS)

Hamilton, Brian S.; Chung, Changik; Cyphers, Soreen Y.; Rinaldi, Vera D.; Marcano, Valerie C.; Whittaker, Gary R.

2014-01-01

Highlights: • Biochemical and cell biological analysis of HAI-2 as an inhibitor of influenza HA cleavage activation. • Biochemical and cell biological analysis of HAI-2 as an inhibitor of influenza virus infection. • Comparative analysis of HAI-2 for vesicular stomatitis virus and human parainfluenza virus type-1. • Analysis of the activity of HAI-2 in a mouse model of influenza. - Abstract: Influenza virus remains a significant concern to public health, with the continued potential for a high fatality pandemic. Vaccination and antiviral therapeutics are effective measures to circumvent influenza virus infection, however, multiple strains have emerged that are resistant to the antiviral therapeutics currently on the market. With this considered, investigation of alternative antiviral therapeutics is being conducted. One such approach is to inhibit cleavage activation of the influenza virus hemagglutinin (HA), which is an essential step in the viral replication cycle that permits viral-endosome fusion. Therefore, targeting trypsin-like, host proteases responsible for HA cleavage in vivo may prove to be an effective therapeutic. Hepatocyte growth factor activator inhibitor 2 (HAI-2) is naturally expressed in the respiratory tract and is a potent inhibitor of trypsin-like serine proteases, some of which have been determined to cleave HA. In this study, we demonstrate that HAI-2 is an effective inhibitor of cleavage of HA from the human-adapted H1 and H3 subtypes. HAI-2 inhibited influenza virus H1N1 infection in cell culture, and HAI-2 administration showed protection in a mouse model of influenza. HAI-2 has the potential to be an effective, alternative antiviral therapeutic for influenza

A human monoclonal antibody derived from a vaccinated volunteer recognizes heterosubtypically a novel epitope on the hemagglutinin globular head of H1 and H9 influenza A viruses

International Nuclear Information System (INIS)

Boonsathorn, Naphatsawan; Panthong, Sumolrat; Koksunan, Sarawut; Chittaganpitch, Malinee; Phuygun, Siripaporn; Waicharoen, Sunthareeya; Prachasupap, Apichai; Sasaki, Tadahiro; Kubota-Koketsu, Ritsuko; Yasugi, Mayo; Ono, Ken-ichiro; Arai, Yasuha

2014-01-01

Highlights: • A human monoclonal antibody against influenza virus was produced from a volunteer. • The antibody was generated from the PBMCs of the volunteer using the fusion method. • The antibody neutralized heterosubtypically group 1 influenza A viruses (H1 and H9). • The antibody targeted a novel epitope in globular head region of the hemagglutinin. • Sequences of the identified epitope are highly conserved among H1 and H9 subtypes. - Abstract: Most neutralizing antibodies elicited during influenza virus infection or by vaccination have a narrow spectrum because they usually target variable epitopes in the globular head region of hemagglutinin (HA). In this study, we describe a human monoclonal antibody (HuMAb), 5D7, that was prepared from the peripheral blood lymphocytes of a vaccinated volunteer using the fusion method. The HuMAb heterosubtypically neutralizes group 1 influenza A viruses, including seasonal H1N1, 2009 pandemic H1N1 (H1N1pdm) and avian H9N2, with a strong hemagglutinin inhibition activity. Selection of an escape mutant showed that the HuMAb targets a novel conformational epitope that is located in the HA head region but is distinct from the receptor binding site. Furthermore, Phe114Ile substitution in the epitope made the HA unrecognizable by the HuMAb. Amino acid residues in the predicted epitope region are also highly conserved in the HAs of H1N1 and H9N2. The HuMAb reported here may be a potential candidate for the development of therapeutic/prophylactic antibodies against H1 and H9 influenza viruses

A human monoclonal antibody derived from a vaccinated volunteer recognizes heterosubtypically a novel epitope on the hemagglutinin globular head of H1 and H9 influenza A viruses

Energy Technology Data Exchange (ETDEWEB)

Boonsathorn, Naphatsawan; Panthong, Sumolrat [Medical Life Sciences Institute, Department of Medical Sciences, Ministry of Public Health, Muang, Nonthaburi (Thailand); Japan Science and Technology Agency/Japan International Cooperation Agency, Science and Technology Research Partnership for Sustainable Development (JST/JICA, SATREPS), Tokyo (Japan); Koksunan, Sarawut [Medical Life Sciences Institute, Department of Medical Sciences, Ministry of Public Health, Muang, Nonthaburi (Thailand); Chittaganpitch, Malinee; Phuygun, Siripaporn; Waicharoen, Sunthareeya [National Institute of Health, Department of Medical Sciences, Ministry of Public Health, Muang, Nonthaburi (Thailand); Prachasupap, Apichai [Medical Life Sciences Institute, Department of Medical Sciences, Ministry of Public Health, Muang, Nonthaburi (Thailand); Japan Science and Technology Agency/Japan International Cooperation Agency, Science and Technology Research Partnership for Sustainable Development (JST/JICA, SATREPS), Tokyo (Japan); Sasaki, Tadahiro [Department of Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka (Japan); Japan Science and Technology Agency/Japan International Cooperation Agency, Science and Technology Research Partnership for Sustainable Development (JST/JICA, SATREPS), Tokyo (Japan); Kubota-Koketsu, Ritsuko [Kanonji Institute, The Research Foundation for Microbial Diseases of Osaka University, Kanonji, Kagawa (Japan); Yasugi, Mayo [Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Izumisano, Osaka (Japan); Ono, Ken-ichiro [Ina Laboratory, Medical and Biological Laboratories Corporation, Ltd., Ina, Nagano (Japan); Japan Science and Technology Agency/Japan International Cooperation Agency, Science and Technology Research Partnership for Sustainable Development (JST/JICA, SATREPS), Tokyo (Japan); Arai, Yasuha [Department of Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka (Japan); and others

2014-09-26

Highlights: • A human monoclonal antibody against influenza virus was produced from a volunteer. • The antibody was generated from the PBMCs of the volunteer using the fusion method. • The antibody neutralized heterosubtypically group 1 influenza A viruses (H1 and H9). • The antibody targeted a novel epitope in globular head region of the hemagglutinin. • Sequences of the identified epitope are highly conserved among H1 and H9 subtypes. - Abstract: Most neutralizing antibodies elicited during influenza virus infection or by vaccination have a narrow spectrum because they usually target variable epitopes in the globular head region of hemagglutinin (HA). In this study, we describe a human monoclonal antibody (HuMAb), 5D7, that was prepared from the peripheral blood lymphocytes of a vaccinated volunteer using the fusion method. The HuMAb heterosubtypically neutralizes group 1 influenza A viruses, including seasonal H1N1, 2009 pandemic H1N1 (H1N1pdm) and avian H9N2, with a strong hemagglutinin inhibition activity. Selection of an escape mutant showed that the HuMAb targets a novel conformational epitope that is located in the HA head region but is distinct from the receptor binding site. Furthermore, Phe114Ile substitution in the epitope made the HA unrecognizable by the HuMAb. Amino acid residues in the predicted epitope region are also highly conserved in the HAs of H1N1 and H9N2. The HuMAb reported here may be a potential candidate for the development of therapeutic/prophylactic antibodies against H1 and H9 influenza viruses.

Neuraminidase stalk length and additional glycosylation of the hemagglutinin influence the virulence of influenza H5N1 viruses for mice.

Science.gov (United States)

Matsuoka, Yumiko; Swayne, David E; Thomas, Colleen; Rameix-Welti, Marie-Anne; Naffakh, Nadia; Warnes, Christine; Altholtz, Melanie; Donis, Ruben; Subbarao, Kanta

2009-05-01

Following circulation of avian influenza H5 and H7 viruses in poultry, the hemagglutinin (HA) can acquire additional glycosylation sites, and the neuraminidase (NA) stalk becomes shorter. We investigated whether these features play a role in the pathogenesis of infection in mammalian hosts. From 1996 to 2007, H5N1 viruses with a short NA stalk have become widespread in several avian species. Compared to viruses with a long-stalk NA, viruses with a short-stalk NA showed a decreased capacity to elute from red blood cells and an increased virulence in mice, but not in chickens. The presence of additional HA glycosylation sites had less of an effect on virulence than did NA stalk length. The short-stalk NA of H5N1 viruses circulating in Asia may contribute to virulence in humans.

Neuraminidase Stalk Length and Additional Glycosylation of the Hemagglutinin Influence the Virulence of Influenza H5N1 Viruses for Mice▿

Science.gov (United States)

Matsuoka, Yumiko; Swayne, David E.; Thomas, Colleen; Rameix-Welti, Marie-Anne; Naffakh, Nadia; Warnes, Christine; Altholtz, Melanie; Donis, Ruben; Subbarao, Kanta

2009-01-01

Following circulation of avian influenza H5 and H7 viruses in poultry, the hemagglutinin (HA) can acquire additional glycosylation sites, and the neuraminidase (NA) stalk becomes shorter. We investigated whether these features play a role in the pathogenesis of infection in mammalian hosts. From 1996 to 2007, H5N1 viruses with a short NA stalk have become widespread in several avian species. Compared to viruses with a long-stalk NA, viruses with a short-stalk NA showed a decreased capacity to elute from red blood cells and an increased virulence in mice, but not in chickens. The presence of additional HA glycosylation sites had less of an effect on virulence than did NA stalk length. The short-stalk NA of H5N1 viruses circulating in Asia may contribute to virulence in humans. PMID:19225004

Viral hemagglutinin is involved in promoting the internalisation of Staphylococcus aureus into human pneumocytes during influenza A H1N1 virus infection.

Science.gov (United States)

Passariello, Claudio; Nencioni, Lucia; Sgarbanti, Rossella; Ranieri, Danilo; Torrisi, Maria Rosaria; Ripa, Sandro; Garaci, Enrico; Palamara, Anna Teresa

2011-02-01

Secondary pneumonia caused by Staphylococcus aureus is reemerging as a primary cause of excess mortality associated with infection by the influenza A virus. We have investigated in vitro the cellular and molecular mechanisms underlying this synergism. Experimental data show a significant increase in the efficiency of internalisation of S. aureus into cultured pneumocytes during the early phases of viral infection, while a relevant increase in the efficiency of adhesion is evident only later during viral infection, suggesting that the 2 effects are based on different molecular mechanisms. Data reported in this paper show that S. aureus cells can bind the viral hemagglutinin (HA) and that this binding promotes enhanced bacterial internalisation by 2 mechanisms: binding to HA exposed at the surface of infected cells and binding to free extracellular virions, enabling internalisation at high efficiency also in cells that are not infected by the virus. The affinity of binding that involves S. aureus and HA was shown to be enhanced by the reducing extracellular environment that the virus can generate. Copyright © 2010 Elsevier GmbH. All rights reserved.

Identifying antigenicity-associated sites in highly pathogenic H5N1 influenza virus hemagglutinin by using sparse learning.

OpenAIRE

Cai, Zhipeng; Yang, Jialiang; Zhang, Tong; Long, Li-Ping; Boon, Adrianus C; Webby, Richard J; Wan, Xiu-Feng

2012-01-01

Since the isolation of A/goose/Guangdong/1/1996 (H5N1) in farmed geese in southern China, highly pathogenic H5N1 avian influenza viruses have posed a continuous threat to both public and animal health. The non-synonymous mutation of the H5 hemagglutinin (HA) gene has resulted in antigenic drift, leading to difficulties in both clinical diagnosis and vaccine strain selection. Characterizing H5N1's antigenic profiles would help resolve these problems. In this study, a novel sparse learning meth...

Evolutionary trends of A(H1N1 influenza virus hemagglutinin since 1918.

Directory of Open Access Journals (Sweden)

Jun Shen

2009-11-01

Full Text Available The Pandemic (H1N1 2009 is spreading to numerous countries and causing many human deaths. Although the symptoms in humans are mild at present, fears are that further mutations in the virus could lead to a potentially more dangerous outbreak in subsequent months. As the primary immunity-eliciting antigen, hemagglutinin (HA is the major agent for host-driven antigenic drift in A(H3N2 virus. However, whether and how the evolution of HA is influenced by existing immunity is poorly understood for A(H1N1. Here, by analyzing hundreds of A(H1N1 HA sequences since 1918, we show the first evidence that host selections are indeed present in A(H1N1 HAs. Among a subgroup of human A(H1N1 HAs between 1918 approximately 2008, we found strong diversifying (positive selection at HA(1 156 and 190. We also analyzed the evolutionary trends at HA(1 190 and 225 that are critical determinants for receptor-binding specificity of A(H1N1 HA. Different A(H1N1 viruses appeared to favor one of these two sites in host-driven antigenic drift: epidemic A(H1N1 HAs favor HA(1 190 while the 1918 pandemic and swine HAs favor HA(1 225. Thus, our results highlight the urgency to understand the interplay between antigenic drift and receptor binding in HA evolution, and provide molecular signatures for monitoring future antigenically drifted 2009 pandemic and seasonal A(H1N1 influenza viruses.

Molecular Characterizations of Surface Proteins Hemagglutinin and Neuraminidase from Recent H5Nx Avian Influenza Viruses

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Yang, Hua; Carney, Paul J.; Mishin, Vasiliy P.; Guo, Zhu; Chang, Jessie C.; Wentworth, David E.; Gubareva, Larisa V.; Stevens, James; Schultz-Cherry, S.

2016-04-06

During 2014, a subclade 2.3.4.4 highly pathogenic avian influenza (HPAI) A(H5N8) virus caused poultry outbreaks around the world. In late 2014/early 2015, the virus was detected in wild birds in Canada and the United States, and these viruses also gave rise to reassortant progeny, composed of viral RNA segments (vRNAs) from both Eurasian and North American lineages. In particular, viruses were found with N1, N2, and N8 neuraminidase vRNAs, and these are collectively referred to as H5Nx viruses. In the United States, more than 48 million domestic birds have been affected. Here we present a detailed structural and biochemical analysis of the surface antigens of H5N1, H5N2, and H5N8 viruses in addition to those of a recent human H5N6 virus. Our results with recombinant hemagglutinin reveal that these viruses have a strict avian receptor binding preference, while recombinantly expressed neuraminidases are sensitive to FDA-approved and investigational antivirals. Although H5Nx viruses currently pose a low risk to humans, it is important to maintain surveillance of these circulating viruses and to continually assess future changes that may increase their pandemic potential.

IMPORTANCEThe H5Nx viruses emerging in North America, Europe, and Asia pose a great public health concern. Here we report a molecular and structural study of the major surface proteins of several H5Nx influenza viruses. Our results improve the understanding of these new viruses and provide important information on their receptor preferences and susceptibilities to antivirals, which are central to pandemic risk assessment.

Identifying antigenicity associated sites in highly pathogenic H5N1 influenza virus hemagglutinin by using sparse learning

OpenAIRE

Cai, Zhipeng; Ducatez, Mariette F.; Yang, Jialiang; Zhang, Tong; Long, Li-Ping; Boon, Adrianus C.; Webby, Richard J.; Wan, Xiu-Feng

2012-01-01

Since the isolation of A/goose/Guangdong/1/1996 (H5N1) in farmed geese in southern China, highly pathogenic H5N1 avian influenza viruses have posed a continuous threat to both public and animal health. The non-synonymous mutation of the H5 hemagglutinin gene has resulted in antigenic drift, leading to difficulties in both clinical diagnosis and vaccine strain selection. Characterizing H5N1’s antigenic profiles would help resolve these problems. In this study, a novel sparse learning method wa...

Establishment of the cross-clade antigen detection system for H5 subtype influenza viruses using peptide monoclonal antibodies specific for influenza virus H5 hemagglutinin.

Science.gov (United States)

Takahashi, Hitoshi; Nagata, Shiho; Odagiri, Takato; Kageyama, Tsutomu

2018-04-15

The H5 subtype of highly pathogenic avian influenza (H5 HPAI) viruses is a threat to both animal and human public health and has the potential to cause a serious future pandemic in humans. Thus, specific and rapid detection of H5 HPAI viruses is required for infection control in humans. To develop a simple and rapid diagnostic system to detect H5 HPAI viruses with high specificity and sensitivity, we attempted to prepare monoclonal antibodies (mAbs) that specifically recognize linear epitopes in hemagglutinin (HA) of H5 subtype viruses. Nine mAb clones were obtained from mice immunized with a synthetic partial peptide of H5 HA molecules conserved among various H5 HPAI viruses. The antigen-capture enzyme-linked immunosorbent assay using the most suitable combination of these mAbs, which bound specifically to lysed H5 HA under an optimized detergent condition, was specific for H5 viruses and could broadly detect H5 viruses in multiple different clades. Taken together, these peptide mAbs, which recognize linear epitopes in a highly conserved region of H5 HA, may be useful for specific and highly sensitive detection of H5 HPAI viruses and can help in the rapid diagnosis of human, avian, and animal H5 virus infections. Copyright © 2018 Elsevier Inc. All rights reserved.

Enhancement of the safety of live influenza vaccine by attenuating mutations from cold-adapted hemagglutinin

International Nuclear Information System (INIS)

Lee, Yoon Jae; Jang, Yo Han; Kim, Paul; Lee, Yun Ha; Lee, Young Jae; Byun, Young Ho; Lee, Kwang-Hee; Kim, Kyusik; Seong, Baik Lin

2016-01-01

In our previous study, X-31ca-based H5N1 LAIVs, in particular, became more virulent in mice than the X-31ca MDV, possibly by the introduction of the surface antigens of highly pathogenic H5N1 influenza virus, implying that additional attenuation is needed in this cases to increase the safety level of the vaccine. In this report we suggest an approach to further increase the safety of LAIV through additional cold-adapted mutations in the hemagglutinin. The cold-adaptation of X-31 virus resulted in four amino acid mutations in the HA. We generated a panel of 7:1 reassortant viruses each carrying the hemagglutinins with individual single amino acid mutations. We examined their phenotypes and found a major attenuating mutation, N81K. This attenuation marker conferred additional temperature-sensitive and attenuation phenotype to the LAIV. Our data indicate that the cold-adapted mutation in the HA confers additional attenuation to the LAIV strain, without compromising its productivity and immune response. - Highlights: • Cold-adaptation process induced four amino acid mutations in the HA of X-31 virus. • The four mutations in the HA also contributed to attenuation of the X-31ca virus • N81K mutation was the most significant marker for the attenuation of X-31ca virus. • Introduction of N81K mutation into H3N2 LAIV further attenuated the vaccine. • This approach provides a useful guideline for enhancing the safety of the LAIVs.

Enhancement of the safety of live influenza vaccine by attenuating mutations from cold-adapted hemagglutinin

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Lee, Yoon Jae [Graduate Program in Biomaterials Science and Engineering, College of Life Science and Biotechnology, Yonsei University, Seoul (Korea, Republic of); Laboratory of Molecular Medicine, Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul (Korea, Republic of); Vaccine Translational Research Center, Yonsei University, Seoul (Korea, Republic of); Jang, Yo Han [Laboratory of Molecular Medicine, Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul (Korea, Republic of); Kim, Paul; Lee, Yun Ha; Lee, Young Jae [Laboratory of Molecular Medicine, Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul (Korea, Republic of); Vaccine Translational Research Center, Yonsei University, Seoul (Korea, Republic of); Byun, Young Ho; Lee, Kwang-Hee; Kim, Kyusik [Laboratory of Molecular Medicine, Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul (Korea, Republic of); Seong, Baik Lin, E-mail: blseong@yonsei.ac.kr [Graduate Program in Biomaterials Science and Engineering, College of Life Science and Biotechnology, Yonsei University, Seoul (Korea, Republic of); Laboratory of Molecular Medicine, Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul (Korea, Republic of); Vaccine Translational Research Center, Yonsei University, Seoul (Korea, Republic of)

2016-04-15

In our previous study, X-31ca-based H5N1 LAIVs, in particular, became more virulent in mice than the X-31ca MDV, possibly by the introduction of the surface antigens of highly pathogenic H5N1 influenza virus, implying that additional attenuation is needed in this cases to increase the safety level of the vaccine. In this report we suggest an approach to further increase the safety of LAIV through additional cold-adapted mutations in the hemagglutinin. The cold-adaptation of X-31 virus resulted in four amino acid mutations in the HA. We generated a panel of 7:1 reassortant viruses each carrying the hemagglutinins with individual single amino acid mutations. We examined their phenotypes and found a major attenuating mutation, N81K. This attenuation marker conferred additional temperature-sensitive and attenuation phenotype to the LAIV. Our data indicate that the cold-adapted mutation in the HA confers additional attenuation to the LAIV strain, without compromising its productivity and immune response. - Highlights: • Cold-adaptation process induced four amino acid mutations in the HA of X-31 virus. • The four mutations in the HA also contributed to attenuation of the X-31ca virus • N81K mutation was the most significant marker for the attenuation of X-31ca virus. • Introduction of N81K mutation into H3N2 LAIV further attenuated the vaccine. • This approach provides a useful guideline for enhancing the safety of the LAIVs.

Universal antibodies against the highly conserved influenza fusion peptide cross-neutralize several subtypes of influenza A virus

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Hashem, Anwar M. [Centre for Vaccine Evaluation, Biologics and Genetic Therapies Directorate, HPFB, Health Canada, Ottawa, ON (Canada); Department of Microbiology, Faculty of Medicine, King Abdulaziz University, Jeddah (Saudi Arabia); Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON (Canada); Van Domselaar, Gary [National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, MB (Canada); Li, Changgui; Wang, Junzhi [National Institute for the Control of Pharmaceutical and Biological Products, Beijing (China); She, Yi-Min; Cyr, Terry D. [Centre for Vaccine Evaluation, Biologics and Genetic Therapies Directorate, HPFB, Health Canada, Ottawa, ON (Canada); Sui, Jianhua [Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Department of Medicine, Harvard Medical School, 44 Binney Street, Boston, MA 02115 (United States); He, Runtao [National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, MB (Canada); Marasco, Wayne A. [Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Department of Medicine, Harvard Medical School, 44 Binney Street, Boston, MA 02115 (United States); Li, Xuguang, E-mail: Sean.Li@hc-sc.gc.ca [Centre for Vaccine Evaluation, Biologics and Genetic Therapies Directorate, HPFB, Health Canada, Ottawa, ON (Canada); Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON (Canada)

2010-12-10

Research highlights: {yields} The fusion peptide is the only universally conserved epitope in all influenza viral hemagglutinins. {yields} Anti-fusion peptide antibodies are universal antibodies that cross-react with all influenza HA subtypes. {yields} The universal antibodies cross-neutralize different influenza A subtypes. {yields} The universal antibodies inhibit the fusion process between the viruses and the target cells. -- Abstract: The fusion peptide of influenza viral hemagglutinin plays a critical role in virus entry by facilitating membrane fusion between the virus and target cells. As the fusion peptide is the only universally conserved epitope in all influenza A and B viruses, it could be an attractive target for vaccine-induced immune responses. We previously reported that antibodies targeting the first 14 amino acids of the N-terminus of the fusion peptide could bind to virtually all influenza virus strains and quantify hemagglutinins in vaccines produced in embryonated eggs. Here we demonstrate that these universal antibodies bind to the viral hemagglutinins in native conformation presented in infected mammalian cell cultures and neutralize multiple subtypes of virus by inhibiting the pH-dependant fusion of viral and cellular membranes. These results suggest that this unique, highly-conserved linear sequence in viral hemagglutinin is exposed sufficiently to be attacked by the antibodies during the course of infection and merits further investigation because of potential importance in the protection against diverse strains of influenza viruses.

Universal antibodies against the highly conserved influenza fusion peptide cross-neutralize several subtypes of influenza A virus

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Hashem, Anwar M.; Van Domselaar, Gary; Li, Changgui; Wang, Junzhi; She, Yi-Min; Cyr, Terry D.; Sui, Jianhua; He, Runtao; Marasco, Wayne A.; Li, Xuguang

2010-01-01

Research highlights: → The fusion peptide is the only universally conserved epitope in all influenza viral hemagglutinins. → Anti-fusion peptide antibodies are universal antibodies that cross-react with all influenza HA subtypes. → The universal antibodies cross-neutralize different influenza A subtypes. → The universal antibodies inhibit the fusion process between the viruses and the target cells. -- Abstract: The fusion peptide of influenza viral hemagglutinin plays a critical role in virus entry by facilitating membrane fusion between the virus and target cells. As the fusion peptide is the only universally conserved epitope in all influenza A and B viruses, it could be an attractive target for vaccine-induced immune responses. We previously reported that antibodies targeting the first 14 amino acids of the N-terminus of the fusion peptide could bind to virtually all influenza virus strains and quantify hemagglutinins in vaccines produced in embryonated eggs. Here we demonstrate that these universal antibodies bind to the viral hemagglutinins in native conformation presented in infected mammalian cell cultures and neutralize multiple subtypes of virus by inhibiting the pH-dependant fusion of viral and cellular membranes. These results suggest that this unique, highly-conserved linear sequence in viral hemagglutinin is exposed sufficiently to be attacked by the antibodies during the course of infection and merits further investigation because of potential importance in the protection against diverse strains of influenza viruses.

A simple Pichia pastoris fermentation and downstream processing strategy for making recombinant pandemic Swine Origin Influenza a virus Hemagglutinin protein.

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Athmaram, T N; Singh, Anil Kumar; Saraswat, Shweta; Srivastava, Saurabh; Misra, Princi; Kameswara Rao, M; Gopalan, N; Rao, P V L

2013-02-01

The present Influenza vaccine manufacturing process has posed a clear impediment to initiation of rapid mass vaccination against spreading pandemic influenza. New vaccine strategies are therefore needed that can accelerate the vaccine production. Pichia offers several advantages for rapid and economical bulk production of recombinant proteins and, hence, can be attractive alternative for producing an effective influenza HA based subunit vaccine. The recombinant Pichia harboring the transgene was subjected to fed-batch fermentation at 10 L scale. A simple fermentation and downstream processing strategy is developed for high-yield secretory expression of the recombinant Hemagglutinin protein of pandemic Swine Origin Influenza A virus using Pichia pastoris via fed-batch fermentation. Expression and purification were optimized and the expressed recombinant Hemagglutinin protein was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis, Western blot and MALDI-TOF analysis. In this paper, we describe a fed-batch fermentation protocol for the secreted production of Swine Influenza A Hemagglutinin protein in the P. pastoris GS115 strain. We have shown that there is a clear relationship between product yield and specific growth rate. The fed-batch fermentation and downstream processing methods optimized in the present study have immense practical application for high-level production of the recombinant H1N1 HA protein in a cost effective way using P. pastoris.

Impact of Mutations in the Hemagglutinin of H10N7 Viruses Isolated from Seals on Virus Replication in Avian and Human Cells

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Anne Dittrich

2018-02-01

Full Text Available Wild birds are the reservoir for low-pathogenic avian influenza viruses, which are frequently transmitted to domestic birds and occasionally to mammals. In 2014, an H10N7 virus caused severe mortality in harbor seals in northeastern Europe. Although the hemagglutinin (HA of this virus was closely related to H10 of avian H10N4 virus, it possessed unique nonsynonymous mutations, particularly in the HA1 subunit in or adjacent to the receptor binding domain and proteolytic cleavage site. Here, the impact of these mutations on virus replication was studied in vitro. Using reverse genetics, an avian H10N4 virus was cloned, and nine recombinant viruses carrying one of eight unique mutations or the complete HA from the seal virus were rescued. Receptor binding affinity, replication in avian and mammalian cell cultures, cell-to-cell spread, and HA cleavability of these recombinant viruses were studied. Results show that wild-type recombinant H10N4 virus has high affinity to avian-type sialic acid receptors and no affinity to mammalian-type receptors. The H10N7 virus exhibits dual receptor binding affinity. Interestingly, Q220L (H10 numbering in the rim of the receptor binding pocket increased the affinity of the H10N4 virus to mammal-type receptors and completely abolished the affinity to avian-type receptors. No remarkable differences in cell-to-cell spread or HA cleavability were observed. All viruses, including the wild-type H10N7 virus, replicated at higher levels in chicken cells than in human cells. These results indicate that H10N7 acquired adaptive mutations (e.g., Q220L to enhance replication in mammals and retained replication efficiency in the original avian host.

Impact of Mutations in the Hemagglutinin of H10N7 Viruses Isolated from Seals on Virus Replication in Avian and Human Cells.

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Dittrich, Anne; Scheibner, David; Salaheldin, Ahmed H; Veits, Jutta; Gischke, Marcel; Mettenleiter, Thomas C; Abdelwhab, Elsayed M

2018-02-14

Wild birds are the reservoir for low-pathogenic avian influenza viruses, which are frequently transmitted to domestic birds and occasionally to mammals. In 2014, an H10N7 virus caused severe mortality in harbor seals in northeastern Europe. Although the hemagglutinin (HA) of this virus was closely related to H10 of avian H10N4 virus, it possessed unique nonsynonymous mutations, particularly in the HA1 subunit in or adjacent to the receptor binding domain and proteolytic cleavage site. Here, the impact of these mutations on virus replication was studied in vitro. Using reverse genetics, an avian H10N4 virus was cloned, and nine recombinant viruses carrying one of eight unique mutations or the complete HA from the seal virus were rescued. Receptor binding affinity, replication in avian and mammalian cell cultures, cell-to-cell spread, and HA cleavability of these recombinant viruses were studied. Results show that wild-type recombinant H10N4 virus has high affinity to avian-type sialic acid receptors and no affinity to mammalian-type receptors. The H10N7 virus exhibits dual receptor binding affinity. Interestingly, Q220L (H10 numbering) in the rim of the receptor binding pocket increased the affinity of the H10N4 virus to mammal-type receptors and completely abolished the affinity to avian-type receptors. No remarkable differences in cell-to-cell spread or HA cleavability were observed. All viruses, including the wild-type H10N7 virus, replicated at higher levels in chicken cells than in human cells. These results indicate that H10N7 acquired adaptive mutations (e.g., Q220L) to enhance replication in mammals and retained replication efficiency in the original avian host.

Isolation of recombinant phage antibodies targeting the hemagglutinin cleavage site of highly pathogenic avian influenza virus.

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Jinhua Dong

Full Text Available Highly pathogenic avian influenza (HPAI H5N1 viruses, which have emerged in poultry and other wildlife worldwide, contain a characteristic multi-basic cleavage site (CS in the hemagglutinin protein (HA. Because this arginine-rich CS is unique among influenza virus subtypes, antibodies against this site have the potential to specifically diagnose pathogenic H5N1. By immunizing mice with the CS peptide and screening a phage display library, we isolated four antibody Fab fragment clones that specifically bind the antigen peptide and several HPAI H5N1 HA proteins in different clades. The soluble Fab fragments expressed in Escherichia coli bound the CS peptide and the H5N1 HA protein with nanomolar affinity. In an immunofluorescence assay, these Fab fragments stained cells infected with HPAI H5N1 but not those infected with a less virulent strain. Lastly, all the Fab clones could detect the CS peptide and H5N1 HA protein by open sandwich ELISA. Thus, these recombinant Fab fragments will be useful novel reagents for the rapid and specific detection of HPAI H5N1 virus.

Analyses of Evolutionary Characteristics of the Hemagglutinin-Esterase Gene of Influenza C Virus during a Period of 68 Years Reveals Evolutionary Patterns Different from Influenza A and B Viruses

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Yuki Furuse

2016-11-01

Full Text Available Infections with the influenza C virus causing respiratory symptoms are common, particularly among children. Since isolation and detection of the virus are rarely performed, compared with influenza A and B viruses, the small number of available sequences of the virus makes it difficult to analyze its evolutionary dynamics. Recently, we reported the full genome sequence of 102 strains of the virus. Here, we exploited the data to elucidate the evolutionary characteristics and phylodynamics of the virus compared with influenza A and B viruses. Along with our data, we obtained public sequence data of the hemagglutinin-esterase gene of the virus; the dataset consists of 218 unique sequences of the virus collected from 14 countries between 1947 and 2014. Informatics analyses revealed that (1 multiple lineages have been circulating globally; (2 there have been weak and infrequent selective bottlenecks; (3 the evolutionary rate is low because of weak positive selection and a low capability to induce mutations; and (4 there is no significant positive selection although a few mutations affecting its antigenicity have been induced. The unique evolutionary dynamics of the influenza C virus must be shaped by multiple factors, including virological, immunological, and epidemiological characteristics.

Kallistatin Ameliorates Influenza Virus Pathogenesis by Inhibition of Kallikrein-Related Peptidase 1-Mediated Cleavage of Viral Hemagglutinin

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Leu, Chia-Hsing; Yang, Mei-Lin; Chung, Nai-Hui; Huang, Yen-Jang; Su, Yu-Chu; Chen, Yi-Cheng; Lin, Chia-Cheng; Shieh, Gia-Shing; Chang, Meng-Ya; Wang, Shainn-Wei; Chang, Yao; Chao, Julie; Chao, Lee

2015-01-01

Proteolytic cleavage of the hemagglutinin (HA) of influenza virus by host trypsin-like proteases is required for viral infectivity. Some serine proteases are capable of cleaving influenza virus HA, whereas some serine protease inhibitors (serpins) inhibit the HA cleavage in various cell types. Kallikrein-related peptidase 1 (KLK1, also known as tissue kallikrein) is a widely distributed serine protease. Kallistatin, a serpin synthesized mainly in the liver and rapidly secreted into the circulation, forms complexes with KLK1 and inhibits its activity. Here, we investigated the roles of KLK1 and kallistatin in influenza virus infection. We show that the levels of KLK1 increased, whereas those of kallistatin decreased, in the lungs of mice during influenza virus infection. KLK1 cleaved H1, H2, and H3 HA molecules and consequently enhanced viral production. In contrast, kallistatin inhibited KLK1-mediated HA cleavage and reduced viral production. Cells transduced with the kallistatin gene secreted kallistatin extracellularly, which rendered them more resistant to influenza virus infection. Furthermore, lentivirus-mediated kallistatin gene delivery protected mice against lethal influenza virus challenge by reducing the viral load, inflammation, and injury in the lung. Taking the data together, we determined that KLK1 and kallistatin contribute to the pathogenesis of influenza virus by affecting the cleavage of the HA peptide and inflammatory responses. This study provides a proof of principle for the potential therapeutic application of kallistatin or other KLK1 inhibitors for influenza. Since proteolytic activation also enhances the infectivity of some other viruses, kallistatin and other kallikrein inhibitors may be explored as antiviral agents against these viruses. PMID:26149981

Epitope Dampening Monotypic Measles Virus Hemagglutinin Glycoprotein Results in Resistance to Cocktail of Monoclonal Antibodies

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Lech, Patrycja J.; Tobin, Gregory J.; Bushnell, Ruth; Gutschenritter, Emily; Pham, Linh D.; Nace, Rebecca; Verhoeyen, Els; Cosset, François-Loïc; Muller, Claude P.; Russell, Stephen J.; Nara, Peter L.

2013-01-01

The measles virus (MV) is serologically monotypic. Life-long immunity is conferred by a single attack of measles or following vaccination with the MV vaccine. This is contrary to viruses such as influenza, which readily develop resistance to the immune system and recur. A better understanding of factors that restrain MV to one serotype may allow us to predict if MV will remain monotypic in the future and influence the design of novel MV vaccines and therapeutics. MV hemagglutinin (H) glycoprotein, binds to cellular receptors and subsequently triggers the fusion (F) glycoprotein to fuse the virus into the cell. H is also the major target for neutralizing antibodies. To explore if MV remains monotypic due to a lack of plasticity of the H glycoprotein, we used the technology of Immune Dampening to generate viruses with rationally designed N-linked glycosylation sites and mutations in different epitopes and screened for viruses that escaped monoclonal antibodies (mAbs). We then combined rationally designed mutations with naturally selected mutations to generate a virus resistant to a cocktail of neutralizing mAbs targeting four different epitopes simultaneously. Two epitopes were protected by engineered N-linked glycosylations and two epitopes acquired escape mutations via two consecutive rounds of artificial selection in the presence of mAbs. Three of these epitopes were targeted by mAbs known to interfere with receptor binding. Results demonstrate that, within the epitopes analyzed, H can tolerate mutations in different residues and additional N-linked glycosylations to escape mAbs. Understanding the degree of change that H can tolerate is important as we follow its evolution in a host whose immunity is vaccine induced by genotype A strains instead of multiple genetically distinct wild-type MVs. PMID:23300970

Structure of the parainfluenza virus 5 (PIV5 hemagglutinin-neuraminidase (HN ectodomain.

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Brett D Welch

Full Text Available Paramyxoviruses cause a wide variety of human and animal diseases. They infect host cells using the coordinated action of two surface glycoproteins, the receptor binding protein (HN, H, or G and the fusion protein (F. HN binds sialic acid on host cells (hemagglutinin activity and hydrolyzes these receptors during viral egress (neuraminidase activity, NA. Additionally, receptor binding is thought to induce a conformational change in HN that subsequently triggers major refolding in homotypic F, resulting in fusion of virus and target cell membranes. HN is an oligomeric type II transmembrane protein with a short cytoplasmic domain and a large ectodomain comprising a long helical stalk and large globular head domain containing the enzymatic functions (NA domain. Extensive biochemical characterization has revealed that HN-stalk residues determine F specificity and activation. However, the F/HN interaction and the mechanisms whereby receptor binding regulates F activation are poorly defined. Recently, a structure of Newcastle disease virus (NDV HN ectodomain revealed the heads (NA domains in a "4-heads-down" conformation whereby two of the heads form a symmetrical interaction with two sides of the stalk. The interface includes stalk residues implicated in triggering F, and the heads sterically shield these residues from interaction with F (at least on two sides. Here we report the x-ray crystal structure of parainfluenza virus 5 (PIV5 HN ectodomain in a "2-heads-up/2-heads-down" conformation where two heads (covalent dimers are in the "down position," forming a similar interface as observed in the NDV HN ectodomain structure, and two heads are in an "up position." The structure supports a model in which the heads of HN transition from down to up upon receptor binding thereby releasing steric constraints and facilitating the interaction between critical HN-stalk residues and F.

A Phylogeny-Based Global Nomenclature System and Automated Annotation Tool for H1 Hemagglutinin Genes from Swine Influenza A Viruses

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Macken, Catherine A.; Lewis, Nicola S.; Van Reeth, Kristien; Brown, Ian H.; Swenson, Sabrina L.; Simon, Gaëlle; Saito, Takehiko; Berhane, Yohannes; Ciacci-Zanella, Janice; Pereda, Ariel; Davis, C. Todd; Donis, Ruben O.; Webby, Richard J.

2016-01-01

ABSTRACT The H1 subtype of influenza A viruses (IAVs) has been circulating in swine since the 1918 human influenza pandemic. Over time, and aided by further introductions from nonswine hosts, swine H1 viruses have diversified into three genetic lineages. Due to limited global data, these H1 lineages were named based on colloquial context, leading to a proliferation of inconsistent regional naming conventions. In this study, we propose rigorous phylogenetic criteria to establish a globally consistent nomenclature of swine H1 virus hemagglutinin (HA) evolution. These criteria applied to a data set of 7,070 H1 HA sequences led to 28 distinct clades as the basis for the nomenclature. We developed and implemented a web-accessible annotation tool that can assign these biologically informative categories to new sequence data. The annotation tool assigned the combined data set of 7,070 H1 sequences to the correct clade more than 99% of the time. Our analyses indicated that 87% of the swine H1 viruses from 2010 to the present had HAs that belonged to 7 contemporary cocirculating clades. Our nomenclature and web-accessible classification tool provide an accurate method for researchers, diagnosticians, and health officials to assign clade designations to HA sequences. The tool can be updated readily to track evolving nomenclature as new clades emerge, ensuring continued relevance. A common global nomenclature facilitates comparisons of IAVs infecting humans and pigs, within and between regions, and can provide insight into the diversity of swine H1 influenza virus and its impact on vaccine strain selection, diagnostic reagents, and test performance, thereby simplifying communication of such data. IMPORTANCE A fundamental goal in the biological sciences is the definition of groups of organisms based on evolutionary history and the naming of those groups. For influenza A viruses (IAVs) in swine, understanding the hemagglutinin (HA) genetic lineage of a circulating strain aids

"Boom" and "Bust" cycles in virus growth suggest multiple selective forces in influenza a evolution

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Marquart Mary E

2011-04-01

Full Text Available Abstract Background Influenza A virus evolution in humans is driven at least in part by mutations allowing the virus to escape antibody neutralization. Little is known about the evolution of influenza in birds, a major reservoir of influenza A. Methods Neutralizing polyclonal antiserum was raised in chicken against reassortant influenza virus, CalX, bearing the hemagglutinin (HA and neuraminidase (NA of A/California/7/2004 [H3N2]. CalX was serially passaged in the presence of anti-CalX polyclonal IgY to derive viruses capable of growth in the presence of antibody. Results Polyclonal chicken antibody neutralized both HA activity and infection by CalX, but had no effect on a strain bearing an earlier human H3 and an irrelevant neuraminidase (A/Memphis/71-Bellamy/42 [H3N1]. Surprisingly, most of the antibody-resistant viruses were still at least partially sensitive to neutralization of HA activity and viral infection. Although mutant HA genes bearing changes that might affect antibody neutralization were identified, the vast majority of HA sequences obtained were identical to wild type, and no individual mutant sequence was found in more than one passage, suggesting that those mutations that were observed did not confer sufficient selective advantage to come to dominate the population. Different passages yielded infectious foci of varying size and plaques of varying size and morphology. Yields of infectious virus and relative frequency of different morphologies changed markedly from passage to passage. Sequences of bulk, uncloned PCR products from antibody-resistant passages indicated changes in the PB2 and PA proteins with respect to the wild type virus. Conclusions Each antibody-selected passage consisted of a variety of different cocirculating populations, rather than pure populations of virus able to escape antibody by changes in antibody epitopes. The ability to escape antibody is apparently due to changes in genes encoding the viral

Diagnostic potential of recombinant scFv antibodies generated against hemagglutinin protein of influenza A virus

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Roopali eRajput

2015-09-01

Full Text Available Human influenza A viruses have been the cause of enormous socio-economic losses worldwide. In order to combat such a notorious pathogen, hemagglutinin protein (HA has been a preferred target for generation of neutralizing-antibodies, as potent therapeutic/ diagnostic agents. In the present study, recombinant anti-HA single chain variable fragment (scFv antibodies were constructed using the phage display technology to aid in diagnosis and treatment of human influenza A virus infections. Spleen cells of mice hyper-immunized with A/New Caledonia/20/99 (H1N1 virus were used as the source for recombinant antibody (rAb production. The antigen-binding phages were quantified after 6 rounds of bio-panning against A/New Caledonia/20/99 (H1N1, A/California/07/2009 (H1N1-like, or A/Udorn/307/72(H3N2 viruses. The phage yield was maximum for the A/New Caledonia/20/99 (H1N1, however, considerable cross-reactivity was observed for the other virus strains as well. The HA-specific polyclonal rAb preparation was subjected to selection of single clones for identification of high reactive relatively conserved epitopes. The high affinity rAbs were tested against certain known conserved HA epitopes by peptide ELISA. Three recombinant mAbs showed reactivity with both the H1N1 strains and one (C5 showed binding with all the three viral strains. The C5 antibody was thus used for development of an ELISA test for diagnosis of influenza virus infection. Based on the sample size in the current analysis, the ELISA test demonstrated 83.9% sensitivity and 100% specificity. Thus, the ELISA, developed in our study, may prove as a cheaper alternative to the presently used real time RT-PCR test for detection of human influenza A viruses in clinical specimens, which will be beneficial, especially in the developing countries. Since, the two antibodies identified in this study are reactive to conserved HA epitopes; these may prove as potential therapeutic agents as well.

The recombinant EHV-1 vector producing CDV hemagglutinin as potential vaccine against canine distemper.

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Pan, Zihao; Liu, Jin; Ma, Jiale; Jin, Qiuli; Yao, Huochun; Osterrieder, Nikolaus

2017-10-01

Canine distemper virus (CDV), is a pantropic agent of morbillivirus that causes fetal disease in dogs. Base on a broad host rang of CDV, the continued vaccines inoculation is unavoidable to pose gene recombination risk in vaccine virus and wild virus. The current study presents the construction of novel vectors, using equine herpesvirus type 1 (EHV-1) expressing the canine distemper virus (CDV). The recent field strain hemagglutinin protein and nucleoprotein were used for the construction of the viral vector vaccines. Based on the Bacterial artificial chromosome (BAC) genomes of EHV-1 RacH strain, the recombinant EHV-1 vaccine virus encoding CDV hemagglutinin protein (EHV-H) or CDV nucleoprotein (EHV-N) was constructed separately. The constructed BACs were rescued after 72 h post infection, and the expression of H or N in the recombinant viruses was confirmed by western-blotting. Furthermore, high levels of neutralizing antibodies were induced persistently following vaccination in the groups EHV-H&EHV-N and EHV-H, but the EHV-N group. The groups of vaccinated EHV-H and EHV-H&EHV-N pups were monitored for clinical signs, whereas the vaccinated EHV-N group developed moderate symptoms. The present study demonstrated that EHV-1 based recombinant virus carrying CDV H could be a promising vaccine candidate against canine distemper. Copyright © 2017. Published by Elsevier Ltd.

Playing Hide and Seek: How Glycosylation of the Influenza Virus Hemagglutinin Can Modulate the Immune Response to Infection

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Michelle D. Tate

2014-03-01

Full Text Available Seasonal influenza A viruses (IAV originate from pandemic IAV and have undergone changes in antigenic structure, including addition of glycans to the hemagglutinin (HA glycoprotein. The viral HA is the major target recognized by neutralizing antibodies and glycans have been proposed to shield antigenic sites on HA, thereby promoting virus survival in the face of widespread vaccination and/or infection. However, addition of glycans can also interfere with the receptor binding properties of HA and this must be compensated for by additional mutations, creating a fitness barrier to accumulation of glycosylation sites. In addition, glycans on HA are also recognized by phylogenetically ancient lectins of the innate immune system and the benefit provided by evasion of humoral immunity is balanced by attenuation of infection. Therefore, a fine balance must exist regarding the optimal pattern of HA glycosylation to offset competing pressures associated with recognition by innate defenses, evasion of humoral immunity and maintenance of virus fitness. In this review, we examine HA glycosylation patterns of IAV associated with pandemic and seasonal influenza and discuss recent advancements in our understanding of interactions between IAV glycans and components of innate and adaptive immunity.

Playing Hide and Seek: How Glycosylation of the Influenza Virus Hemagglutinin Can Modulate the Immune Response to Infection

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Tate, Michelle D.; Job, Emma R.; Deng, Yi-Mo; Gunalan, Vithiagaran; Maurer-Stroh, Sebastian; Reading, Patrick C.

2014-01-01

Seasonal influenza A viruses (IAV) originate from pandemic IAV and have undergone changes in antigenic structure, including addition of glycans to the hemagglutinin (HA) glycoprotein. The viral HA is the major target recognized by neutralizing antibodies and glycans have been proposed to shield antigenic sites on HA, thereby promoting virus survival in the face of widespread vaccination and/or infection. However, addition of glycans can also interfere with the receptor binding properties of HA and this must be compensated for by additional mutations, creating a fitness barrier to accumulation of glycosylation sites. In addition, glycans on HA are also recognized by phylogenetically ancient lectins of the innate immune system and the benefit provided by evasion of humoral immunity is balanced by attenuation of infection. Therefore, a fine balance must exist regarding the optimal pattern of HA glycosylation to offset competing pressures associated with recognition by innate defenses, evasion of humoral immunity and maintenance of virus fitness. In this review, we examine HA glycosylation patterns of IAV associated with pandemic and seasonal influenza and discuss recent advancements in our understanding of interactions between IAV glycans and components of innate and adaptive immunity. PMID:24638204

Systemic and oral immunogenicity of hemagglutinin protein of rinderpest virus expressed by transgenic peanut plants in a mouse model

International Nuclear Information System (INIS)

Khandelwal, Abha; Renukaradhya, G.J.; Rajasekhar, M.; Sita, G. Lakshmi; Shaila, M.S.

2004-01-01

Rinderpest causes a devastating disease, often fatal, in wild and domestic ruminants. It has been eradicated successfully using a live, attenuated vaccine from most part of the world leaving a few foci of disease in parts of Africa, the Middle East, and South Asia. We have developed transgenic peanut (Arachis hypogaea L.) plants expressing hemagglutinin (H) protein of rinderpest virus (RPV), which is antigenically authentic. In this work, we have evaluated the immunogenicity of peanut-expressed H protein using mouse model, administered parenterally as well as orally. Intraperitoneal immunization of mice with the transgenic peanut extract elicited antibody response specific to H. These antibodies neutralized virus infectivity in vitro. Oral immunization of mice with transgenic peanut induced H-specific serum IgG and IgA antibodies. The systemic and oral immunogenicity of plant-derived H in absence of any adjuvant indicates the potential of edible vaccine for rinderpest

Protection of pigs against pandemic swine origin H1N1 influenza A virus infection by hemagglutinin- or neuraminidase-expressing attenuated pseudorabies virus recombinants.

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Klingbeil, Katharina; Lange, Elke; Blohm, Ulrike; Teifke, Jens P; Mettenleiter, Thomas C; Fuchs, Walter

2015-03-02

Influenza is an important respiratory disease of pigs, and may lead to novel human pathogens like the 2009 pandemic H1N1 swine-origin influenza virus (SoIV). Therefore, improved influenza vaccines for pigs are required. Recently, we demonstrated that single intranasal immunization with a hemagglutinin (HA)-expressing pseudorabies virus recombinant of vaccine strain Bartha (PrV-Ba) protected pigs from H1N1 SoIV challenge (Klingbeil et al., 2014). Now we investigated enhancement of efficacy by prime-boost vaccination and/or intramuscular administration. Furthermore, a novel PrV-Ba recombinant expressing codon-optimized N1 neuraminidase (NA) was included. In vitro replication of this virus was only slightly affected compared to parental virus. Unlike HA, the abundantly expressed NA was efficiently incorporated into PrV particles. Immunization of pigs with the two PrV recombinants, either singly or in combination, induced B cell proliferation and the expected SoIV-specific antibodies, whose titers increased substantially after boost vaccination. After immunization of animals with either PrV recombinant H1N1 SoIV challenge virus replication was significantly reduced compared to PrV-Ba vaccinated or naïve controls. Protective efficacy of HA-expressing PrV was higher than of NA-expressing PrV, and not significantly enhanced by combination. Despite higher serum antibody titers obtained after intramuscular immunization, transmission of challenge virus to naïve contact animals was only prevented after intranasal prime-boost vaccination with HA-expressing PrV-Ba. Copyright © 2015 Elsevier B.V. All rights reserved.

Protective immunity provided by HLA-A2 epitopes for fusion and hemagglutinin proteins of measles virus

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Oh, Sang Kon; Stegman, Brian; Pendleton, C. David; Ota, Martin O.; Pan, C.-H.; Griffin, Diane E.; Burke, Donald S.; Berzofsky, Jay A.

2006-01-01

Natural infection and vaccination with a live-attenuated measles virus (MV) induce CD8 + T-cell-mediated immune responses that may play a central role in controlling MV infection. In this study, we show that newly identified human HLA-A2 epitopes from MV hemagglutinin (H) and fusion (F) proteins induced protective immunity in HLA-A2 transgenic mice challenged with recombinant vaccinia viruses expressing F or H protein. HLA-A2 epitopes were predicted and synthesized. Five and four peptides from H and F, respectively, bound to HLA-A2 molecules in a T2-binding assay, and four from H and two from F could induce peptide-specific CD8 + T cell responses in HLA-A2 transgenic mice. Further experiments proved that three peptides from H (H9-567, H10-250, and H10-516) and one from F protein (F9-57) were endogenously processed and presented on HLA-A2 molecules. All peptides tested in this study are common to 5 different strains of MV including Edmonston. In both A2K b and HHD-2 mice, the identified peptide epitopes induced protective immunity against recombinant vaccinia viruses expressing H or F. Because F and H proteins induce neutralizing antibodies, they are major components of new vaccine strategies, and therefore data from this study will contribute to the development of new vaccines against MV infection

Anti-Hemagglutinin Antibody Derived Lead Peptides for Inhibitors of Influenza Virus Binding.

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Henry Memczak

Full Text Available Antibodies against spike proteins of influenza are used as a tool for characterization of viruses and therapeutic approaches. However, development, production and quality control of antibodies is expensive and time consuming. To circumvent these difficulties, three peptides were derived from complementarity determining regions of an antibody heavy chain against influenza A spike glycoprotein. Their binding properties were studied experimentally, and by molecular dynamics simulations. Two peptide candidates showed binding to influenza A/Aichi/2/68 H3N2. One of them, termed PeB, with the highest affinity prevented binding to and infection of target cells in the micromolar region without any cytotoxic effect. PeB matches best the conserved receptor binding site of hemagglutinin. PeB bound also to other medical relevant influenza strains, such as human-pathogenic A/California/7/2009 H1N1, and avian-pathogenic A/Mute Swan/Rostock/R901/2006 H7N1. Strategies to improve the affinity and to adapt specificity are discussed and exemplified by a double amino acid substituted peptide, obtained by substitutional analysis. The peptides and their derivatives are of great potential for drug development as well as biosensing.

Quantitative characterization of glycan-receptor binding of H9N2 influenza A virus hemagglutinin.

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Karunya Srinivasan

Full Text Available Avian influenza subtypes such as H5, H7 and H9 are yet to adapt to the human host so as to establish airborne transmission between humans. However, lab-generated reassorted viruses possessing hemagglutinin (HA and neuraminidase (NA genes from an avian H9 isolate and other genes from a human-adapted (H3 or H1 subtype acquired two amino acid changes in HA and a single amino acid change in NA that confer respiratory droplet transmission in ferrets. We previously demonstrated for human-adapted H1, H2 and H3 subtypes that quantitative binding affinity of their HA to α2→6 sialylated glycan receptors correlates with respiratory droplet transmissibility of the virus in ferrets. Such a relationship remains to be established for H9 HA. In this study, we performed a quantitative biochemical characterization of glycan receptor binding properties of wild-type and mutant forms of representative H9 HAs that were previously used in context of reassorted viruses in ferret transmission studies. We demonstrate here that distinct molecular interactions in the glycan receptor-binding site of different H9 HAs affect the glycan-binding specificity and affinity. Further we show that α2→6 glycan receptor-binding affinity of a mutant H9 HA carrying Thr-189→Ala amino acid change correlates with the respiratory droplet transmission in ferrets conferred by this change. Our findings contribute to a framework for monitoring the evolution of H9 HA by understanding effects of molecular changes in HA on glycan receptor-binding properties.

Identification of Low- and High-Impact Hemagglutinin Amino Acid Substitutions That Drive Antigenic Drift of Influenza A(H1N1 Viruses.

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William T Harvey

2016-04-01

Full Text Available Determining phenotype from genetic data is a fundamental challenge. Identification of emerging antigenic variants among circulating influenza viruses is critical to the vaccine virus selection process, with vaccine effectiveness maximized when constituents are antigenically similar to circulating viruses. Hemagglutination inhibition (HI assay data are commonly used to assess influenza antigenicity. Here, sequence and 3-D structural information of hemagglutinin (HA glycoproteins were analyzed together with corresponding HI assay data for former seasonal influenza A(H1N1 virus isolates (1997-2009 and reference viruses. The models developed identify and quantify the impact of eighteen amino acid substitutions on the antigenicity of HA, two of which were responsible for major transitions in antigenic phenotype. We used reverse genetics to demonstrate the causal effect on antigenicity for a subset of these substitutions. Information on the impact of substitutions allowed us to predict antigenic phenotypes of emerging viruses directly from HA gene sequence data and accuracy was doubled by including all substitutions causing antigenic changes over a model incorporating only the substitutions with the largest impact. The ability to quantify the phenotypic impact of specific amino acid substitutions should help refine emerging techniques that predict the evolution of virus populations from one year to the next, leading to stronger theoretical foundations for selection of candidate vaccine viruses. These techniques have great potential to be extended to other antigenically variable pathogens.

Identification of Low- and High-Impact Hemagglutinin Amino Acid Substitutions That Drive Antigenic Drift of Influenza A(H1N1) Viruses

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Harvey, William T.; Benton, Donald J.; Gregory, Victoria; Hall, James P. J.; Daniels, Rodney S.; Bedford, Trevor; Haydon, Daniel T.; Hay, Alan J.; McCauley, John W.; Reeve, Richard

2016-01-01

Determining phenotype from genetic data is a fundamental challenge. Identification of emerging antigenic variants among circulating influenza viruses is critical to the vaccine virus selection process, with vaccine effectiveness maximized when constituents are antigenically similar to circulating viruses. Hemagglutination inhibition (HI) assay data are commonly used to assess influenza antigenicity. Here, sequence and 3-D structural information of hemagglutinin (HA) glycoproteins were analyzed together with corresponding HI assay data for former seasonal influenza A(H1N1) virus isolates (1997–2009) and reference viruses. The models developed identify and quantify the impact of eighteen amino acid substitutions on the antigenicity of HA, two of which were responsible for major transitions in antigenic phenotype. We used reverse genetics to demonstrate the causal effect on antigenicity for a subset of these substitutions. Information on the impact of substitutions allowed us to predict antigenic phenotypes of emerging viruses directly from HA gene sequence data and accuracy was doubled by including all substitutions causing antigenic changes over a model incorporating only the substitutions with the largest impact. The ability to quantify the phenotypic impact of specific amino acid substitutions should help refine emerging techniques that predict the evolution of virus populations from one year to the next, leading to stronger theoretical foundations for selection of candidate vaccine viruses. These techniques have great potential to be extended to other antigenically variable pathogens. PMID:27057693

Large-scale FMO-MP3 calculations on the surface proteins of influenza virus, hemagglutinin (HA) and neuraminidase (NA)

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Mochizuki, Yuji; Yamashita, Katsumi; Fukuzawa, Kaori; Takematsu, Kazutomo; Watanabe, Hirofumi; Taguchi, Naoki; Okiyama, Yoshio; Tsuboi, Misako; Nakano, Tatsuya; Tanaka, Shigenori

2010-06-01

Two proteins on the influenza virus surface have been well known. One is hemagglutinin (HA) associated with the infection to cells. The fragment molecular orbital (FMO) calculations were performed on a complex consisting of HA trimer and two Fab-fragments at the third-order Møller-Plesset perturbation (MP3) level. The numbers of residues and 6-31G basis functions were 2351 and 201276, and thus a massively parallel-vector computer was utilized to accelerate the processing. This FMO-MP3 job was completed in 5.8 h with 1024 processors. Another protein is neuraminidase (NA) involved in the escape from infected cells. The FMO-MP3 calculation was also applied to analyze the interactions between oseltamivir and surrounding residues in pharmacophore.

Synonymous Mutations at the Beginning of the Influenza A Virus Hemagglutinin Gene Impact Experimental Fitness.

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Canale, Aneth S; Venev, Sergey V; Whitfield, Troy W; Caffrey, Daniel R; Marasco, Wayne A; Schiffer, Celia A; Kowalik, Timothy F; Jensen, Jeffrey D; Finberg, Robert W; Zeldovich, Konstantin B; Wang, Jennifer P; Bolon, Daniel N A

2018-04-13

The fitness effects of synonymous mutations can provide insights into biological and evolutionary mechanisms. We analyzed the experimental fitness effects of all single-nucleotide mutations, including synonymous substitutions, at the beginning of the influenza A virus hemagglutinin (HA) gene. Many synonymous substitutions were deleterious both in bulk competition and for individually isolated clones. Investigating protein and RNA levels of a subset of individually expressed HA variants revealed that multiple biochemical properties contribute to the observed experimental fitness effects. Our results indicate that a structural element in the HA segment viral RNA may influence fitness. Examination of naturally evolved sequences in human hosts indicates a preference for the unfolded state of this structural element compared to that found in swine hosts. Our overall results reveal that synonymous mutations may have greater fitness consequences than indicated by simple models of sequence conservation, and we discuss the implications of this finding for commonly used evolutionary tests and analyses. Copyright © 2018. Published by Elsevier Ltd.

Chicken galectin-1B inhibits Newcastle disease virus adsorption and replication through binding to hemagglutinin-neuraminidase (HN) glycoprotein.

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Sun, Junfeng; Han, Zongxi; Qi, Tianming; Zhao, Ran; Liu, Shengwang

2017-12-08

Galectin-1 is an important immunoregulatory factor and can mediate the host-pathogen interaction via binding glycans on the surface of various viruses. We previously reported that avian respiratory viruses, including lentogenic Newcastle disease virus (NDV), can induce up-regulation of chicken galectin (CG)-1B in the primary target organ. In this study, we investigated whether CG-1B participated in the infectious process of NDV in chickens. We demonstrated that velogenic NDV induced up-regulation of CG-1B in target organs. We also found that CG-1B directly bound to NDV virions and inhibited their hemagglutination activity in vitro We confirmed that CG-1B interacted with NDV hemagglutinin-neuraminidase (HN) glycoprotein, in which the specific G4 N -glycans significantly contributed to the interaction between CG-1B and HN glycoprotein. The presence of extracellular CG-1B, rather than the internalization process, inhibited adsorption of NDV. The interaction between intracellular CG-1B and NDV HN glycoproteins inhibited cell-surface expression of HN glycoprotein and reduced the titer of progeny virus in NDV-infected DF-1 cells. Significantly, the replication of parental and HN glycosylation mutant viruses in CG-1B knockdown and overexpression cells demonstrated that the replication of NDV was correlated with the expression of CG-1B in a specific glycan-dependent manner. Collectively, our results indicate that CG-1B has anti-NDV activity by binding to N -glycans on HN glycoprotein. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Avian Influenza Virus Infection of Immortalized Human Respiratory Epithelial Cells Depends upon a Delicate Balance between Hemagglutinin Acid Stability and Endosomal pH.

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Daidoji, Tomo; Watanabe, Yohei; Ibrahim, Madiha S; Yasugi, Mayo; Maruyama, Hisataka; Masuda, Taisuke; Arai, Fumihito; Ohba, Tomoyuki; Honda, Ayae; Ikuta, Kazuyoshi; Nakaya, Takaaki

2015-04-24

The highly pathogenic avian influenza (AI) virus, H5N1, is a serious threat to public health worldwide. Both the currently circulating H5N1 and previously circulating AI viruses recognize avian-type receptors; however, only the H5N1 is highly infectious and virulent in humans. The mechanism(s) underlying this difference in infectivity remains unclear. The aim of this study was to clarify the mechanisms responsible for the difference in infectivity between the current and previously circulating strains. Primary human small airway epithelial cells (SAECs) were transformed with the SV40 large T-antigen to establish a series of clones (SAEC-Ts). These clones were then used to test the infectivity of AI strains. Human SAEC-Ts could be broadly categorized into two different types based on their susceptibility (high or low) to the viruses. SAEC-T clones were poorly susceptible to previously circulating AI but were completely susceptible to the currently circulating H5N1. The hemagglutinin (HA) of the current H5N1 virus showed greater membrane fusion activity at higher pH levels than that of previous AI viruses, resulting in broader cell tropism. Moreover, the endosomal pH was lower in high susceptibility SAEC-T clones than that in low susceptibility SAEC-T clones. Taken together, the results of this study suggest that the infectivity of AI viruses, including H5N1, depends upon a delicate balance between the acid sensitivity of the viral HA and the pH within the endosomes of the target cell. Thus, one of the mechanisms underlying H5N1 pathogenesis in humans relies on its ability to fuse efficiently with the endosomes in human airway epithelial cells. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Acid Stability of the Hemagglutinin Protein Regulates H5N1 Influenza Virus Pathogenicity

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DuBois, Rebecca M.; Zaraket, Hassan; Reddivari, Muralidhar; Heath, Richard J.; White, Stephen W.; Russell, Charles J. (Tennessee-HSC); (SJCH)

2012-12-10

Highly pathogenic avian influenza viruses of the H5N1 subtype continue to threaten agriculture and human health. Here, we use biochemistry and x-ray crystallography to reveal how amino-acid variations in the hemagglutinin (HA) protein contribute to the pathogenicity of H5N1 influenza virus in chickens. HA proteins from highly pathogenic (HP) A/chicken/Hong Kong/YU562/2001 and moderately pathogenic (MP) A/goose/Hong Kong/437-10/1999 isolates of H5N1 were found to be expressed and cleaved in similar amounts, and both proteins had similar receptor-binding properties. However, amino-acid variations at positions 104 and 115 in the vestigial esterase sub-domain of the HA1 receptor-binding domain (RBD) were found to modulate the pH of HA activation such that the HP and MP HA proteins are activated for membrane fusion at pH 5.7 and 5.3, respectively. In general, an increase in H5N1 pathogenicity in chickens was found to correlate with an increase in the pH of HA activation for mutant and chimeric HA proteins in the observed range of pH 5.2 to 6.0. We determined a crystal structure of the MP HA protein at 2.50 {angstrom} resolution and two structures of HP HA at 2.95 and 3.10 {angstrom} resolution. Residues 104 and 115 that modulate the acid stability of the HA protein are situated at the N- and C-termini of the 110-helix in the vestigial esterase sub-domain, which interacts with the B loop of the HA2 stalk domain. Interactions between the 110-helix and the stalk domain appear to be important in regulating HA protein acid stability, which in turn modulates influenza virus replication and pathogenesis. Overall, an optimal activation pH of the HA protein is found to be necessary for high pathogenicity by H5N1 influenza virus in avian species.

Immunization with plasmid DNA encoding the hemagglutinin and the nucleoprotein confers robust protection against a lethal canine distemper virus challenge.

Science.gov (United States)

Dahl, Lotte; Jensen, Trine Hammer; Gottschalck, Elisabeth; Karlskov-Mortensen, Peter; Jensen, Tove Dannemann; Nielsen, Line; Andersen, Mads Klindt; Buckland, Robin; Wild, T Fabian; Blixenkrone-Møller, Merete

2004-09-09

We have investigated the protective effect of immunization of a highly susceptible natural host of canine distemper virus (CDV) with DNA plasmids encoding the viral nucleoprotein (N) and hemagglutinin (H). The combined intradermal and intramuscular routes of immunization elicited high virus-neutralizing serum antibody titres in mink (Mustela vison). To mimic natural exposure, we also conducted challenge infection by horizontal transmission from infected contact animals. Other groups received a lethal challenge infection by administration to the mucosae of the respiratory tract and into the muscle. One of the mink vaccinated with N plasmid alone developed severe disease after challenge. In contrast, vaccination with the H plasmid together with the N plasmid conferred solid protection against disease and we were unable to detect CDV infection in PBMCs or in different tissues after challenge. Our findings show that DNA immunization by the combined intradermal and intramuscular routes can confer solid protective immunity against naturally transmitted morbillivirus infection and disease.

Characterization of Hemagglutinin Negative Botulinum Progenitor Toxins

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Suzanne R. Kalb

2017-06-01

Full Text Available Botulism is a disease involving intoxication with botulinum neurotoxins (BoNTs, toxic proteins produced by Clostridium botulinum and other clostridia. The 150 kDa neurotoxin is produced in conjunction with other proteins to form the botulinum progenitor toxin complex (PTC, alternating in size from 300 kDa to 500 kDa. These progenitor complexes can be classified into hemagglutinin positive or hemagglutinin negative, depending on the ability of some of the neurotoxin-associated proteins (NAPs to cause hemagglutination. The hemagglutinin positive progenitor toxin complex consists of BoNT, nontoxic non-hemagglutinin (NTNH, and three hemagglutinin proteins; HA-70, HA-33, and HA-17. Hemagglutinin negative progenitor toxin complexes contain BoNT and NTNH as the minimally functional PTC (M-PTC, but not the three hemagglutinin proteins. Interestingly, the genome of hemagglutinin negative progenitor toxin complexes comprises open reading frames (orfs which encode for three proteins, but the existence of these proteins has not yet been extensively demonstrated. In this work, we demonstrate that these three proteins exist and form part of the PTC for hemagglutinin negative complexes. Several hemagglutinin negative strains producing BoNT/A, /E, and /F were found to contain the three open reading frame proteins. Additionally, several BoNT/A-containing bivalent strains were examined, and NAPs from both genes, including the open reading frame proteins, were associated with BoNT/A. The open reading frame encoded proteins are more easily removed from the botulinum complex than the hemagglutinin proteins, but are present in several BoNT/A and /F toxin preparations. These are not easily removed from the BoNT/E complex, however, and are present even in commercially-available purified BoNT/E complex.

Canine distemper virus neutralization activity is low in human serum and it is sensitive to an amino acid substitution in the hemagglutinin protein.

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Zhang, Xinsheng; Wallace, Olivia L; Domi, Arban; Wright, Kevin J; Driscoll, Jonathan; Anzala, Omu; Sanders, Eduard J; Kamali, Anatoli; Karita, Etienne; Allen, Susan; Fast, Pat; Gilmour, Jill; Price, Matt A; Parks, Christopher L

2015-08-01

Serum was analyzed from 146 healthy adult volunteers in eastern Africa to evaluate measles virus (MV) and canine distemper virus (CDV) neutralizing antibody (nAb) prevalence and potency. MV plaque reduction neutralization test (PRNT) results indicated that all sera were positive for MV nAbs. Furthermore, the 50% neutralizing dose (ND50) for the majority of sera corresponded to antibody titers induced by MV vaccination. CDV nAbs titers were low and generally were detected in sera with high MV nAb titers. A mutant CDV was generated that was less sensitive to neutralization by human serum. The mutant virus genome had 10 nucleotide substitutions, which coded for single amino acid substitutions in the fusion (F) and hemagglutinin (H) glycoproteins and two substitutions in the large polymerase (L) protein. The H substitution occurred in a conserved region involved in receptor interactions among morbilliviruses, implying that this region is a target for cross-reactive neutralizing antibodies. Copyright © 2015 Elsevier Inc. All rights reserved.

The V domain of dog PVRL4 (nectin-4) mediates canine distemper virus entry and virus cell-to-cell spread.

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Delpeut, Sebastien; Noyce, Ryan S; Richardson, Christopher D

2014-04-01

The entry of canine distemper virus (CDV) is a multistep process that involves the attachment of CDV hemagglutinin (H) to its cellular receptor, followed by fusion between virus and cell membranes. Our laboratory recently identified PVRL4 (nectin-4) to be the epithelial receptor for measles and canine distemper viruses. In this study, we demonstrate that the V domain of PVRL4 is critical for CDV entry and virus cell-to-cell spread. Furthermore, four key amino acid residues within the V domain of dog PVRL4 and two within the CDV hemagglutinin were shown to be essential for receptor-mediated virus entry. Copyright © 2014 Elsevier Inc. All rights reserved.

Computational design of protein interactions: designing proteins that neutralize influenza by inhibiting its hemagglutinin surface protein

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Fleishman, Sarel

2012-02-01

Molecular recognition underlies all life processes. Design of interactions not seen in nature is a test of our understanding of molecular recognition and could unlock the vast potential of subtle control over molecular interaction networks, allowing the design of novel diagnostics and therapeutics for basic and applied research. We developed the first general method for designing protein interactions. The method starts by computing a region of high affinity interactions between dismembered amino acid residues and the target surface and then identifying proteins that can harbor these residues. Designs are tested experimentally for binding the target surface and successful ones are affinity matured using yeast cell surface display. Applied to the conserved stem region of influenza hemagglutinin we designed two unrelated proteins that, following affinity maturation, bound hemagglutinin at subnanomolar dissociation constants. Co-crystal structures of hemagglutinin bound to the two designed binders were within 1Angstrom RMSd of their models, validating the accuracy of the design strategy. One of the designed proteins inhibits the conformational changes that underlie hemagglutinin's cell-invasion functions and blocks virus infectivity in cell culture, suggesting that such proteins may in future serve as diagnostics and antivirals against a wide range of pathogenic influenza strains. We have used this method to obtain experimentally validated binders of several other target proteins, demonstrating the generality of the approach. We discuss the combination of modeling and high-throughput characterization of design variants which has been key to the success of this approach, as well as how we have used the data obtained in this project to enhance our understanding of molecular recognition. References: Science 332:816 JMB, in press Protein Sci 20:753

Determinants of glycan receptor specificity of H2N2 influenza A virus hemagglutinin.

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Viswanathan, Karthik; Koh, Xiaoying; Chandrasekaran, Aarthi; Pappas, Claudia; Raman, Rahul; Srinivasan, Aravind; Shriver, Zachary; Tumpey, Terrence M; Sasisekharan, Ram

2010-10-29

The H2N2 subtype of influenza A virus was responsible for the Asian pandemic of 1957-58. However, unlike other subtypes that have caused pandemics such as H1N1 and H3N2, which continue to circulate among humans, H2N2 stopped circulating in the human population in 1968. Strains of H2 subtype still continue to circulate in birds and occasionally pigs and could be reintroduced into the human population through antigenic drift or shift. Such an event is a potential global health concern because of the waning population immunity to H2 hemagglutinin (HA). The first step in such a cross-species transmission and human adaptation of influenza A virus is the ability for its surface glycoprotein HA to bind to glycan receptors expressed in the human upper respiratory epithelia. Recent structural and biochemical studies have focused on understanding the glycan receptor binding specificity of the 1957-58 pandemic H2N2 HA. However, there has been considerable HA sequence divergence in the recent avian-adapted H2 strains from the pandemic H2N2 strain. Using a combination of structural modeling, quantitative glycan binding and human respiratory tissue binding methods, we systematically identify mutations in the HA from a recent avian-adapted H2N2 strain (A/Chicken/PA/2004) that make its quantitative glycan receptor binding affinity (defined using an apparent binding constant) comparable to that of a prototypic pandemic H2N2 (A/Albany/6/58) HA.

Determinants of glycan receptor specificity of H2N2 influenza A virus hemagglutinin.

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Karthik Viswanathan

Full Text Available The H2N2 subtype of influenza A virus was responsible for the Asian pandemic of 1957-58. However, unlike other subtypes that have caused pandemics such as H1N1 and H3N2, which continue to circulate among humans, H2N2 stopped circulating in the human population in 1968. Strains of H2 subtype still continue to circulate in birds and occasionally pigs and could be reintroduced into the human population through antigenic drift or shift. Such an event is a potential global health concern because of the waning population immunity to H2 hemagglutinin (HA. The first step in such a cross-species transmission and human adaptation of influenza A virus is the ability for its surface glycoprotein HA to bind to glycan receptors expressed in the human upper respiratory epithelia. Recent structural and biochemical studies have focused on understanding the glycan receptor binding specificity of the 1957-58 pandemic H2N2 HA. However, there has been considerable HA sequence divergence in the recent avian-adapted H2 strains from the pandemic H2N2 strain. Using a combination of structural modeling, quantitative glycan binding and human respiratory tissue binding methods, we systematically identify mutations in the HA from a recent avian-adapted H2N2 strain (A/Chicken/PA/2004 that make its quantitative glycan receptor binding affinity (defined using an apparent binding constant comparable to that of a prototypic pandemic H2N2 (A/Albany/6/58 HA.

New Orf virus (Parapoxvirus) recombinant expressing H5 hemagglutinin protects mice against H5N1 and H1N1 influenza A virus.

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Rohde, Jörg; Amann, Ralf; Rziha, Hanns-Joachim

2013-01-01

Previously we demonstrated the versatile utility of the Parapoxvirus Orf virus (ORFV) as a vector platform for the development of potent recombinant vaccines. In this study we present the generation of new ORFV recombinants expressing the hemagglutinin (HA) or nucleoprotein (NP) of the highly pathogenic avian influenza virus (HPAIV) H5N1. Correct foreign gene expression was examined in vitro by immunofluorescence, Western blotting and flow cytometry. The protective potential of both recombinants was evaluated in the mouse challenge model. Despite adequate expression of NP, the recombinant D1701-V-NPh5 completely failed to protect mice from lethal challenge. However, the H5 HA-expressing recombinant D1701-V-HAh5n mediated solid protection in a dose-dependent manner. Two intramuscular (i.m.) injections of the HA-expressing recombinant protected all animals from lethal HPAIV infection without loss of body weight. Notably, the immunized mice resisted cross-clade H5N1 and heterologous H1N1 (strain PR8) influenza virus challenge. In vivo antibody-mediated depletion of CD4-positive and/or CD8-posititve T-cell subpopulations during immunization and/or challenge infection implicated the relevance of CD4-positive T-cells for induction of protective immunity by D1701-V-HAh5n, whereas the absence of CD8-positive T-cells did not significantly influence protection. In summary, this study validates the potential of the ORFV vectored vaccines also to combat HPAIV.

New Orf virus (Parapoxvirus recombinant expressing H5 hemagglutinin protects mice against H5N1 and H1N1 influenza A virus.

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Jörg Rohde

Full Text Available Previously we demonstrated the versatile utility of the Parapoxvirus Orf virus (ORFV as a vector platform for the development of potent recombinant vaccines. In this study we present the generation of new ORFV recombinants expressing the hemagglutinin (HA or nucleoprotein (NP of the highly pathogenic avian influenza virus (HPAIV H5N1. Correct foreign gene expression was examined in vitro by immunofluorescence, Western blotting and flow cytometry. The protective potential of both recombinants was evaluated in the mouse challenge model. Despite adequate expression of NP, the recombinant D1701-V-NPh5 completely failed to protect mice from lethal challenge. However, the H5 HA-expressing recombinant D1701-V-HAh5n mediated solid protection in a dose-dependent manner. Two intramuscular (i.m. injections of the HA-expressing recombinant protected all animals from lethal HPAIV infection without loss of body weight. Notably, the immunized mice resisted cross-clade H5N1 and heterologous H1N1 (strain PR8 influenza virus challenge. In vivo antibody-mediated depletion of CD4-positive and/or CD8-posititve T-cell subpopulations during immunization and/or challenge infection implicated the relevance of CD4-positive T-cells for induction of protective immunity by D1701-V-HAh5n, whereas the absence of CD8-positive T-cells did not significantly influence protection. In summary, this study validates the potential of the ORFV vectored vaccines also to combat HPAIV.

Circulating avian influenza viruses closely related to the 1918 virus have pandemic potential

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Watanabe, Tokiko; Zhong, Gongxun; Russell, Colin A.; Nakajima, Noriko; Hatta, Masato; Hanson, Anthony; McBride, Ryan; Burke, David F.; Takahashi, Kenta; Fukuyama, Satoshi; Tomita, Yuriko; Maher, Eileen A.; Watanabe, Shinji; Imai, Masaki; Neumann, Gabriele; Hasegawa, Hideki; Paulson, James C.; Smith, Derek J.; Kawaoka, Yoshihiro

2014-01-01

Summary Wild birds harbor a large gene pool of influenza A viruses that have the potential to cause influenza pandemics. Foreseeing and understanding this potential is important for effective surveillance. Our phylogenetic and geographic analyses revealed the global prevalence of avian influenza virus genes whose proteins differ only a few amino acids from the 1918 pandemic influenza virus, suggesting that 1918-like pandemic viruses may emerge in the future. To assess this risk, we generated and characterized a virus composed of avian influenza viral segments with high homology to the 1918 virus. This virus exhibited higher pathogenicity in mice and ferrets than an authentic avian influenza virus. Further, acquisition of seven amino acid substitutions in the viral polymerases and the hemagglutinin surface glycoprotein conferred respiratory droplet transmission to the 1918-like avian virus in ferrets, demonstrating that contemporary avian influenza viruses with 1918 virus-like proteins may have pandemic potential. PMID:24922572

Conserved synthetic peptides from the hemagglutinin of influenza viruses induce broad humoral and T-cell responses in a pig model.

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Júlia Vergara-Alert

Full Text Available Outbreaks involving either H5N1 or H1N1 influenza viruses (IV have recently become an increasing threat to cause potential pandemics. Pigs have an important role in this aspect. As reflected in the 2009 human H1N1 pandemia, they may act as a vehicle for mixing and generating new assortments of viruses potentially pathogenic to animals and humans. Lack of universal vaccines against the highly variable influenza virus forces scientists to continuously design vaccines à la carte, which is an expensive and risky practice overall when dealing with virulent strains. Therefore, we focused our efforts on developing a broadly protective influenza vaccine based on the Informational Spectrum Method (ISM. This theoretical prediction allows the selection of highly conserved peptide sequences from within the hemagglutinin subunit 1 protein (HA1 from either H5 or H1 viruses which are located in the flanking region of the HA binding site and with the potential to elicit broader immune responses than conventional vaccines. Confirming the theoretical predictions, immunization of conventional farm pigs with the synthetic peptides induced humoral responses in every single pig. The fact that the induced antibodies were able to recognize in vitro heterologous influenza viruses such as the pandemic H1N1 virus (pH1N1, two swine influenza field isolates (SwH1N1 and SwH3N2 and a H5N1 highly pathogenic avian virus, confirm the broad recognition of the antibodies induced. Unexpectedly, all pigs also showed T-cell responses that not only recognized the specific peptides, but also the pH1N1 virus. Finally, a partial effect on the kinetics of virus clearance was observed after the intranasal infection with the pH1N1 virus, setting forth the groundwork for the design of peptide-based vaccines against influenza viruses. Further insights into the understanding of the mechanisms involved in the protection afforded will be necessary to optimize future vaccine formulations.

Phylogeography of Influenza A(H3N2) Virus in Peru, 2010-2012.

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Pollett, Simon; Nelson, Martha I; Kasper, Matthew; Tinoco, Yeny; Simons, Mark; Romero, Candice; Silva, Marita; Lin, Xudong; Halpin, Rebecca A; Fedorova, Nadia; Stockwell, Timothy B; Wentworth, David; Holmes, Edward C; Bausch, Daniel G

2015-08-01

It remains unclear whether lineages of influenza A(H3N2) virus can persist in the tropics and seed temperate areas. We used viral gene sequence data sampled from Peru to test this source-sink model for a Latin American country. Viruses were obtained during 2010-2012 from influenza surveillance cohorts in Cusco, Tumbes, Puerto Maldonado, and Lima. Specimens positive for influenza A(H3N2) virus were randomly selected and underwent hemagglutinin sequencing and phylogeographic analyses. Analysis of 389 hemagglutinin sequences from Peru and 2,192 global sequences demonstrated interseasonal extinction of Peruvian lineages. Extensive mixing occurred with global clades, but some spatial structure was observed at all sites; this structure was weakest in Lima and Puerto Maldonado, indicating that these locations may experience greater viral traffic. The broad diversity and co-circulation of many simultaneous lineages of H3N2 virus in Peru suggests that this country should not be overlooked as a potential source for novel pandemic strains.

Phylogeography of Influenza A(H3N2) Virus in Peru, 2010–2012

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Nelson, Martha I.; Kasper, Matthew; Tinoco, Yeny; Simons, Mark; Romero, Candice; Silva, Marita; Lin, Xudong; Halpin, Rebecca A.; Fedorova, Nadia; Stockwell, Timothy B.; Wentworth, David; Holmes, Edward C.; Bausch, Daniel G.

2015-01-01

It remains unclear whether lineages of influenza A(H3N2) virus can persist in the tropics and seed temperate areas. We used viral gene sequence data sampled from Peru to test this source–sink model for a Latin American country. Viruses were obtained during 2010–2012 from influenza surveillance cohorts in Cusco, Tumbes, Puerto Maldonado, and Lima. Specimens positive for influenza A(H3N2) virus were randomly selected and underwent hemagglutinin sequencing and phylogeographic analyses. Analysis of 389 hemagglutinin sequences from Peru and 2,192 global sequences demonstrated interseasonal extinction of Peruvian lineages. Extensive mixing occurred with global clades, but some spatial structure was observed at all sites; this structure was weakest in Lima and Puerto Maldonado, indicating that these locations may experience greater viral traffic. The broad diversity and co-circulation of many simultaneous lineages of H3N2 virus in Peru suggests that this country should not be overlooked as a potential source for novel pandemic strains. PMID:26196599

A combination in-ovo vaccine for avian influenza virus and Newcastle disease virus.

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Steel, John; Burmakina, Svetlana V; Thomas, Colleen; Spackman, Erica; García-Sastre, Adolfo; Swayne, David E; Palese, Peter

2008-01-24

The protection of poultry from H5N1 highly pathogenic avian influenza A (HPAI) and Newcastle disease virus (NDV) can be achieved through vaccination, as part of a broader disease control strategy. We have previously generated a recombinant influenza virus expressing, (i) an H5 hemagglutinin protein, modified by the removal of the polybasic cleavage peptide and (ii) the ectodomain of the NDV hemagglutinin-neuraminidase (HN) protein in the place of the ectodomain of influenza neuraminidase (Park MS, et al. Proc Natl Acad Sci USA 2006;103(21):8203-8). Here we show this virus is attenuated in primary normal human bronchial epithelial (NHBE) cell culture, and demonstrate protection of C57BL/6 mice from lethal challenge with an H5 HA-containing influenza virus through immunisation with the recombinant virus. In addition, in-ovo vaccination of 18-day-old embryonated chicken eggs provided 90% and 80% protection against highly stringent lethal challenge by NDV and H5N1 virus, respectively. We propose that this virus has potential as a safe in-ovo live, attenuated, bivalent avian influenza and Newcastle disease virus vaccine.

Structural Basis for a Switch in Receptor Binding Specificity of Two H5N1 Hemagglutinin Mutants

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Xueyong Zhu

2015-11-01

Full Text Available Avian H5N1 influenza viruses continue to spread in wild birds and domestic poultry with sporadic infection in humans. Receptor binding specificity changes are a prerequisite for H5N1 viruses and other zoonotic viruses to be transmitted among humans. Previous reported hemagglutinin (HA mutants from ferret-transmissible H5N1 viruses of A/Vietnam/1203/2004 and A/Indonesia/5/2005 showed slightly increased, but still very weak, binding to human receptors. From mutagenesis and glycan array studies, we previously identified two H5N1 HA mutants that could more effectively switch receptor specificity to human-like α2-6-linked sialosides with avidity comparable to wild-type H5 HA binding to avian-like α2-3-linked sialosides. Here, crystal structures of these two H5 HA mutants free and in complex with human and avian glycan receptor analogs reveal the structural basis for their preferential binding to human receptors. These findings suggest continuous surveillance should be maintained to monitor and assess human-to-human transmission potential of H5N1 viruses.

H1N1 influenza viruses varying widely in hemagglutinin stability transmit efficiently from swine to swine and to ferrets.

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Marion Russier

2017-03-01

Full Text Available A pandemic-capable influenza virus requires a hemagglutinin (HA surface glycoprotein that is immunologically unseen by most people and is capable of supporting replication and transmission in humans. HA stabilization has been linked to 2009 pH1N1 pandemic potential in humans and H5N1 airborne transmissibility in the ferret model. Swine have served as an intermediate host for zoonotic influenza viruses, yet the evolutionary pressure exerted by this host on HA stability was unknown. For over 70 contemporary swine H1 and H3 isolates, we measured HA activation pH to range from pH 5.1 to 5.9 for H1 viruses and pH 5.3 to 5.8 for H3 viruses. Thus, contemporary swine isolates vary widely in HA stability, having values favored by both avian (pH >5.5 and human and ferret (pH ≤5.5 species. Using an early 2009 pandemic H1N1 (pH1N1 virus backbone, we generated three viruses differing by one HA residue that only altered HA stability: WT (pH 5.5, HA1-Y17H (pH 6.0, and HA2-R106K (pH 5.3. All three replicated in pigs and transmitted from pig-to-pig and pig-to-ferret. WT and R106 viruses maintained HA genotype and phenotype after transmission. Y17H (pH 6.0 acquired HA mutations that stabilized the HA protein to pH 5.8 after transmission to pigs and 5.5 after transmission to ferrets. Overall, we found swine support a broad range of HA activation pH for contact transmission and many recent swine H1N1 and H3N2 isolates have stabilized (human-like HA proteins. This constitutes a heightened pandemic risk and underscores the importance of ongoing surveillance and control efforts for swine viruses.

Deliberate reduction of hemagglutinin and neuraminidase expression of influenza virus leads to an ultraprotective live vaccine in mice.

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Yang, Chen; Skiena, Steven; Futcher, Bruce; Mueller, Steffen; Wimmer, Eckard

2013-06-04

A long-held dogma posits that strong presentation to the immune system of the dominant influenza virus glycoprotein antigens neuraminidase (NA) and hemagglutinin (HA) is paramount for inducing protective immunity against influenza virus infection. We have deliberately violated this dogma by constructing a recombinant influenza virus strain of A/PR8/34 (H1N1) in which expression of NA and HA genes was suppressed. We down-regulated NA and HA expression by recoding the respective genes with suboptimal codon pair bias, thereby introducing hundreds of nucleotide changes while preserving their codon use and protein sequence. The variants PR8-NA(Min), PR8-HA(Min), and PR8-(NA+HA)(Min) (Min, minimal expression) were used to assess the contribution of reduced glycoprotein expression to growth in tissue culture and pathogenesis in BALB/c mice. All three variants proliferated in Madin-Darby canine kidney cells to nearly the degree as WT PR8. In mice, however, they expressed explicit attenuation phenotypes, as revealed by their LD50 values: PR8, 32 plaque-forming units (PFU); HA(Min), 1.7 × 10(3) PFU; NA(Min), 2.4 × 10(5) PFU; (NA+HA)(Min), ≥3.16 × 10(6) PFU. Remarkably, (NA+HA)(Min) was attenuated >100,000-fold, with NA(Min) the major contributor to attenuation. In vaccinated mice (NA+HA)(Min) was highly effective in providing long-lasting protective immunity against lethal WT challenge at a median protective dose (PD50) of 2.4 PFU. Moreover, at a PD50 of only 147 or 237, (NA+HA)(Min) conferred protection against heterologous lethal challenges with two mouse-adapted H3N2 viruses. We conclude that the suppression of HA and NA is a unique strategy in live vaccine development.

Comparative study of the hemagglutinin and neuraminidase genes of influenza A virus H3N2, H9N2, and H5N1 subtypes using bioinformatics techniques.

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Ahn, Insung; Son, Hyeon S

2007-07-01

To investigate the genomic patterns of influenza A virus subtypes, such as H3N2, H9N2, and H5N1, we collected 1842 sequences of the hemagglutinin and neuraminidase genes from the NCBI database and parsed them into 7 categories: accession number, host species, sampling year, country, subtype, gene name, and sequence. The sequences that were isolated from the human, avian, and swine populations were extracted and stored in a MySQL database for intensive analysis. The GC content and relative synonymous codon usage (RSCU) values were calculated using JAVA codes. As a result, correspondence analysis of the RSCU values yielded the unique codon usage pattern (CUP) of each subtype and revealed no extreme differences among the human, avian, and swine isolates. H5N1 subtype viruses exhibited little variation in CUPs compared with other subtypes, suggesting that the H5N1 CUP has not yet undergone significant changes within each host species. Moreover, some observations may be relevant to CUP variation that has occurred over time among the H3N2 subtype viruses isolated from humans. All the sequences were divided into 3 groups over time, and each group seemed to have preferred synonymous codon patterns for each amino acid, especially for arginine, glycine, leucine, and valine. The bioinformatics technique we introduce in this study may be useful in predicting the evolutionary patterns of pandemic viruses.

Glycosylation of Hemagglutinin and Neuraminidase of Influenza A Virus as Signature for Ecological Spillover and Adaptation among Influenza Reservoirs

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Paul Kim

2018-04-01

Full Text Available Glycosylation of the hemagglutinin (HA and neuraminidase (NA of the influenza provides crucial means for immune evasion and viral fitness in a host population. However, the time-dependent dynamics of each glycosylation sites have not been addressed. We monitored the potential N-linked glycosylation (NLG sites of over 10,000 HA and NA of H1N1 subtype isolated from human, avian, and swine species over the past century. The results show a shift in glycosylation sites as a hallmark of 1918 and 2009 pandemics, and also for the 1976 “abortive pandemic”. Co-segregation of particular glycosylation sites was identified as a characteristic of zoonotic transmission from animal reservoirs, and interestingly, of “reverse zoonosis” of human viruses into swine populations as well. After the 2009 pandemic, recent isolates accrued glycosylation at canonical sites in HA, reflecting gradual seasonal adaptation, and a novel glycosylation in NA as an independent signature for adaptation among humans. Structural predictions indicated a remarkably pleiotropic influence of glycans on multiple HA epitopes for immune evasion, without sacrificing the receptor binding of HA or the activity of NA. The results provided the rationale for establishing the ecological niche of influenza viruses among the reservoir and could be implemented for influenza surveillance and improving pandemic preparedness.

Glycosylation of Hemagglutinin and Neuraminidase of Influenza A Virus as Signature for Ecological Spillover and Adaptation among Influenza Reservoirs

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Kim, Paul; Jang, Yo Han; Kwon, Soon Bin; Lee, Chung Min; Han, Gyoonhee; Seong, Baik Lin

2018-01-01

Glycosylation of the hemagglutinin (HA) and neuraminidase (NA) of the influenza provides crucial means for immune evasion and viral fitness in a host population. However, the time-dependent dynamics of each glycosylation sites have not been addressed. We monitored the potential N-linked glycosylation (NLG) sites of over 10,000 HA and NA of H1N1 subtype isolated from human, avian, and swine species over the past century. The results show a shift in glycosylation sites as a hallmark of 1918 and 2009 pandemics, and also for the 1976 “abortive pandemic”. Co-segregation of particular glycosylation sites was identified as a characteristic of zoonotic transmission from animal reservoirs, and interestingly, of “reverse zoonosis” of human viruses into swine populations as well. After the 2009 pandemic, recent isolates accrued glycosylation at canonical sites in HA, reflecting gradual seasonal adaptation, and a novel glycosylation in NA as an independent signature for adaptation among humans. Structural predictions indicated a remarkably pleiotropic influence of glycans on multiple HA epitopes for immune evasion, without sacrificing the receptor binding of HA or the activity of NA. The results provided the rationale for establishing the ecological niche of influenza viruses among the reservoir and could be implemented for influenza surveillance and improving pandemic preparedness. PMID:29642453

Recombinant Vaccinia Virus: Immunization against Multiple Pathogens

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Perkus, Marion E.; Piccini, Antonia; Lipinskas, Bernard R.; Paoletti, Enzo

1985-09-01

The coding sequences for the hepatitis B virus surface antigen, the herpes simplex virus glycoprotein D, and the influenza virus hemagglutinin were inserted into a single vaccinia virus genome. Rabbits inoculated intravenously or intradermally with this polyvalent vaccinia virus recombinant produced antibodies reactive to all three authentic foreign antigens. In addition, the feasibility of multiple rounds of vaccination with recombinant vaccinia virus was demonstrated.

Unique Infectious Strategy of H5N1 Avian Influenza Virus Is Governed by the Acid-Destabilized Property of Hemagglutinin.

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Daidoji, Tomo; Watanabe, Yohei; Arai, Yasuha; Kajikawa, Junichi; Hirose, Ryohei; Nakaya, Takaaki

Highly pathogenic avian influenza (HPAI) H5N1 virus emerged in 1997 as a zoonotic disease in Hong Kong. It has since spread to Asia and Europe and is a serious threat to both the poultry industry and human health. For effective surveillance and possible prevention/control of HPAI H5N1 viruses, it is necessary to understand the molecular mechanism underlying HPAI H5N1 pathogenesis. The hemagglutinin (HA) protein of influenza A viruses (IAVs) is one of the major determinants of host adaptation, transmissibility, and viral virulence. The main function of the HA protein is to facilitate viral entry and viral genome release within host cells before infection. To achieve viral infection, IAVs belonging to different subtypes or strains induce viral-cell membrane fusion at different endosomal pH levels after internalization through endocytosis. However, host-specific endosomal pH also affects induction of membrane fusion followed by infection. The HA protein of HPAI H5N1 has a higher pH threshold for membrane fusion than the HA protein of classical avian influenza viruses. Although this particular property of HA (which governs viral infection) is prone to deactivation in the avian intestine or in an ambient environment, it facilitates efficient infection of host cells, resulting in a broad host tropism, regardless of the pH in the host endosome. Accumulated knowledge, together with further research, about the HA-governed mechanism underlying HPAI H5N1 virulence (i.e., receptor tropism and pH-dependent viral-cell membrane fusion) will be helpful for developing effective surveillance strategies and for prevention/control of HPAI H5N1 infection.

Initial characterization of Vaccinia Virus B4 suggests a role in virus spread

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Burles, Kristin; Irwin, Chad R.; Burton, Robyn-Lee [Li Ka Shing Institute of Virology, Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada T6G 2S2 (Canada); Schriewer, Jill [Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, St. Louis, MO (United States); Evans, David H. [Li Ka Shing Institute of Virology, Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada T6G 2S2 (Canada); Buller, R. Mark [Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, St. Louis, MO (United States); Barry, Michele, E-mail: michele.barry@ualberta.ca [Li Ka Shing Institute of Virology, Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada T6G 2S2 (Canada)

2014-05-15

Currently, little is known about the ankyrin/F-box protein B4. Here, we report that B4R-null viruses exhibited reduced plaque size in tissue culture, and decreased ability to spread, as assessed by multiple-step growth analysis. Electron microscopy indicated that B4R-null viruses still formed mature and extracellular virions; however, there was a slight decrease of virions released into the media following deletion of B4R. Deletion of B4R did not affect the ability of the virus to rearrange actin; however, VACV811, a large vaccinia virus deletion mutant missing 55 open reading frames, had decreased ability to produce actin tails. Using ectromelia virus, a natural mouse pathogen, we demonstrated that virus devoid of EVM154, the B4R homolog, showed decreased spread to organs and was attenuated during infection. This initial characterization suggests that B4 may play a role in virus spread, and that other unidentified mediators of actin tail formation may exist in vaccinia virus. - Highlights: • B4R-null viruses show reduced plaque size, and decreased ability to spread. • B4R-null viruses formed mature and extracellular virions; and rearranged actin. • Virus devoid of EVM154, the B4R homolog, was attenuated during infection. • Initial characterization suggests that B4 may play a role in virus spread. • Unidentified mediators of actin tail formation may exist in vaccinia virus.

Initial characterization of Vaccinia Virus B4 suggests a role in virus spread

International Nuclear Information System (INIS)

Burles, Kristin; Irwin, Chad R.; Burton, Robyn-Lee; Schriewer, Jill; Evans, David H.; Buller, R. Mark; Barry, Michele

2014-01-01

Currently, little is known about the ankyrin/F-box protein B4. Here, we report that B4R-null viruses exhibited reduced plaque size in tissue culture, and decreased ability to spread, as assessed by multiple-step growth analysis. Electron microscopy indicated that B4R-null viruses still formed mature and extracellular virions; however, there was a slight decrease of virions released into the media following deletion of B4R. Deletion of B4R did not affect the ability of the virus to rearrange actin; however, VACV811, a large vaccinia virus deletion mutant missing 55 open reading frames, had decreased ability to produce actin tails. Using ectromelia virus, a natural mouse pathogen, we demonstrated that virus devoid of EVM154, the B4R homolog, showed decreased spread to organs and was attenuated during infection. This initial characterization suggests that B4 may play a role in virus spread, and that other unidentified mediators of actin tail formation may exist in vaccinia virus. - Highlights: • B4R-null viruses show reduced plaque size, and decreased ability to spread. • B4R-null viruses formed mature and extracellular virions; and rearranged actin. • Virus devoid of EVM154, the B4R homolog, was attenuated during infection. • Initial characterization suggests that B4 may play a role in virus spread. • Unidentified mediators of actin tail formation may exist in vaccinia virus

Computational Analysis of the Interaction Energies between Amino Acid Residues of the Measles Virus Hemagglutinin and Its Receptors

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Fengqi Xu

2018-05-01

Full Text Available Measles virus (MV causes an acute and highly devastating contagious disease in humans. Employing the crystal structures of three human receptors, signaling lymphocyte-activation molecule (SLAM, CD46, and Nectin-4, in complex with the measles virus hemagglutinin (MVH, we elucidated computationally the details of binding energies between the amino acid residues of MVH and those of the receptors with an ab initio fragment molecular orbital (FMO method. The calculated inter-fragment interaction energies (IFIEs revealed a number of significantly interacting amino acid residues of MVH that played essential roles in binding to the receptors. As predicted from previously reported experiments, some important amino-acid residues of MVH were shown to be common but others were specific to interactions with the three receptors. Particularly, some of the (non-polar hydrophobic residues of MVH were found to be attractively interacting with multiple receptors, thus indicating the importance of the hydrophobic pocket for intermolecular interactions (especially in the case of Nectin-4. In contrast, the electrostatic interactions tended to be used for specific molecular recognition. Furthermore, we carried out FMO calculations for in silico experiments of amino acid mutations, finding reasonable agreements with virological experiments concerning the substitution effect of residues. Thus, the present study demonstrates that the electron-correlated FMO method is a powerful tool to search exhaustively for amino acid residues that contribute to interactions with receptor molecules. It is also applicable for designing inhibitors of MVH and engineered MVs for cancer therapy.

New world bats harbor diverse influenza A viruses.

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Suxiang Tong

Full Text Available Aquatic birds harbor diverse influenza A viruses and are a major viral reservoir in nature. The recent discovery of influenza viruses of a new H17N10 subtype in Central American fruit bats suggests that other New World species may similarly carry divergent influenza viruses. Using consensus degenerate RT-PCR, we identified a novel influenza A virus, designated as H18N11, in a flat-faced fruit bat (Artibeus planirostris from Peru. Serologic studies with the recombinant H18 protein indicated that several Peruvian bat species were infected by this virus. Phylogenetic analyses demonstrate that, in some gene segments, New World bats harbor more influenza virus genetic diversity than all other mammalian and avian species combined, indicative of a long-standing host-virus association. Structural and functional analyses of the hemagglutinin and neuraminidase indicate that sialic acid is not a ligand for virus attachment nor a substrate for release, suggesting a unique mode of influenza A virus attachment and activation of membrane fusion for entry into host cells. Taken together, these findings indicate that bats constitute a potentially important and likely ancient reservoir for a diverse pool of influenza viruses.

Phocine Distemper Virus in Seals, East Coast, United States, 2006

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Earle, J.A. Philip; Melia, Mary M.; Doherty, Nadine V.; Nielsen, Ole

2011-01-01

In 2006 and 2007, elevated numbers of deaths among seals, constituting an unusual mortality event, occurred off the coasts of Maine and Massachusetts, United States. We isolated a virus from seal tissue and confirmed it as phocine distemper virus (PDV). We compared the viral hemagglutinin, phosphoprotein, and fusion (F) and matrix (M) protein gene sequences with those of viruses from the 1988 and 2002 PDV epizootics. The virus showed highest similarity with a PDV 1988 Netherlands virus, which raises the possibility that the 2006 isolate from the United States might have emerged independently from 2002 PDVs and that multiple lineages of PDV might be circulating among enzootically infected North American seals. Evidence from comparison of sequences derived from different tissues suggested that mutations in the F and M genes occur in brain tissue that are not present in lung, liver, or blood, which suggests virus persistence in the central nervous system. PMID:21291591

Potent peptidic fusion inhibitors of influenza virus

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Kadam, Rameshwar U.; Juraszek, Jarek; Brandenburg, Boerries; Buyck, Christophe; Schepens, Wim B. G.; Kesteleyn, Bart; Stoops, Bart; Vreeken, Rob J.; Vermond, Jan; Goutier, Wouter; Tang, Chan; Vogels, Ronald; Friesen, Robert H. E.; Goudsmit, Jaap; van Dongen, Maria J. P.; Wilson, Ian A.

2017-09-28

Influenza therapeutics with new targets and mechanisms of action are urgently needed to combat potential pandemics, emerging viruses, and constantly mutating strains in circulation. We report here on the design and structural characterization of potent peptidic inhibitors of influenza hemagglutinin. The peptide design was based on complementarity-determining region loops of human broadly neutralizing antibodies against the hemagglutinin (FI6v3 and CR9114). The optimized peptides exhibit nanomolar affinity and neutralization against influenza A group 1 viruses, including the 2009 H1N1 pandemic and avian H5N1 strains. The peptide inhibitors bind to the highly conserved stem epitope and block the low pH–induced conformational rearrangements associated with membrane fusion. These peptidic compounds and their advantageous biological properties should accelerate the development of new small molecule– and peptide-based therapeutics against influenza virus.

Fusion peptide of influenza hemagglutinin requires a fixed angle boomerang structure for activity.

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Lai, Alex L; Park, Heather; White, Judith M; Tamm, Lukas K

2006-03-03

The fusion peptide of influenza hemagglutinin is crucial for cell entry of this virus. Previous studies showed that this peptide adopts a boomerang-shaped structure in lipid model membranes at the pH of membrane fusion. To examine the role of the boomerang in fusion, we changed several residues proposed to stabilize the kink in this structure and measured fusion. Among these, mutants E11A and W14A expressed hemagglutinins with hemifusion and no fusion activities, and F9A and N12A had no effect on fusion, respectively. Binding enthalpies and free energies of mutant peptides to model membranes and their ability to perturb lipid bilayer structures correlated well with the fusion activities of the parent full-length molecules. The structure of W14A determined by NMR and site-directed spin labeling features a flexible kink that points out of the membrane, in sharp contrast to the more ordered boomerang of the wild-type, which points into the membrane. A specific fixed angle boomerang structure is thus required to support membrane fusion.

Centralized Consensus Hemagglutinin Genes Induce Protective Immunity against H1, H3 and H5 Influenza Viruses.

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Richard J Webby

Full Text Available With the exception of the live attenuated influenza vaccine there have been no substantial changes in influenza vaccine strategies since the 1940's. Here we report an alternative vaccine approach that uses Adenovirus-vectored centralized hemagglutinin (HA genes as vaccine antigens. Consensus H1-Con, H3-Con and H5-Con HA genes were computationally derived. Mice were immunized with Ad vaccines expressing the centralized genes individually. Groups of mice were vaccinated with 1 X 1010, 5 X 107 and 1 X 107 virus particles per mouse to represent high, intermediate and low doses, respectively. 100% of the mice that were vaccinated with the high dose vaccine were protected from heterologous lethal challenges within each subtype. In addition to 100% survival, there were no signs of weight loss and disease in 7 out of 8 groups of high dose vaccinated mice. Lower doses of vaccine showed a reduction of protection in a dose-dependent manner. However, even the lowest dose of vaccine provided significant levels of protection against the divergent influenza strains, especially considering the stringency of the challenge virus. In addition, we found that all doses of H5-Con vaccine were capable of providing complete protection against mortality when challenged with lethal doses of all 3 H5N1 influenza strains. This data demonstrates that centralized H1-Con, H3-Con and H5-Con genes can be effectively used to completely protect mice against many diverse strains of influenza. Therefore, we believe that these Ad-vectored centralized genes could be easily translated into new human vaccines.

Resistance of Sendai virus (HVJ) F-protein against gamma irradiation

International Nuclear Information System (INIS)

Hosokawa, Yasushi

1980-01-01

Sendai virus envelope proteins (hemagglutinin and F-protein), containing disulfide bonds in their molecules, were almost caused neither cleavage of protein molecules nor disappearance of antigenisitics by 10 5 R of γ-irradiation, but the liberated inner proteins (P, NP and M-protein) were severed into low molecules. By irradiation, hemagglutinin and F-protein of the virions were polymerized or aggregated together in such a way that the aggregate was not dissociated by SDS-2ME treatment on Slab gel electrophoresis, but in the liberated states hemagglutinin alone was aggregated and F-protein was separated with little loss of its anigenisity. From above results, the interaction between hemagglutinin and F-protein, and the utility of irradiation for isolation of F-protein were discussed. (author)

Limited Antigenic Diversity in Contemporary H7 Avian-Origin Influenza A Viruses from North America

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Xu, Yifei; Bailey, Elizabeth; Spackman, Erica; Li, Tao; Wang, Hui; Long, Li-Ping; Baroch, John A.; Cunningham, Fred L.; Lin, Xiaoxu; Jarman, Richard G.; DeLiberto, Thomas J.; Wan, Xiu-Feng

2016-01-01

Subtype H7 avian–origin influenza A viruses (AIVs) have caused at least 500 confirmed human infections since 2003 and culling of >75 million birds in recent years. Here we antigenically and genetically characterized 93 AIV isolates from North America (85 from migratory waterfowl [1976–2010], 7 from domestic poultry [1971–2012], and 1 from a seal [1980]). The hemagglutinin gene of these H7 viruses are separated from those from Eurasia. Gradual accumulation of nucleotide and amino acid substitutions was observed in the hemagglutinin of H7 AIVs from waterfowl and domestic poultry. Genotype characterization suggested that H7 AIVs in wild birds form diverse and transient internal gene constellations. Serologic analyses showed that the 93 isolates cross-reacted with each other to different extents. Antigenic cartography showed that the average antigenic distance among them was 1.14 units (standard deviation [SD], 0.57 unit) and that antigenic diversity among the H7 isolates we tested was limited. Our results suggest that the continuous genetic evolution has not led to significant antigenic diversity for H7 AIVs from North America. These findings add to our understanding of the natural history of IAVs and will inform public health decision-making regarding the threat these viruses pose to humans and poultry. PMID:26858078

The V domain of dog PVRL4 (nectin-4) mediates canine distemper virus entry and virus cell-to-cell spread

International Nuclear Information System (INIS)

Delpeut, Sebastien; Noyce, Ryan S.; Richardson, Christopher D.

2014-01-01

The entry of canine distemper virus (CDV) is a multistep process that involves the attachment of CDV hemagglutinin (H) to its cellular receptor, followed by fusion between virus and cell membranes. Our laboratory recently identified PVRL4 (nectin-4) to be the epithelial receptor for measles and canine distemper viruses. In this study, we demonstrate that the V domain of PVRL4 is critical for CDV entry and virus cell-to-cell spread. Furthermore, four key amino acid residues within the V domain of dog PVRL4 and two within the CDV hemagglutinin were shown to be essential for receptor-mediated virus entry. - Highlights: • PVRL4 (nectin-4) is the epithelial cell receptor for measles and canine distemper viruses. • V domain of PVRL4 is critical for CDV entry, cell-to-cell spread, and syncytia formation. • Chimeric PVRL1 backbone substituted with the V domain of PVRL4 can function as a receptor. • Amino acids (F132/P133/A134/G135) within the V domain are essential for PVRL4 receptor activity. • Amino acids (P493/Y539) within CDV H protein are essential for PVRL4 receptor interaction

The V domain of dog PVRL4 (nectin-4) mediates canine distemper virus entry and virus cell-to-cell spread

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Delpeut, Sebastien; Noyce, Ryan S. [The Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 1X5 (Canada); IWK Health Centre, Canadian Center for Vaccinology, Goldbloom Pavilion, Halifax, Nova Scotia, Canada B3H 1X5 (Canada); Richardson, Christopher D., E-mail: chris.richardson@dal.ca [The Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 1X5 (Canada); IWK Health Centre, Canadian Center for Vaccinology, Goldbloom Pavilion, Halifax, Nova Scotia, Canada B3H 1X5 (Canada); The Department of Pediatrics, Dalhousie University, Halifax, Nova Scotia (Canada)

2014-04-15

The entry of canine distemper virus (CDV) is a multistep process that involves the attachment of CDV hemagglutinin (H) to its cellular receptor, followed by fusion between virus and cell membranes. Our laboratory recently identified PVRL4 (nectin-4) to be the epithelial receptor for measles and canine distemper viruses. In this study, we demonstrate that the V domain of PVRL4 is critical for CDV entry and virus cell-to-cell spread. Furthermore, four key amino acid residues within the V domain of dog PVRL4 and two within the CDV hemagglutinin were shown to be essential for receptor-mediated virus entry. - Highlights: • PVRL4 (nectin-4) is the epithelial cell receptor for measles and canine distemper viruses. • V domain of PVRL4 is critical for CDV entry, cell-to-cell spread, and syncytia formation. • Chimeric PVRL1 backbone substituted with the V domain of PVRL4 can function as a receptor. • Amino acids (F132/P133/A134/G135) within the V domain are essential for PVRL4 receptor activity. • Amino acids (P493/Y539) within CDV H protein are essential for PVRL4 receptor interaction.

Acquisition of a novel eleven amino acid insertion directly N-terminal to a tetrabasic cleavage site confers intracellular cleavage of an H7N7 influenza virus hemagglutinin

International Nuclear Information System (INIS)

Hamilton, Brian S.; Sun, Xiangjie; Chung, Changik; Whittaker, Gary R.

2012-01-01

A critical feature of highly pathogenic avian influenza viruses (H5N1 and H7N7) is the efficient intracellular cleavage of the hemagglutinin (HA) protein. H7N7 viruses also exist in equine species, and a unique feature of the equine H7N7 HA is the presence of an eleven amino acid insertion directly N-terminal to a tetrabasic cleavage site. Here, we show that three histidine residues within the unique insertion of the equine H7N7 HA are essential for intracellular cleavage. An asparagine residue within the insertion-derived glycosylation site was also found to be essential for intracellular cleavage. The presence of the histidine residues also appear to be involved in triggering fusion, since mutation of the histidine residues resulted in a destabilizing effect. Importantly, the addition of a tetrabasic site and the eleven amino acid insertion conferred efficient intracellular cleavage to the HA of an H7N3 low pathogenicity avian influenza virus. Our studies show that acquisition of the eleven amino acid insertion offers an alternative mechanism for intracellular cleavage of influenza HA.

Acquisition of a novel eleven amino acid insertion directly N-terminal to a tetrabasic cleavage site confers intracellular cleavage of an H7N7 influenza virus hemagglutinin

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Hamilton, Brian S.; Sun, Xiangjie; Chung, Changik [Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca NY 14853 (United States); New York Center of Excellence for Influenza Research and Surveillance, University of Rochester Medical Center, Rochester NY 14627 (United States); Whittaker, Gary R., E-mail: grw7@cornell.edu [Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca NY 14853 (United States); New York Center of Excellence for Influenza Research and Surveillance, University of Rochester Medical Center, Rochester NY 14627 (United States)

2012-12-05

A critical feature of highly pathogenic avian influenza viruses (H5N1 and H7N7) is the efficient intracellular cleavage of the hemagglutinin (HA) protein. H7N7 viruses also exist in equine species, and a unique feature of the equine H7N7 HA is the presence of an eleven amino acid insertion directly N-terminal to a tetrabasic cleavage site. Here, we show that three histidine residues within the unique insertion of the equine H7N7 HA are essential for intracellular cleavage. An asparagine residue within the insertion-derived glycosylation site was also found to be essential for intracellular cleavage. The presence of the histidine residues also appear to be involved in triggering fusion, since mutation of the histidine residues resulted in a destabilizing effect. Importantly, the addition of a tetrabasic site and the eleven amino acid insertion conferred efficient intracellular cleavage to the HA of an H7N3 low pathogenicity avian influenza virus. Our studies show that acquisition of the eleven amino acid insertion offers an alternative mechanism for intracellular cleavage of influenza HA.

Molecular surveillance of low pathogenic avian influenza viruses in wild birds across the United States: inferences from the hemagglutinin gene.

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Antoinette J Piaggio

Full Text Available A United States interagency avian influenza surveillance plan was initiated in 2006 for early detection of highly pathogenic avian influenza viruses (HPAIV in wild birds. The plan included a variety of wild bird sampling strategies including the testing of fecal samples from aquatic areas throughout the United States from April 2006 through December 2007. Although HPAIV was not detected through this surveillance effort we were able to obtain 759 fecal samples that were positive for low pathogenic avian influenza virus (LPAIV. We used 136 DNA sequences obtained from these samples along with samples from a public influenza sequence database for a phylogenetic assessment of hemagglutinin (HA diversity in the United States. We analyzed sequences from all HA subtypes except H5, H7, H14 and H15 to examine genetic variation, exchange between Eurasia and North America, and geographic distribution of LPAIV in wild birds in the United States. This study confirms intercontinental exchange of some HA subtypes (including a newly documented H9 exchange event, as well as identifies subtypes that do not regularly experience intercontinental gene flow but have been circulating and evolving in North America for at least the past 20 years. These HA subtypes have high levels of genetic diversity with many lineages co-circulating within the wild birds of North America. The surveillance effort that provided these samples demonstrates that such efforts, albeit labor-intensive, provide important information about the ecology of LPAIV circulating in North America.

Hemagglutinin 222D/G polymorphism facilitates fast intra-host evolution of pandemic (H1N1 2009 influenza A viruses.

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Nora Seidel

Full Text Available The amino acid substitution of aspartic acid to glycine in hemagglutinin (HA in position 222 (HA-D222G as well as HA-222D/G polymorphism of pandemic (H1N1 2009 influenza viruses (A(H1N1pdm09 were frequently reported in severe influenza in humans and mice. Their impact on viral pathogenicity and the course of influenza has been discussed controversially and the underlying mechanism remained unclarified. In the present study, BALB/c mice, infected with the once mouse lung- and cell-passaged A(H1N1pdm09 isolate A/Jena/5258/09 (mpJena/5258, developed severe pneumonia. From day 2 to 3 or 4 post infection (p.i. symptoms (body weight loss and clinical score continuously worsened. After a short disease stagnation or even recovery phase in most mice, severity of disease further increased on days 6 and 7 p.i. Thereafter, surviving mice recovered. A 45 times higher virus titer maximum in the lung than in the trachea on day 2 p.i. and significantly higher tracheal virus titers compared to lung on day 6 p.i. indicated changes in the organ tropism during infection. Sequence analysis revealed an HA-222D/G polymorphism. HA-D222 and HA-G222 variants co-circulated in lung and trachea. Whereas, HA-D222 variant predominated in the lung, HA-G222 became the major variant in the trachea after day 4 p.i. This was accompanied by lower neutralizing antibody titers and broader receptor recognition including terminal sialic acid α-2,3-linked galactose, which is abundant on mouse trachea epithelial cells. Plaque-purified HA-G222-mpJena/5258 virus induced severe influenza with maximum symptom on day 6 p.i. These results demonstrated for the first time that HA-222D/G quasispecies of A(H1N1pdm09 caused severe biphasic influenza because of fast viral intra-host evolution, which enabled partial antibody escape and minor changes in receptor binding.

A novel eight amino acid insertion contributes to the hemagglutinin cleavability and the virulence of a highly pathogenic avian influenza A (H7N3) virus in mice

International Nuclear Information System (INIS)

Sun, Xiangjie; Belser, Jessica A.; Tumpey, Terrence M.

2016-01-01

In 2012, an avian influenza A H7N3 (A/Mexico/InDRE7218/2012; Mx/7218) virus was responsible for two confirmed cases of human infection and led to the death or culling of more than 22 million chickens in Jalisco, Mexico. Interestingly, this virus acquired an 8-amino acid (aa)-insertion (..PENPK-DRKSRHRR-TR/GLF) near the hemagglutinin (HA) cleavage site by nonhomologous recombination with host rRNA. It remains unclear which specific residues at the cleavage site contribute to the virulence of H7N3 viruses in mammals. Using loss-of-function approaches, we generated a series of cleavage site mutant viruses by reverse genetics and characterized the viruses in vitro and in vivo. We found that the 8-aa insertion and the arginine at position P4 of the Mx/7218 HA cleavage site are essential for intracellular HA cleavage in 293T cells, but have no effect on the pH of membrane fusion. However, we identified a role for the histidine residue at P5 position in viral fusion pH. In mice, the 8-aa insertion is required for Mx/7218 virus virulence; however, the basic residues upstream of the P4 position are dispensable for virulence. Overall, our study provides the first line of evidence that the insertion in the Mx/7218 virus HA cleavage site confers its intracellular cleavability, and consequently contributes to enhanced virulence in mice. - Highlights: • An avian influenza H7N3 virus acquired a unique 8-amino acid (aa) insertion. • The role of specific basic residues in the HA insertion in viral pathogenesis was determined. • In mice, the 8-aa insertion is required for H7N3 virus virulence. • The R residue at position P4 is essential for HA intracellular cleavage and virus virulence.

A novel eight amino acid insertion contributes to the hemagglutinin cleavability and the virulence of a highly pathogenic avian influenza A (H7N3) virus in mice

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Sun, Xiangjie; Belser, Jessica A.; Tumpey, Terrence M., E-mail: tft9@cdc.gov

2016-01-15

In 2012, an avian influenza A H7N3 (A/Mexico/InDRE7218/2012; Mx/7218) virus was responsible for two confirmed cases of human infection and led to the death or culling of more than 22 million chickens in Jalisco, Mexico. Interestingly, this virus acquired an 8-amino acid (aa)-insertion (..PENPK-DRKSRHRR-TR/GLF) near the hemagglutinin (HA) cleavage site by nonhomologous recombination with host rRNA. It remains unclear which specific residues at the cleavage site contribute to the virulence of H7N3 viruses in mammals. Using loss-of-function approaches, we generated a series of cleavage site mutant viruses by reverse genetics and characterized the viruses in vitro and in vivo. We found that the 8-aa insertion and the arginine at position P4 of the Mx/7218 HA cleavage site are essential for intracellular HA cleavage in 293T cells, but have no effect on the pH of membrane fusion. However, we identified a role for the histidine residue at P5 position in viral fusion pH. In mice, the 8-aa insertion is required for Mx/7218 virus virulence; however, the basic residues upstream of the P4 position are dispensable for virulence. Overall, our study provides the first line of evidence that the insertion in the Mx/7218 virus HA cleavage site confers its intracellular cleavability, and consequently contributes to enhanced virulence in mice. - Highlights: • An avian influenza H7N3 virus acquired a unique 8-amino acid (aa) insertion. • The role of specific basic residues in the HA insertion in viral pathogenesis was determined. • In mice, the 8-aa insertion is required for H7N3 virus virulence. • The R residue at position P4 is essential for HA intracellular cleavage and virus virulence.

Applying Unique Molecular Identifiers in Next Generation Sequencing Reveals a Constrained Viral Quasispecies Evolution under Cross-Reactive Antibody Pressure Targeting Long Alpha Helix of Hemagglutinin

Science.gov (United States)

Hauck, Nastasja C.; Kirpach, Josiane; Kiefer, Christina; Farinelle, Sophie; Morris, Stephen A.; Muller, Claude P.; Lu, I-Na

2018-01-01

To overcome yearly efforts and costs for the production of seasonal influenza vaccines, new approaches for the induction of broadly protective and long-lasting immune responses have been developed in the past decade. To warrant safety and efficacy of the emerging crossreactive vaccine candidates, it is critical to understand the evolution of influenza viruses in response to these new immune pressures. Here we applied unique molecular identifiers in next generation sequencing to analyze the evolution of influenza quasispecies under in vivo antibody pressure targeting the hemagglutinin (HA) long alpha helix (LAH). Our vaccine targeting LAH of hemagglutinin elicited significant seroconversion and protection against homologous and heterologous influenza virus strains in mice. The vaccine not only significantly reduced lung viral titers, but also induced a well-known bottleneck effect by decreasing virus diversity. In contrast to the classical bottleneck effect, here we showed a significant increase in the frequency of viruses with amino acid sequences identical to that of vaccine targeting LAH domain. No escape mutant emerged after vaccination. These results not only support the potential of a universal influenza vaccine targeting the conserved LAH domains, but also clearly demonstrate that the well-established bottleneck effect on viral quasispecies evolution does not necessarily generate escape mutants. PMID:29587397

Applying Unique Molecular Identifiers in Next Generation Sequencing Reveals a Constrained Viral Quasispecies Evolution under Cross-Reactive Antibody Pressure Targeting Long Alpha Helix of Hemagglutinin

Directory of Open Access Journals (Sweden)

Nastasja C. Hauck

2018-03-01

Full Text Available To overcome yearly efforts and costs for the production of seasonal influenza vaccines, new approaches for the induction of broadly protective and long-lasting immune responses have been developed in the past decade. To warrant safety and efficacy of the emerging crossreactive vaccine candidates, it is critical to understand the evolution of influenza viruses in response to these new immune pressures. Here we applied unique molecular identifiers in next generation sequencing to analyze the evolution of influenza quasispecies under in vivo antibody pressure targeting the hemagglutinin (HA long alpha helix (LAH. Our vaccine targeting LAH of hemagglutinin elicited significant seroconversion and protection against homologous and heterologous influenza virus strains in mice. The vaccine not only significantly reduced lung viral titers, but also induced a well-known bottleneck effect by decreasing virus diversity. In contrast to the classical bottleneck effect, here we showed a significant increase in the frequency of viruses with amino acid sequences identical to that of vaccine targeting LAH domain. No escape mutant emerged after vaccination. These results not only support the potential of a universal influenza vaccine targeting the conserved LAH domains, but also clearly demonstrate that the well-established bottleneck effect on viral quasispecies evolution does not necessarily generate escape mutants.

Domain architecture and oligomerization properties of the paramyxovirus PIV 5 hemagglutinin-neuraminidase (HN) protein.

Science.gov (United States)

Yuan, Ping; Leser, George P; Demeler, Borries; Lamb, Robert A; Jardetzky, Theodore S

2008-09-01

The mechanism by which the paramyxovirus hemagglutinin-neuraminidase (HN) protein couples receptor binding to activation of virus entry remains to be fully understood, but the HN stalk is thought to play an important role in the process. We have characterized ectodomain constructs of the parainfluenza virus 5 HN to understand better the underlying architecture and oligomerization properties that may influence HN functions. The PIV 5 neuraminidase (NA) domain is monomeric whereas the ectodomain forms a well-defined tetramer. The HN stalk also forms tetramers and higher order oligomers with high alpha-helical content. Together, the data indicate that the globular NA domains form weak intersubunit interactions at the end of the HN stalk tetramer, while stabilizing the stalk and overall oligomeric state of the ectodomain. Electron microscopy of the HN ectodomain reveals flexible arrangements of the NA and stalk domains, which may be important for understanding how these two HN domains impact virus entry.

An Immunosensor Based on Antibody Binding Fragments Attached to Gold Nanoparticles for the Detection of Peptides Derived from Avian Influenza Hemagglutinin H5

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Urszula Jarocka

2014-08-01

Full Text Available This paper concerns the development of an immunosensor for detection of peptides derived from avian influenza hemagglutinin H5. Its preparation consists of successive gold electrode modification steps: (i modification with 1,6-hexanedithiol and gold colloidal nanoparticles; (ii immobilization of antibody-binding fragments (Fab’ of anti-hemagglutinin H5 monoclonal antibodies Mab 6-9-1 via S-Au covalent bonds; and (iii covering the remaining free space on the electrode surfaces with bovine serum albumin. The interactions between Fab’ fragments and hemagglutinin (HA variants have been explored with electrochemical impedance spectroscopy (EIS in the presence of [Fe(CN6]3−/4− as an electroactive marker. The immunosensor was able to recognize three different His-tagged variants of recombinant hemagglutinin from H5N1 viruses: H1 subunit (17–340 residues of A/swan/Poland/305-135V08/2006, the long HA (17–530 residues A/Bar-headed Goose/Qinghai/12/2005 and H1 subunit (1–345 residues of A/Vietnam/1194/2004. The strongest response has been observed for the long variant with detection limit of 2.2 pg/mL and dynamic range from 4.0 to 20.0 pg/mL.

A global phylogenetic analysis in order to determine the host species and geography dependent features present in the evolution of avian H9N2 influenza hemagglutinin

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Andrew R. Dalby

2014-10-01

Full Text Available A complete phylogenetic analysis of all of the H9N2 hemagglutinin sequences that were collected between 1966 and 2012 was carried out in order to build a picture of the geographical and host specific evolution of the hemagglutinin protein. To improve the quality and applicability of the output data the sequences were divided into subsets based upon location and host species.The phylogenetic analysis of hemagglutinin reveals that the protein has distinct lineages between China and the Middle East, and that wild birds in both regions retain a distinct form of the H9 molecule, from the same lineage as the ancestral hemagglutinin. The results add further evidence to the hypothesis that the current predominant H9N2 hemagglutinin lineage might have originated in Southern China. The study also shows that there are sampling problems that affect the reliability of this and any similar analysis. This raises questions about the surveillance of H9N2 and the need for wider sampling of the virus in the environment.The results of this analysis are also consistent with a model where hemagglutinin has predominantly evolved by neutral drift punctuated by occasional selection events. These selective events have produced the current pattern of distinct lineages in the Middle East, Korea and China. This interpretation is in agreement with existing studies that have shown that there is widespread intra-country sequence evolution.

Swine Influenza Virus (H1N2) Characterization and Transmission in Ferrets, Chile.

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Bravo-Vasquez, Nicolás; Karlsson, Erik A; Jimenez-Bluhm, Pedro; Meliopoulos, Victoria; Kaplan, Bryan; Marvin, Shauna; Cortez, Valerie; Freiden, Pamela; Beck, Melinda A; Hamilton-West, Christopher; Schultz-Cherry, Stacey

2017-02-01

Phylogenetic analysis of the influenza hemagglutinin gene (HA) has suggested that commercial pigs in Chile harbor unique human seasonal H1-like influenza viruses, but further information, including characterization of these viruses, was unavailable. We isolated influenza virus (H1N2) from a swine in a backyard production farm in Central Chile and demonstrated that the HA gene was identical to that in a previous report. Its HA and neuraminidase genes were most similar to human H1 and N2 viruses from the early 1990s and internal segments were similar to influenza A(H1N1)pdm09 virus. The virus replicated efficiently in vitro and in vivo and transmitted in ferrets by respiratory droplet. Antigenically, it was distinct from other swine viruses. Hemagglutination inhibition analysis suggested that antibody titers to the swine Chilean H1N2 virus were decreased in persons born after 1990. Further studies are needed to characterize the potential risk to humans, as well as the ecology of influenza in swine in South America.

Expression of defective measles virus genes in brain tissues of patients with subacute sclerosing panencephalitis

International Nuclear Information System (INIS)

Baczko, K.; Liebert, U.G.; Billeter, M.; Cattaneo, R.; Budka, H.; Ter Meulen, V.

1986-01-01

The persistence of measles virus in selected areas of the brains of four patients with subacute sclerosing panencephalitis (SSPE) was characterized by immunohistological and biochemical techniques. The five measles virus structural proteins were never simultaneously detectable in any of the bran sections. Nucleocapsid proteins and phosphoproteins were found in every diseased brain area, whereas hemagglutinin protein was detected in two cases, fusion protein was detected in three cases, and matrix protein was detected in only one case. Also, it could be shown that the amounts of measles virus RNA in the brains differed from patient to patient and in the different regions investigated. In all patients, plus-strand RNAs specific for these five viral genes could be detected. However, the amounts of fusion and hemagglutinin mRNAs were low compared with the amounts in lytically infected cells. The presence of particular measles virus RNAs in SSPE-infected brains did not always correlate with mRNA activity. In in vitro translations, the matrix protein was produced in only one case, and the hemagglutinin protein was produced in none. These results indicate that measles virus persistence in SSPE is correlated with different defects of several genes which probably prevent assembly of viral particles in SSPE-infected brain tissue

Newcastle disease virus-based H5 influenza vaccine protects chickens from lethal challenge with a highly pathogenic H5N2 avian influenza virus.

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Ma, Jingjiao; Lee, Jinhwa; Liu, Haixia; Mena, Ignacio; Davis, A Sally; Sunwoo, Sun Young; Lang, Yuekun; Duff, Michael; Morozov, Igor; Li, Yuhao; Yang, Jianmei; García-Sastre, Adolfo; Richt, Juergen A; Ma, Wenjun

2017-01-01

Since December 2014, Eurasian-origin, highly pathogenic avian influenza H5 viruses including H5N1, H5N2, and H5N8 subtypes (called H5N x viruses), which belong to the H5 clade 2.3.4.4, have been detected in U.S. wild birds. Subsequently, highly pathogenic H5N2 and H5N8 viruses have caused outbreaks in U.S. domestic poultry. Vaccination is one of the most effective ways to control influenza outbreaks and protect animal and public health. Newcastle disease virus (NDV)-based influenza vaccines have been demonstrated to be efficacious and safe in poultry. Herein, we developed an NDV-based H5 vaccine (NDV-H5) that expresses a codon-optimized ectodomain of the hemagglutinin from the A/chicken/Iowa/04-20/2015 (H5N2) virus and evaluated its efficacy in chickens. Results showed that both live and inactivated NDV-H5 vaccines induced hemagglutinin inhibition antibody titers against the H5N2 virus in immunized chickens after prime and booster, and both NDV-H5 vaccines completely protected chickens from lethal challenge with the highly pathogenic H5N2 A/turkey/Minnesota/9845-4/2015 virus. No clinical signs and only minimal virus shedding was observed in both vaccinated groups. In contrast, all mock-vaccinated, H5N2-infected chickens shed virus and died within 5 days post challenge. Furthermore, one dose of the live NDV-H5 vaccine also provided protection of 90% chickens immunized by coarse spraying; after exposure to H5N2 challenge, sera from vaccinated surviving chickens neutralized both highly pathogenic H5N1 and H5N8 viruses. Taken together, our results suggest that the NDV-based H5 vaccine is able to protect chickens against intercontinental highly pathogenic H5N x viruses and can be used by mass application to protect the poultry industry.

Stable lentiviral transformation of CHO cells for the expression of the hemagglutinin H5 of avian influenza virus in suspension culture

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Alaín González Pose

2014-09-01

Full Text Available Avian influenza virus H5N1 has caused extensive damage worldwide among poultry and humans. Effective expression systems are needed for the production of viral proteins required for monitoring this devastating disease. The present study deals with the establishment of a stable expression system for the hemagglutinin H5 (HAH5 of avian influenza virus using CHO cells in suspension culture transduced with a recombinant lentiviral vector. The synthetic gene coding the HAH5 protein was inserted in a lentiviral vector with the aim of performing a stable transduction of CHO cells. After the selection of recombinant clones, the one with the highest expression level was adapted to suspension culture and the HAH5 protein was purified by immunoaffinity chromatography from the culture supernatant. There were no significant differences when this protein, purified or direct from the culture supernatant of CHO or SiHa cells, was utilized in an immunologic assay using positive and negative sera as reference. It was also demonstrated that the HAH5 protein in its purified form is able to bind anti-HAH5 antibodies generated with proper and non-proper folded proteins. The results demonstrate that the CHO cell line stably transduced with a lentiviral vector coding the sequence of the HAH5 protein and cultured in suspension can be a suitable expression system to obtain this protein for diagnostic purpose in a consistent and reliable manner.

Heterogeneity within the hemagglutinin genes of canine distemper virus (CDV) strains detected in Italy

DEFF Research Database (Denmark)

Martella, V.; Cirone, F.; Elia, G.

2006-01-01

Canine distemper virus (CDV) is a highly contagious viral pathogen causing lethal disease in dogs and other mammalians. A high degree of genetic variation is found between recent CDV strains and the old CDV isolates used in the vaccines and such genetic variation is regarded as a possible cause....... These results suggest that at least three different CDV lineages are present in Italy. Keywords: Canine distemper virus; Dogs; Lineages; H gene...

Expression of H5 hemagglutinin vaccine antigen in common duckweed (Lemna minor) protects against H5N1 high pathogenicity avian influenza virus challenge in immunized chickens.

Science.gov (United States)

Bertran, Kateri; Thomas, Colleen; Guo, Xuan; Bublot, Michel; Pritchard, Nikki; Regan, Jeffrey T; Cox, Kevin M; Gasdaska, John R; Dickey, Lynn F; Kapczynski, Darrell R; Swayne, David E

2015-07-09

A synthetic hemagglutinin (HA) gene from the highly pathogenic avian influenza (HPAI) virus A/chicken/Indonesia/7/2003 (H5N1) (Indo/03) was expressed in aquatic plant Lemna minor (rLemna-HA). In Experiment 1, efficacy of rLemna-HA was tested on birds immunized with 0.2μg or 2.3 μg HA and challenged with 10(6) mean chicken embryo infectious doses (EID50) of homologous virus strain. Both dosages of rLemna-HA conferred clinical protection and dramatically reduced viral shedding. Almost all the birds immunized with either dosage of rLemna-HA elicited HA antibody titers against Indo/03 antigen, suggesting an association between levels of anti-Indo/03 antibodies and protection. In Experiment 2, efficacy of rLemna-HA was tested on birds immunized with 0.9 μg or 2.2 μg HA and challenged with 10(6) EID50 of heterologous H5N1 virus strains A/chicken/Vietnam/NCVD-421/2010 (VN/10) or A/chicken/West Java/PWT-WIJ/2006 (PWT/06). Birds challenged with VN/10 exhibited 100% survival regardless of immunization dosage, while birds challenged with PWT/06 had 50% and 30% mortality at 0.9 μg HA and 2.2 μg HA, respectively. For each challenge virus, viral shedding titers from 2.2 μg HA vaccinated birds were significantly lower than those from 0.9μg HA vaccinated birds, and titers from both immunized groups were in turn significantly lower than those from sham vaccinated birds. Even if immunized birds elicited HA titers against the vaccine antigen Indo/03, only the groups challenged with VN/10 developed humoral immunity against the challenge antigen. None (rLemna-HA 0.9 μg HA) and 40% (rLemna-HA 2.2 μg HA) of the immunized birds challenged with PWT/06 elicited pre-challenge antibody titers, respectively. In conclusion, Lemna-expressed HA demonstrated complete protective immunity against homologous challenge and suboptimal protection against heterologous challenge, the latter being similar to results from inactivated whole virus vaccines. Transgenic duckweed-derived HA could be a

MVA recombinants expressing the fusion and hemagglutinin genes of PPRV protects goats against virulent challenge.

Science.gov (United States)

Chandran, Dev; Reddy, Kolli Bhaktavatsala; Vijayan, Shahana Pallichera; Sugumar, Parthasarthy; Rani, Gudavalli Sudha; Kumar, Ponsekaran Santha; Rajendra, Lingala; Srinivasan, Villuppanoor Alwar

2010-09-01

Peste des Petits Ruminants (PPR) is a highly contagious animal disease caused by the Peste des Petits Ruminants virus (PPRV) belonging to the genus morbillivirus and family Paramyxoviridae. The disease results in high morbidity and mortality in goats, sheep and in some small wild ruminants. The presence of large number of small ruminants reared in endemic areas makes PPR a notorious disease threatening the livelihood of poor farmers. Conventional vaccination using a live, attenuated vaccine gives adequate protection but cannot be used in case of eradication of the disease due to difficulty in differentiation of infected animals from the vaccinated ones.In the present study, we constructed two recombinant viruses using attenuated Modified Vaccinia virus Ankara virus (MVA) namely MVA-F and MVA-H expressing the full length PPRV fusion (F) and hemagglutinin (H) glycoproteins, respectively. Goats were vaccinated intramuscularly with 105 plaque forming units (PFU) each of the recombinant viruses and a live attenuated vaccine (RAKSHA PPR) and challenged 4 months later with PPRV challenge virus (10(3) goat LD(50)). All goats were completely protected from the clinical disease. This study gave an indication that mass vaccination of small ruminants with either of the above or both recombinant inexpensive virus vaccines could help in possible eradication of PPRV from endemic countries like India and subsequent seromonitoring of the disease for differentiation of infected animals from vaccinated ones.

Fatal canine distemper virus infection of giant pandas in China.

Science.gov (United States)

Feng, Na; Yu, Yicong; Wang, Tiecheng; Wilker, Peter; Wang, Jianzhong; Li, Yuanguo; Sun, Zhe; Gao, Yuwei; Xia, Xianzhu

2016-06-16

We report an outbreak of canine distemper virus (CDV) infection among endangered giant pandas (Ailuropoda melanoleuca). Five of six CDV infected giant pandas died. The surviving giant panda was previously vaccinated against CDV. Genomic sequencing of CDV isolated from one of the infected pandas (giant panda/SX/2014) suggests it belongs to the Asia-1 cluster. The hemagglutinin protein of the isolated virus and virus sequenced from lung samples originating from deceased giant pandas all possessed the substitutions V26M, T213A, K281R, S300N, P340Q, and Y549H. The presence of the Y549H substitution is notable as it is found at the signaling lymphocytic activation molecule (SLAM) receptor-binding site and has been implicated in the emergence of highly pathogenic CDV and host switching. These findings demonstrate that giant pandas are susceptible to CDV and suggest that surveillance and vaccination among all captive giant pandas are warranted to support conservation efforts for this endangered species.

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

The recombinant globular head domain of the measles virus hemagglutinin protein as a subunit vaccine against measles.

Science.gov (United States)

Lobanova, Liubov M; Eng, Nelson F; Satkunarajah, Malathy; Mutwiri, George K; Rini, James M; Zakhartchouk, Alexander N

2012-04-26

Despite the availability of live attenuated measles virus (MV) vaccines, a large number of measles-associated deaths occur among infants in developing countries. The development of a measles subunit vaccine may circumvent the limitations associated with the current live attenuated vaccines and eventually contribute to global measles eradication. Therefore, the goal of this study was to test the feasibility of producing the recombinant globular head domain of the MV hemagglutinin (H) protein by stably transfected human cells and to examine the ability of this recombinant protein to elicit MV-specific immune responses. The recombinant protein was purified from the culture supernatant of stably transfected HEK293T cells secreting a tagged version of the protein. Two subcutaneous immunizations with the purified recombinant protein alone resulted in the production of MV-specific serum IgG and neutralizing antibodies in mice. Formulation of the protein with adjuvants (polyphosphazene or alum) further enhanced the humoral immune response and in addition resulted in the induction of cell-mediated immunity as measured by the production of MV H-specific interferon gamma (IFN-γ) and interleukin 5 (IL-5) by in vitro re-stimulated splenocytes. Furthermore, the inclusion of polyphosphazene into the vaccine formulation induced a mixed Th1/Th2-type immune response. In addition, the purified recombinant protein retained its immunogenicity even after storage at 37°C for 2 weeks. Copyright © 2012 Elsevier Ltd. All rights reserved.

In vitro evolution of H5N1 avian influenza virus toward human-type receptor specificity

DEFF Research Database (Denmark)

Chen, Li-Mei; Blixt, Klas Ola; Stevens, James

2012-01-01

Acquisition of a2-6 sialoside receptor specificity by a2-3 specific highly-pathogenic avian influenza viruses (H5N1) is thought to be a prerequisite for efficient transmission in humans. By in vitro selection for binding a2-6 sialosides, we identified four variant viruses with amino acid....... Unlike the wild type H5N1, this mutant virus was transmitted by direct contact in the ferret model although not by airborne respiratory droplets. However, a reassortant virus with the mutant hemagglutinin, a human N2 neuraminidase and internal genes from an H5N1 virus was partially transmitted via...... respiratory droplets. The complex changes required for airborne transmissibility in ferrets suggest that extensive evolution is needed for H5N1 transmissibility in humans....

Development and Characterization of Canine Distemper Virus Monoclonal Antibodies.

Science.gov (United States)

Liu, Yuxiu; Hao, Liying; Li, Xiangdong; Wang, Linxiao; Zhang, Jianpo; Deng, Junhua; Tian, Kegong

2017-06-01

Five canine distemper virus monoclonal antibodies were developed by immunizing BALB/c mice with a traditional vaccine strain Snyder Hill. Among these monoclonal antibodies, four antibodies recognized both field and vaccine strains of canine distemper virus without neutralizing ability. One monoclonal antibody, 1A4, against hemagglutinin protein of canine distemper virus was found to react only with vaccine strain virus but not field isolates, and showed neutralizing activity to vaccine strain virus. These monoclonal antibodies could be very useful tools in the study of the pathogenesis of canine distemper virus and the development of diagnostic reagents.

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....

Detection of closed influenza virus hemagglutinin fusion peptide structures in membranes by backbone {sup 13}CO-{sup 15}N rotational-echo double-resonance solid-state NMR

Energy Technology Data Exchange (ETDEWEB)

Ghosh, Ujjayini; Xie Li; Weliky, David P., E-mail: weliky@chemistry.msu.edu [Michigan State University, Department of Chemistry (United States)

2013-02-15

The influenza virus fusion peptide is the N-terminal {approx}20 residues of the HA2 subunit of the hemagglutinin protein and this peptide plays a key role in the fusion of the viral and endosomal membranes during initial infection of a cell. The fusion peptide adopts N-helix/turn/C-helix structure in both detergent and membranes with reports of both open and closed interhelical topologies. In the present study, backbone {sup 13}CO-{sup 15}N REDOR solid-state NMR was applied to the membrane-associated fusion peptide to detect the distribution of interhelical distances. The data clearly showed a large fraction of closed and semi-closed topologies and were best-fitted to a mixture of two structures that do not exchange. One of the earlier open structural models may have incorrect G13 dihedral angles derived from TALOS analysis of experimentally correct {sup 13}C shifts.

A Single-Amino-Acid Substitution at Position 225 in Hemagglutinin Alters the Transmissibility of Eurasian Avian-Like H1N1 Swine Influenza Virus in Guinea Pigs.

Science.gov (United States)

Wang, Zeng; Yang, Huanliang; Chen, Yan; Tao, Shiyu; Liu, Liling; Kong, Huihui; Ma, Shujie; Meng, Fei; Suzuki, Yasuo; Qiao, Chuanling; Chen, Hualan

2017-11-01

Efficient transmission from human to human is the prerequisite for an influenza virus to cause a pandemic; however, the molecular determinants of influenza virus transmission are still largely unknown. In this study, we explored the molecular basis for transmission of Eurasian avian-like H1N1 (EAH1N1) swine influenza viruses by comparing two viruses that are genetically similar but differ in their transmissibility in guinea pigs: the A/swine/Guangxi/18/2011 virus (GX/18) is highly transmissible by respiratory droplet in guinea pigs, whereas the A/swine/Heilongjiang/27/2012 virus (HLJ/27) does not transmit in this animal model. We used reverse genetics to generate a series of reassortants and mutants in the GX/18 background and tested their transmissibility in guinea pigs. We found that a single-amino-acid substitution of glycine (G) for glutamic acid (E) at position 225 (E225G) in the HA1 protein completely abolished the respiratory droplet transmission of GX/18, whereas the substitution of E for G at the same position (G225E) in HA1 enabled HLJ/27 to transmit in guinea pigs. We investigated the underlying mechanism and found that viruses bearing 225E in HA1 replicated more rapidly than viruses bearing 225G due to differences in assembly and budding efficiencies. Our study indicates that the amino acid 225E in HA1 plays a key role in EAH1N1 swine influenza virus transmission and provides important information for evaluating the pandemic potential of field influenza virus strains. IMPORTANCE Efficient transmission among humans is a prerequisite for a novel influenza virus to cause a human pandemic. Transmissibility of influenza viruses is a polygenic trait, and understanding the genetic determinants for transmissibility will provide useful insights for evaluating the pandemic potential of influenza viruses in the field. Several amino acids in the hemagglutinin (HA) protein of influenza viruses have been shown to be important for transmissibility, usually by

Structures of Receptor Complexes of a North American H7N2 Influenza Hemagglutinin with a Loop Deletion in the Receptor Binding Site

Energy Technology Data Exchange (ETDEWEB)

Yang, Hua; Chen, Li-Mei; Carney, Paul J.; Donis, Ruben O.; Stevens, James (CDC)

2012-02-21

Human infections with subtype H7 avian influenza viruses have been reported as early as 1979. In 1996, a genetically stable 24-nucleotide deletion emerged in North American H7 influenza virus hemagglutinins, resulting in an eight amino acid deletion in the receptor-binding site. The continuous circulation of these viruses in live bird markets, as well as its documented ability to infect humans, raises the question of how these viruses achieve structural stability and functionality. Here we report a detailed molecular analysis of the receptor binding site of the North American lineage subtype H7N2 virus A/New York/107/2003 (NY107), including complexes with an avian receptor analog (3'-sialyl-N-acetyllactosamine, 3'SLN) and two human receptor analogs (6'-sialyl-N-acetyllactosamine, 6'SLN; sialyllacto-N-tetraose b, LSTb). Structural results suggest a novel mechanism by which residues Arg220 and Arg229 (H3 numbering) are used to compensate for the deletion of the 220-loop and form interactions with the receptor analogs. Glycan microarray results reveal that NY107 maintains an avian-type ({alpha}2-3) receptor binding profile, with only moderate binding to human-type ({alpha}2-6) receptor. Thus despite its dramatically altered receptor binding site, this HA maintains functionality and confirms a need for continued influenza virus surveillance of avian and other animal reservoirs to define their zoonotic potential.

Structures of receptor complexes of a North American H7N2 influenza hemagglutinin with a loop deletion in the receptor binding site.

Directory of Open Access Journals (Sweden)

Hua Yang

2010-09-01

Full Text Available Human infections with subtype H7 avian influenza viruses have been reported as early as 1979. In 1996, a genetically stable 24-nucleotide deletion emerged in North American H7 influenza virus hemagglutinins, resulting in an eight amino acid deletion in the receptor-binding site. The continuous circulation of these viruses in live bird markets, as well as its documented ability to infect humans, raises the question of how these viruses achieve structural stability and functionality. Here we report a detailed molecular analysis of the receptor binding site of the North American lineage subtype H7N2 virus A/New York/107/2003 (NY107, including complexes with an avian receptor analog (3'-sialyl-N-acetyllactosamine, 3'SLN and two human receptor analogs (6'-sialyl-N-acetyllactosamine, 6'SLN; sialyllacto-N-tetraose b, LSTb. Structural results suggest a novel mechanism by which residues Arg220 and Arg229 (H3 numbering are used to compensate for the deletion of the 220-loop and form interactions with the receptor analogs. Glycan microarray results reveal that NY107 maintains an avian-type (alpha2-3 receptor binding profile, with only moderate binding to human-type (alpha2-6 receptor. Thus despite its dramatically altered receptor binding site, this HA maintains functionality and confirms a need for continued influenza virus surveillance of avian and other animal reservoirs to define their zoonotic potential.

Migration and persistence of human influenza A viruses, Vietnam, 2001-2008.

Science.gov (United States)

Le, Mai Quynh; Lam, Ha Minh; Cuong, Vuong Duc; Lam, Tommy Tsan-Yuk; Halpin, Rebecca A; Wentworth, David E; Hien, Nguyen Tran; Thanh, Le Thi; Phuong, Hoang Vu Mai; Horby, Peter; Boni, Maciej F

2013-11-01

Understanding global influenza migration and persistence is crucial for vaccine strain selection. Using 240 new human influenza A virus whole genomes collected in Vietnam during 2001-2008, we looked for persistence patterns and migratory connections between Vietnam and other countries. We found that viruses in Vietnam migrate to and from China, Hong Kong, Taiwan, Cambodia, Japan, South Korea, and the United States. We attempted to reduce geographic bias by generating phylogenies subsampled at the year and country levels. However, migration events in these phylogenies were still driven by the presence or absence of sequence data, indicating that an epidemiologic study design that controls for prevalence is required for robust migration analysis. With whole-genome data, most migration events are not detectable from the phylogeny of the hemagglutinin segment alone, although general migratory relationships between Vietnam and other countries are visible in the hemagglutinin phylogeny. It is possible that virus lineages in Vietnam persisted for >1 year.

DNA vaccines encoding proteins from wild-type and attenuated canine distemper virus protect equally well against wild-type virus challenge.

Science.gov (United States)

Nielsen, Line; Jensen, Trine Hammer; Kristensen, Birte; Jensen, Tove Dannemann; Karlskov-Mortensen, Peter; Lund, Morten; Aasted, Bent; Blixenkrone-Møller, Merete

2012-10-01

Immunity induced by DNA vaccines containing the hemagglutinin (H) and nucleoprotein (N) genes of wild-type and attenuated canine distemper virus (CDV) was investigated in mink (Mustela vison), a highly susceptible natural host of CDV. All DNA-immunized mink seroconverted, and significant levels of virus-neutralizing (VN) antibodies were present on the day of challenge with wild-type CDV. The DNA vaccines also primed the cell-mediated memory responses, as indicated by an early increase in the number of interferon-gamma (IFN-γ)-producing lymphocytes after challenge. Importantly, the wild-type and attenuated CDV DNA vaccines had a long-term protective effect against wild-type CDV challenge. The vaccine-induced immunity induced by the H and N genes from wild-type CDV and those from attenuated CDV was comparable. Because these two DNA vaccines were shown to protect equally well against wild-type virus challenge, it is suggested that the genetic/antigenic heterogeneity between vaccine strains and contemporary wild-type strains are unlikely to cause vaccine failure.

Mumps vaccine virus genome is present in throat swabs obtained from uncomplicated healthy recipients.

Science.gov (United States)

Nagai, T; Nakayama, T

2001-01-08

Seven children were followed for up to 42 days post-vaccination with live mumps vaccine and 37 throat swabs were obtained serially. Viral genomic RNA was detected by reverse transcription-polymerase chain reaction (RT-PCR) in the phosphoprotein (P) and hemagglutinin-neuraminidase (HN) regions. Virus isolation was also attempted. Genomic differentiation of detected mumps virus genome was performed by sequence analysis and/or restriction fragment length polymorphism (RFLP). No adverse reaction was observed in these children. Although mumps virus was not isolated from any of the samples, viral RNA was detected in four samples from three vaccine recipients, 18, 18 and 26, and 7 days after vaccination, respectively. Detected viral RNA was identified as the vaccine strain. Our data suggests that vaccine virus inoculated replicates in the parotid glands but the incidence of virus transmission from recipients to other susceptible subjects should be low.

Influence of vaccine strains on the evolution of canine distemper virus.

Science.gov (United States)

da Fontoura Budaszewski, Renata; Streck, André Felipe; Nunes Weber, Matheus; Maboni Siqueira, Franciele; Muniz Guedes, Rafael Lucas; Wageck Canal, Cláudio

2016-07-01

Canine distemper virus (CDV) is a major dog pathogen belonging to the genus Morbillivirus of the family Paramyxoviridae. CDV causes disease and high mortality in dogs and wild carnivores. Although homologous recombination has been demonstrated in many members of Paramyxoviridae, these events have rarely been reported for CDV. To detect potential recombination events, the complete CDV genomes available in GenBank up to June 2015 were screened using distinct algorithms to detect genetic conversions and incongruent phylogenies. Eight putative recombinant viruses derived from different CDV genotypes and different hosts were detected. The breakpoints of the recombinant strains were primarily located on fusion and hemagglutinin glycoproteins. These results suggest that homologous recombination is a frequent phenomenon in morbillivirus populations under natural replication, and CDV vaccine strains might play an important role in shaping the evolution of this virus.

Immune Escape Variants of H9N2 Influenza Viruses Containing Deletions at the Hemagglutinin Receptor Binding Site Retain Fitness In Vivo and Display Enhanced Zoonotic Characteristics.

Science.gov (United States)

Peacock, Thomas P; Benton, Donald J; James, Joe; Sadeyen, Jean-Remy; Chang, Pengxiang; Sealy, Joshua E; Bryant, Juliet E; Martin, Stephen R; Shelton, Holly; Barclay, Wendy S; Iqbal, Munir

2017-07-15

H9N2 avian influenza viruses are enzootic in poultry across Asia and North Africa, where they pose a threat to human health as both zoonotic agents and potential pandemic candidates. Poultry vaccination against H9N2 viruses has been employed in many regions; however, vaccine effectiveness is frequently compromised due to antigenic drift arising from amino acid substitutions in the major influenza virus antigen hemagglutinin (HA). Using selection with HA-specific monoclonal antibodies, we previously identified H9N2 antibody escape mutants that contained deletions of amino acids in the 220 loop of the HA receptor binding sites (RBSs). Here we analyzed the impact of these deletions on virus zoonotic infection characteristics and fitness. We demonstrated that mutant viruses with RBS deletions are able to escape polyclonal antiserum binding and are able to infect and be transmitted between chickens. We showed that the deletion mutants have increased binding to human-like receptors and greater replication in primary human airway cells; however, the mutant HAs also displayed reduced pH and thermal stability. In summary, we infer that variant influenza viruses with deletions in the 220 loop could arise in the field due to immune selection pressure; however, due to reduced HA stability, we conclude that these viruses are unlikely to be transmitted from human to human by the airborne route, a prerequisite for pandemic emergence. Our findings underscore the complex interplay between antigenic drift and viral fitness for avian influenza viruses as well as the challenges of predicting which viral variants may pose the greatest threats for zoonotic and pandemic emergence. IMPORTANCE Avian influenza viruses, such as H9N2, cause disease in poultry as well as occasionally infecting humans and are therefore considered viruses with pandemic potential. Many countries have introduced vaccination of poultry to try to control the disease burden; however, influenza viruses are able to

Standardization of an inactivated H17N1 avian influenza vaccine and efficacy against A/Chicken/Italy/13474/99 high-pathogenicity virus infection.

Science.gov (United States)

Di Trani, L; Cordioli, P; Falcone, E; Lombardi, G; Moreno, A; Sala, G; Tollis, M

2003-01-01

The minimum requirements for assessing the immunogenicity of an experimental avian influenza (AI) vaccine prepared from inactivated A/Turkey/Italy/2676/99 (H7N1) low-pathogenicity (LP) AI (LPAI) virus were determined in chickens of different ages. A correlation between the amount of hemagglutinin (HA) per dose of vaccine and the protection against clinical signs of disease and infection by A/Chicken/Italy/13474/99 highly pathogenic (HP) AI (HPAI) virus was established. Depending on the vaccination schedule, one or two administrations of 0.5 microg of hemagglutinin protected chickens against clinical signs and death and completely prevented virus shedding from birds challenged at different times after vaccination.

Seasonal trivalent inactivated influenza vaccine protects against 1918 Spanish influenza virus in ferrets

Science.gov (United States)

The influenza H1N1 pandemic of 1918 was one of the worst medical disasters in human history. Recent studies have demonstrated that the hemagglutinin (HA) protein of the 1918 virus and 2009 H1N1 pandemic virus, the latter now a component of the seasonal trivalent inactivated influenza vaccine (TIV),...

Migration and Persistence of Human Influenza A Viruses, Vietnam, 2001–2008

Science.gov (United States)

Le, Mai Quynh; Lam, Ha Minh; Cuong, Vuong Duc; Lam, Tommy Tsan-Yuk; Halpin, Rebecca A; Wentworth, David E; Hien, Nguyen Tran; Thanh, Le Thi; Phuong, Hoang Vu Mai; Horby, Peter

2013-01-01

Understanding global influenza migration and persistence is crucial for vaccine strain selection. Using 240 new human influenza A virus whole genomes collected in Vietnam during 2001–2008, we looked for persistence patterns and migratory connections between Vietnam and other countries. We found that viruses in Vietnam migrate to and from China, Hong Kong, Taiwan, Cambodia, Japan, South Korea, and the United States. We attempted to reduce geographic bias by generating phylogenies subsampled at the year and country levels. However, migration events in these phylogenies were still driven by the presence or absence of sequence data, indicating that an epidemiologic study design that controls for prevalence is required for robust migration analysis. With whole-genome data, most migration events are not detectable from the phylogeny of the hemagglutinin segment alone, although general migratory relationships between Vietnam and other countries are visible in the hemagglutinin phylogeny. It is possible that virus lineages in Vietnam persisted for >1 year. PMID:24188643

Diversity of the murine antibody response targeting influenza A(H1N1pdm09) hemagglutinin.

Science.gov (United States)

Wilson, Jason R; Tzeng, Wen-Pin; Spesock, April; Music, Nedzad; Guo, Zhu; Barrington, Robert; Stevens, James; Donis, Ruben O; Katz, Jacqueline M; York, Ian A

2014-06-01

We infected mice with the 2009 influenza A pandemic virus (H1N1pdm09), boosted with an inactivated vaccine, and cloned immunoglobulins (Igs) from HA-specific B cells. Based on the redundancy in germline gene utilization, we inferred that between 72-130 unique IgH VDJ and 35 different IgL VJ combinations comprised the anti-HA recall response. The IgH VH1 and IgL VK14 variable gene families were employed most frequently. A representative panel of antibodies were cloned and expressed to confirm reactivity with H1N1pdm09 HA. The majority of the recombinant antibodies were of high avidity and capable of inhibiting H1N1pdm09 hemagglutination. Three of these antibodies were subtype-specific cross-reactive, binding to the HA of A/South Carolina/1/1918(H1N1), and one further reacted with A/swine/Iowa/15/1930(H1N1). These results help to define the genetic diversity of the influenza anti-HA antibody repertoire profile induced following infection and vaccination, which may facilitate the development of influenza vaccines that are more protective and broadly neutralizing. Protection against influenza viruses is mediated mainly by antibodies, and in most cases this antibody response is narrow, only providing protection against closely related viruses. In spite of this limited range of protection, recent findings indicate that individuals immune to one influenza virus may contain antibodies (generally a minority of the overall response) that are more broadly reactive. These findings have raised the possibility that influenza vaccines could induce a more broadly protective response, reducing the need for frequent vaccine strain changes. However, interpretation of these observations is hampered by the lack of quantitative characterization of the antibody repertoire. In this study, we used single-cell cloning of influenza HA-specific B cells to assess the diversity and nature of the antibody response to influenza hemagglutinin in mice. Our findings help to put bounds on the

Fatal canine distemper virus infection of giant pandas in China

Science.gov (United States)

Feng, Na; Yu, Yicong; Wang, Tiecheng; Wilker, Peter; Wang, Jianzhong; Li, Yuanguo; Sun, Zhe; Gao, Yuwei; Xia, Xianzhu

2016-01-01

We report an outbreak of canine distemper virus (CDV) infection among endangered giant pandas (Ailuropoda melanoleuca). Five of six CDV infected giant pandas died. The surviving giant panda was previously vaccinated against CDV. Genomic sequencing of CDV isolated from one of the infected pandas (giant panda/SX/2014) suggests it belongs to the Asia-1 cluster. The hemagglutinin protein of the isolated virus and virus sequenced from lung samples originating from deceased giant pandas all possessed the substitutions V26M, T213A, K281R, S300N, P340Q, and Y549H. The presence of the Y549H substitution is notable as it is found at the signaling lymphocytic activation molecule (SLAM) receptor-binding site and has been implicated in the emergence of highly pathogenic CDV and host switching. These findings demonstrate that giant pandas are susceptible to CDV and suggest that surveillance and vaccination among all captive giant pandas are warranted to support conservation efforts for this endangered species. PMID:27310722

Continuing evolution of H9N2 avian influenza virus in South Korea

Science.gov (United States)

The H9N2 low pathogenic avian influenza (LPAI) has caused great economic losses in Korean poultry industry since the first outbreak in 1996. Although the hemagglutinin gene of early H9N2 viruses were closely related to Chinese Y439-like lineage virus, it evolved into a unique Korean lineage after ...

Cowpox Virus in Llama, Italy

Science.gov (United States)

Brozzi, Alberto; Eleni, Claudia; Polici, Nicola; D’Alterio, Gianlorenzo; Carletti, Fabrizio; Scicluna, Maria Teresa; Castilletti, Concetta; Capobianchi, Maria R.; Di Caro, Antonino; Autorino, Gian Luca; Amaddeo, Demetrio

2011-01-01

Cowpox virus (CPXV) was isolated from skin lesions of a llama on a farm in Italy. Transmission electron microscopy showed brick-shaped particles consistent with orthopoxviruses. CPXV-antibodies were detected in llama and human serum samples; a CPXV isolate had a hemagglutinin sequence identical to CPXV-MonKre08/1–2-3 strains isolated from banded mongooses in Germany. PMID:21801638

Perspective of Use of Antiviral Peptides against Influenza Virus

Czech Academy of Sciences Publication Activity Database

Skaličková, S.; Heger, Z.; Krejčová, L.; Pekárik, V.; Bastl, K.; Janda, Jozef; Kostolanský, F.; Varečková, E.; Zítka, O.; Adam, V.; Kizek, R.

2015-01-01

Roč. 7, č. 10 (2015), s. 5428-5442 ISSN 1999-4915 R&D Projects: GA MŠk ED2.1.00/03.0124 Institutional support: RVO:67985904 Keywords : cationic peptides * hemagglutinin * influenza virus Subject RIV: EE - Microbiology, Virology Impact factor: 3.042, year: 2015

Influenza A virus targets a cGAS-independent STING pathway that controls enveloped RNA viruses.

Science.gov (United States)

Holm, Christian K; Rahbek, Stine H; Gad, Hans Henrik; Bak, Rasmus O; Jakobsen, Martin R; Jiang, Zhaozaho; Hansen, Anne Louise; Jensen, Simon K; Sun, Chenglong; Thomsen, Martin K; Laustsen, Anders; Nielsen, Camilla G; Severinsen, Kasper; Xiong, Yingluo; Burdette, Dara L; Hornung, Veit; Lebbink, Robert Jan; Duch, Mogens; Fitzgerald, Katherine A; Bahrami, Shervin; Mikkelsen, Jakob Giehm; Hartmann, Rune; Paludan, Søren R

2016-02-19

Stimulator of interferon genes (STING) is known be involved in control of DNA viruses but has an unexplored role in control of RNA viruses. During infection with DNA viruses STING is activated downstream of cGAMP synthase (cGAS) to induce type I interferon. Here we identify a STING-dependent, cGAS-independent pathway important for full interferon production and antiviral control of enveloped RNA viruses, including influenza A virus (IAV). Further, IAV interacts with STING through its conserved hemagglutinin fusion peptide (FP). Interestingly, FP antagonizes interferon production induced by membrane fusion or IAV but not by cGAMP or DNA. Similar to the enveloped RNA viruses, membrane fusion stimulates interferon production in a STING-dependent but cGAS-independent manner. Abolishment of this pathway led to reduced interferon production and impaired control of enveloped RNA viruses. Thus, enveloped RNA viruses stimulate a cGAS-independent STING pathway, which is targeted by IAV.

Computation of Hemagglutinin Free Energy Difference by the Confinement Method

Science.gov (United States)

2017-01-01

Hemagglutinin (HA) mediates membrane fusion, a crucial step during influenza virus cell entry. How many HAs are needed for this process is still subject to debate. To aid in this discussion, the confinement free energy method was used to calculate the conformational free energy difference between the extended intermediate and postfusion state of HA. Special care was taken to comply with the general guidelines for free energy calculations, thereby obtaining convergence and demonstrating reliability of the results. The energy that one HA trimer contributes to fusion was found to be 34.2 ± 3.4kBT, similar to the known contributions from other fusion proteins. Although computationally expensive, the technique used is a promising tool for the further energetic characterization of fusion protein mechanisms. Knowledge of the energetic contributions per protein, and of conserved residues that are crucial for fusion, aids in the development of fusion inhibitors for antiviral drugs. PMID:29151344

Selective Bottlenecks Shape Evolutionary Pathways Taken during Mammalian Adaptation of a 1918-like Avian Influenza Virus.

Science.gov (United States)

Moncla, Louise H; Zhong, Gongxun; Nelson, Chase W; Dinis, Jorge M; Mutschler, James; Hughes, Austin L; Watanabe, Tokiko; Kawaoka, Yoshihiro; Friedrich, Thomas C

2016-02-10

Avian influenza virus reassortants resembling the 1918 human pandemic virus can become transmissible among mammals by acquiring mutations in hemagglutinin (HA) and polymerase. Using the ferret model, we trace the evolutionary pathway by which an avian-like virus evolves the capacity for mammalian replication and airborne transmission. During initial infection, within-host HA diversity increased drastically. Then, airborne transmission fixed two polymerase mutations that do not confer a detectable replication advantage. In later transmissions, selection fixed advantageous HA1 variants. Transmission initially involved a "loose" bottleneck, which became strongly selective after additional HA mutations emerged. The stringency and evolutionary forces governing between-host bottlenecks may therefore change throughout host adaptation. Mutations occurred in multiple combinations in transmitted viruses, suggesting that mammalian transmissibility can evolve through multiple genetic pathways despite phenotypic constraints. Our data provide a glimpse into avian influenza virus adaptation in mammals, with broad implications for surveillance on potentially zoonotic viruses. Copyright © 2016 Elsevier Inc. All rights reserved.

Casein Kinase 1α Mediates the Degradation of Receptors for Type I and Type II Interferons Caused by Hemagglutinin of Influenza A Virus.

Science.gov (United States)

Xia, Chuan; Wolf, Jennifer J; Vijayan, Madhuvanthi; Studstill, Caleb J; Ma, Wenjun; Hahm, Bumsuk

2018-04-01

Although influenza A virus (IAV) evades cellular defense systems to effectively propagate in the host, the viral immune-evasive mechanisms are incompletely understood. Our recent data showed that hemagglutinin (HA) of IAV induces degradation of type I IFN receptor 1 (IFNAR1). Here, we demonstrate that IAV HA induces degradation of type II IFN (IFN-γ) receptor 1 (IFNGR1), as well as IFNAR1, via casein kinase 1α (CK1α), resulting in the impairment of cellular responsiveness to both type I and II IFNs. IAV infection or transient HA expression induced degradation of both IFNGR1 and IFNAR1, whereas HA gene-deficient IAV failed to downregulate the receptors. IAV HA caused the phosphorylation and ubiquitination of IFNGR1, leading to the lysosome-dependent degradation of IFNGR1. Influenza viral HA strongly decreased cellular sensitivity to type II IFNs, as it suppressed the activation of STAT1 and the induction of IFN-γ-stimulated genes in response to exogenously supplied recombinant IFN-γ. Importantly, CK1α, but not p38 MAP kinase or protein kinase D2, was proven to be critical for HA-induced degradation of both IFNGR1 and IFNAR1. Pharmacologic inhibition of CK1α or small interfering RNA (siRNA)-based knockdown of CK1α repressed the degradation processes of both IFNGR1 and IFNAR1 triggered by IAV infection. Further, CK1α was shown to be pivotal for proficient replication of IAV. Collectively, the results suggest that IAV HA induces degradation of IFN receptors via CK1α, creating conditions favorable for viral propagation. Therefore, the study uncovers a new immune-evasive pathway of influenza virus. IMPORTANCE Influenza A virus (IAV) remains a grave threat to humans, causing seasonal and pandemic influenza. Upon infection, innate and adaptive immunity, such as the interferon (IFN) response, is induced to protect hosts against IAV infection. However, IAV seems to be equipped with tactics to evade the IFN-mediated antiviral responses, although the detailed

Structural Characterization of the Hemagglutinin Receptor Specificity from the 2009 H1N1 Influenza Pandemic

Energy Technology Data Exchange (ETDEWEB)

Xu, Rui; McBride, Ryan; Nycholat, Corwin M.; Paulson, James C.; Wilson, Ian A. (Scripps)

2012-02-13

Influenza virus hemagglutinin (HA) is the viral envelope protein that mediates viral attachment to host cells and elicits membrane fusion. The HA receptor-binding specificity is a key determinant for the host range and transmissibility of influenza viruses. In human pandemics of the 20th century, the HA normally has acquired specificity for human-like receptors before widespread infection. Crystal structures of the H1 HA from the 2009 human pandemic (A/California/04/2009 [CA04]) in complex with human and avian receptor analogs reveal conserved recognition of the terminal sialic acid of the glycan ligands. However, favorable interactions beyond the sialic acid are found only for {alpha}2-6-linked glycans and are mediated by Asp190 and Asp225, which hydrogen bond with Gal-2 and GlcNAc-3. For {alpha}2-3-linked glycan receptors, no specific interactions beyond the terminal sialic acid are observed. Our structural and glycan microarray analyses, in the context of other high-resolution HA structures with {alpha}2-6- and {alpha}2-3-linked glycans, now elucidate the structural basis of receptor-binding specificity for H1 HAs in human and avian viruses and provide a structural explanation for the preference for {alpha}2-6 siaylated glycan receptors for the 2009 pandemic swine flu virus.

Divergent genetic evolution of hemagglutinin in influenza A H1N1 and A H1N2 subtypes isolated in the south-France since the winter of 2001-2002.

Science.gov (United States)

Al Faress, Shaker; Cartet, Gaëlle; Ferraris, Olivier; Norder, Helene; Valette, Martine; Lina, Bruno

2005-07-01

Influenza A viruses are divided into subtypes based on their hemagglutinin (H1 to H15) and neuraminidase (N1 to N9) glycoproteins. Of these, three A subtypes H1N1, H3N2 and H1N2 circulate in the human population. Influenza A viruses display a high antigenic variability called "antigenic drift" which allows the virus to escape antibody neutralization. Evaluate the mutations apparition that might predict a divergent antigenic evolution of hemagglutinin in influenza A H1N1 and A H1N2 viruses. During the three winters of 2001-2002 to 2003-2004, 58 A H1N1 and 23 A H1N2 subtypes have been isolated from patients with influenza-like illness in the south of France. The HA1 region was analyzed by RT-PCR and subsequently sequenced to compare the HA1 genetic evolution of influenza A H1N1 and A H1N2 subtypes. Our results showed that 28 amino acid substitutions have accumulated in the HA1 region since the circulation of A/New Caledonia/20/99-like viruses in France. Of these, fifteen were located in four antigenic sites (B, C, D and E). Six of them were observed only in the A H1N2 isolates, six only in the A H1N1 isolates and three in both subtypes. Furthermore, nine of twenty two A H1N2 isolates from the winter of 2002-2003 shared a T90A amino acid change which has not been observed in any A H1N1 isolate; resulting in the introduction of a new glycosylation site close to the antigenic site E. This might mask some antigenic E determinants and therefore, modify the A H1N2 antigenicity. The divergent genetic evolution of hemagglutinin may ultimately lead to a significant different antigenicity between A H1N1 and A H1N2 subtypes that would require the introduction of a new subtype in the vaccine batches.

Vaccination with Recombinant Parainfluenza Virus 5 Expressing Neuraminidase Protects against Homologous and Heterologous Influenza Virus Challenge.

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Mooney, Alaina J; Gabbard, Jon D; Li, Zhuo; Dlugolenski, Daniel A; Johnson, Scott K; Tripp, Ralph A; He, Biao; Tompkins, S Mark

2017-12-01

Seasonal human influenza virus continues to cause morbidity and mortality annually, and highly pathogenic avian influenza (HPAI) viruses along with other emerging influenza viruses continue to pose pandemic threats. Vaccination is considered the most effective measure for controlling influenza; however, current strategies rely on a precise vaccine match with currently circulating virus strains for efficacy, requiring constant surveillance and regular development of matched vaccines. Current vaccines focus on eliciting specific antibody responses against the hemagglutinin (HA) surface glycoprotein; however, the diversity of HAs across species and antigenic drift of circulating strains enable the evasion of virus-inhibiting antibody responses, resulting in vaccine failure. The neuraminidase (NA) surface glycoprotein, while diverse, has a conserved enzymatic site and presents an appealing target for priming broadly effective antibody responses. Here we show that vaccination with parainfluenza virus 5 (PIV5), a promising live viral vector expressing NA from avian (H5N1) or pandemic (H1N1) influenza virus, elicited NA-specific antibody and T cell responses, which conferred protection against homologous and heterologous influenza virus challenges. Vaccination with PIV5-N1 NA provided cross-protection against challenge with a heterosubtypic (H3N2) virus. Experiments using antibody transfer indicate that antibodies to NA have an important role in protection. These findings indicate that PIV5 expressing NA may be effective as a broadly protective vaccine against seasonal influenza and emerging pandemic threats. IMPORTANCE Seasonal influenza viruses cause considerable morbidity and mortality annually, while emerging viruses pose potential pandemic threats. Currently licensed influenza virus vaccines rely on the antigenic match of hemagglutinin (HA) for vaccine strain selection, and most vaccines rely on HA inhibition titers to determine efficacy, despite the growing

Canine distemper virus (CDV) in another big cat: should CDV be renamed carnivore distemper virus?

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Terio, Karen A; Craft, Meggan E

2013-09-17

One of the greatest threats to the conservation of wild cat populations may be dogs or, at least, one of their viruses. Canine distemper virus (CDV), a single-stranded RNA virus in the Paramyxoviridae family and genus Morbillivirus, infects and causes disease in a variety of species, not just canids. An outbreak of CDV in wild lions in the Serengeti, Tanzania, in 1994 was a wake-up call for conservationists, as it demonstrated that an infectious disease could swiftly impact a previously healthy felid population. To understand how this virus causes disease in noncanid hosts, researchers have focused on specific mutations in the binding site of the CDV hemagglutinin gene. Now, Seimon et al. provide information on CDV in its latest feline victim, the endangered wild Amur tiger (Panthera tigris altaica) [T. A. Seimon et al., mBio 4(4):e00410-13, 2013, doi:10.1128/mBio.00410-13]. Their findings of CDV strains infecting tigers, in combination with recent information from other felids, paints a different picture, one in which CDV strains from a variety of geographic lineages and with a variety of amino acid residues in the hemagglutinin gene binding site can infect cats and cause disease. Although CDV has been known as a multihost disease since its discovery in domestic dogs in 1905, perhaps it is time to reconsider whether these noncanid species are not just incidental or "spillover" hosts but, rather, a normal part of the complex ecology of this infectious disease.

Canine Distemper Virus (CDV) in Another Big Cat: Should CDV Be Renamed Carnivore Distemper Virus?

Science.gov (United States)

Terio, Karen A.; Craft, Meggan E.

2013-01-01

ABSTRACT One of the greatest threats to the conservation of wild cat populations may be dogs or, at least, one of their viruses. Canine distemper virus (CDV), a single-stranded RNA virus in the Paramyxoviridae family and genus Morbillivirus, infects and causes disease in a variety of species, not just canids. An outbreak of CDV in wild lions in the Serengeti, Tanzania, in 1994 was a wake-up call for conservationists, as it demonstrated that an infectious disease could swiftly impact a previously healthy felid population. To understand how this virus causes disease in noncanid hosts, researchers have focused on specific mutations in the binding site of the CDV hemagglutinin gene. Now, Seimon et al. provide information on CDV in its latest feline victim, the endangered wild Amur tiger (Panthera tigris altaica) [T. A. Seimon et al., mBio 4(4):e00410-13, 2013, doi:10.1128/mBio.00410-13]. Their findings of CDV strains infecting tigers, in combination with recent information from other felids, paints a different picture, one in which CDV strains from a variety of geographic lineages and with a variety of amino acid residues in the hemagglutinin gene binding site can infect cats and cause disease. Although CDV has been known as a multihost disease since its discovery in domestic dogs in 1905, perhaps it is time to reconsider whether these noncanid species are not just incidental or “spillover” hosts but, rather, a normal part of the complex ecology of this infectious disease. PMID:24045642

Broad cross-reactive IgG responses elicited by adjuvanted vaccination with recombinant influenza hemagglutinin (rHA) in ferrets and mice

Science.gov (United States)

Wang, Jiong; Hilchey, Shannon P.; DeDiego, Marta; Perry, Sheldon; Hyrien, Ollivier; Nogales, Aitor; Garigen, Jessica; Amanat, Fatima; Huertas, Nelson; Krammer, Florian; Martinez-Sobrido, Luis; Topham, David J.; Treanor, John J.; Sangster, Mark Y.

2018-01-01

Annual immunization against influenza virus is a large international public health effort. Accumulating evidence suggests that antibody mediated cross-reactive immunity against influenza hemagglutinin (HA) strongly correlates with long-lasting cross-protection against influenza virus strains that differ from the primary infection or vaccination strain. However, the optimal strategies for achieving highly cross-reactive antibodies to the influenza virus HA have not yet to be defined. In the current study, using Luminex-based mPlex-Flu assay, developed by our laboratory, to quantitatively measure influenza specific IgG antibody mediated cross-reactivity, we found that prime-boost-boost vaccination of ferrets with rHA proteins admixed with adjuvant elicited higher magnitude and broader cross-reactive antibody responses than that induced by actual influenza viral infection, and this cross-reactive response likely correlated with increased anti-stalk reactive antibodies. We observed a similar phenomenon in mice receiving three sequential vaccinations with rHA proteins from either A/California/07/2009 (H1N1) or A/Hong Kong/1/1968 (H3N2) viruses admixed with Addavax, an MF59-like adjuvant. Using this same mouse vaccination model, we determined that Addavax plays a more significant role in the initial priming event than in subsequent boosts. We also characterized the generation of cross-reactive antibody secreting cells (ASCs) and memory B cells (MBCs) when comparing vaccination to viral infection. We have also found that adjuvant plays a critical role in the generation of long-lived ASCs and MBCs cross-reactive to influenza viruses as a result of vaccination with rHA of influenza virus, and the observed increase in stalk-reactive antibodies likely contributes to this IgG mediated broad cross-reactivity. PMID:29641537

Evolutionary mechanisms involved in the virulence of infectious salmon anaemia virus (ISAV), a piscine orthomyxovirus

International Nuclear Information System (INIS)

Markussen, Turhan; Jonassen, Christine Monceyron; Numanovic, Sanela; Braaen, Stine; Hjortaas, Monika; Nilsen, Hanne; Mjaaland, Siri

2008-01-01

Infectious salmon anaemia virus (ISAV) is an orthomyxovirus causing a multisystemic, emerging disease in Atlantic salmon. Here we present, for the first time, detailed sequence analyses of the full-genome sequence of a presumed avirulent isolate displaying a full-length hemagglutinin-esterase (HE) gene (HPR0), and compare this with full-genome sequences of 11 Norwegian ISAV isolates from clinically diseased fish. These analyses revealed the presence of a virulence marker right upstream of the putative cleavage site R 267 in the fusion (F) protein, suggesting a Q 266 → L 266 substitution to be a prerequisite for virulence. To gain virulence in isolates lacking this substitution, a sequence insertion near the cleavage site seems to be required. This strongly suggests the involvement of a protease recognition pattern at the cleavage site of the fusion protein as a determinant of virulence, as seen in highly pathogenic influenza A virus H5 or H7 and the paramyxovirus Newcastle disease virus

The public health impact of avian influenza viruses.

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Katz, J M; Veguilla, V; Belser, J A; Maines, T R; Van Hoeven, N; Pappas, C; Hancock, K; Tumpey, T M

2009-04-01

Influenza viruses with novel hemagglutinin and 1 or more accompanying genes derived from avian influenza viruses sporadically emerge in humans and have the potential to result in a pandemic if the virus causes disease and spreads efficiently in a population that lacks immunity to the novel hemagglutinin. Since 1997, multiple avian influenza virus subtypes have been transmitted directly from domestic poultry to humans and have caused a spectrum of human disease, from asymptomatic to severe and fatal. To assess the pandemic risk that avian influenza viruses pose, we have used multiple strategies to better understand the capacity of avian viruses to infect, cause disease, and transmit among mammals, including humans. Seroepidemiologic studies that evaluate the frequency and risk of human infection with avian influenza viruses in populations with exposure to domestic or wild birds can provide a better understanding of the pandemic potential of avian influenza subtypes. Investigations conducted in Hong Kong following the first H5N1 outbreak in humans in 1997 determined that exposure to poultry in live bird markets was a key risk factor for human disease. Among poultry workers, butchering and exposure to sick poultry were risk factors for antibody to H5 virus, which provided evidence for infection. A second risk assessment tool, the ferret, can be used to evaluate the level of virulence and potential for host-to-host transmission of avian influenza viruses in this naturally susceptible host. Avian viruses isolated from humans exhibit a level of virulence and transmissibility in ferrets that generally reflects that seen in humans. The ferret model thus provides a means to monitor emerging avian influenza viruses for pandemic risk, as well as to evaluate laboratory-generated reassortants and mutants to better understand the molecular basis of influenza virus transmissibility. Taken together, such studies provide valuable information with which we can assess the public

Pre-clinical evaluation of a replication-competent recombinant adenovirus serotype 4 vaccine expressing influenza H5 hemagglutinin.

Science.gov (United States)

Alexander, Jeff; Ward, Simone; Mendy, Jason; Manayani, Darly J; Farness, Peggy; Avanzini, Jenny B; Guenther, Ben; Garduno, Fermin; Jow, Lily; Snarsky, Victoria; Ishioka, Glenn; Dong, Xin; Vang, Lo; Newman, Mark J; Mayall, Tim

2012-01-01

Influenza virus remains a significant health and social concern in part because of newly emerging strains, such as avian H5N1 virus. We have developed a prototype H5N1 vaccine using a recombinant, replication-competent Adenovirus serotype 4 (Ad4) vector, derived from the U.S. military Ad4 vaccine strain, to express the hemagglutinin (HA) gene from A/Vietnam/1194/2004 influenza virus (Ad4-H5-Vtn). Our hypothesis is that a mucosally-delivered replicating Ad4-H5-Vtn recombinant vector will be safe and induce protective immunity against H5N1 influenza virus infection and disease pathogenesis. The Ad4-H5-Vtn vaccine was designed with a partial deletion of the E3 region of Ad4 to accommodate the influenza HA gene. Replication and growth kinetics of the vaccine virus in multiple human cell lines indicated that the vaccine virus is attenuated relative to the wild type virus. Expression of the HA transgene in infected cells was documented by flow cytometry, western blot analysis and induction of HA-specific antibody and cellular immune responses in mice. Of particular note, mice immunized intranasally with the Ad4-H5-Vtn vaccine were protected against lethal H5N1 reassortant viral challenge even in the presence of pre-existing immunity to the Ad4 wild type virus. Several non-clinical attributes of this vaccine including safety, induction of HA-specific humoral and cellular immunity, and efficacy were demonstrated using an animal model to support Phase 1 clinical trial evaluation of this new vaccine.

A human-infecting H10N8 influenza virus retains a strong preference for avian-type receptors

NARCIS (Netherlands)

Zhang, Heng; de Vries, Robert P; Tzarum, Netanel; Zhu, Xueyong; Yu, Wenli; McBride, Ryan; Paulson, James C; Wilson, Ian A

2015-01-01

Recent avian-origin H10N8 influenza A viruses that have infected humans pose a potential pandemic threat. Alterations in the viral surface glycoprotein, hemagglutinin (HA), typically are required for influenza A viruses to cross the species barrier for adaptation to a new host, but whether H10N8

pH Optimum of Hemagglutinin-Mediated Membrane Fusion Determines Sensitivity of Influenza A Viruses to the Interferon-Induced Antiviral State and IFITMs.

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Gerlach, Thomas; Hensen, Luca; Matrosovich, Tatyana; Bergmann, Janina; Winkler, Michael; Peteranderl, Christin; Klenk, Hans-Dieter; Weber, Friedemann; Herold, Susanne; Pöhlmann, Stefan; Matrosovich, Mikhail

2017-06-01

The replication and pathogenicity of influenza A viruses (IAVs) critically depend on their ability to tolerate the antiviral interferon (IFN) response. To determine a potential role for the IAV hemagglutinin (HA) in viral sensitivity to IFN, we studied the restriction of IAV infection in IFN-β-treated human epithelial cells by using 2:6 recombinant IAVs that shared six gene segments of A/Puerto Rico/8/1934 virus (PR8) and contained HAs and neuraminidases of representative avian, human, and zoonotic H5N1 and H7N9 viruses. In A549 and Calu-3 cells, viruses displaying a higher pH optimum of HA-mediated membrane fusion, H5N1-PR8 and H7N9-PR8, were less sensitive to the IFN-induced antiviral state than their counterparts with HAs from duck and human viruses, which fused at a lower pH. The association between a high pH optimum of fusion and reduced IFN sensitivity was confirmed by using HA point mutants of A/Hong Kong/1/1968-PR8 that differed solely by their fusion properties. Furthermore, similar effects of the viral fusion pH on IFN sensitivity were observed in experiments with (i) primary human type II alveolar epithelial cells and differentiated cultures of human airway epithelial cells, (ii) nonrecombinant zoonotic and pandemic IAVs, and (iii) preparations of IFN-α and IFN-λ1. A higher pH of membrane fusion and reduced sensitivity to IFN correlated with lower restriction of the viruses in MDCK cells stably expressing the IFN-inducible transmembrane proteins IFITM2 and IFITM3, which are known to inhibit viral fusion. Our results reveal that the pH optimum of HA-driven membrane fusion of IAVs is a determinant of their sensitivity to IFN and IFITM proteins. IMPORTANCE The IFN system constitutes an important innate defense against viral infection. Substantial information is available on how IAVs avoid detection by sensors of the IFN system and disable IFN signaling pathways. Much less is known about the ability of IAVs to tolerate the antiviral activity of IFN

Recombinant rabies virus expressing the H protein of canine distemper virus protects dogs from the lethal distemper challenge.

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Wang, Feng-Xue; Zhang, Shu-Qin; Zhu, Hong-Wei; Yang, Yong; Sun, Na; Tan, Bin; Li, Zhen-Guang; Cheng, Shi-Peng; Fu, Zhen F; Wen, Yong-Jun

2014-12-05

The rabies virus (RV) vector LBNSE expressing foreign antigens have shown considerable promise as vaccines against viral and bacteria diseases, which is effective and safe. We produced a new RV-based vaccine vehicle expressing 1.824 kb hemagglutinin (H) gene of the canine distemper virus (CDV) by reverse genetics technology. The recombinant virus LBNSE-CDV-H retained growth properties similar to those of vector LBNSE both in BSR and mNA cell culture. The H gene of CDV was expressed and detected by immunostaining. To compare the immunogenicity of LBNSE-CDV-H, dogs were immunized with each of these recombinant viruses by intramuscular (i.m.). The dogs were bled at third weeks after the immunization for the measurement of virus neutralizing antibody (VNA) and then challenged with virulent virus (ZJ 7) at fourth weeks. The parent virus (LBNSE) without expression of any foreign molecules was included for comparison. Dogs inoculated with LBNSE-CDV-H showed no any signs of disease and exhibited seroconversion against both RV and CDV H protein. The LBNSE-CDV-H did not cause disease in dogs and conferred protection from challenge with a lethal wild type CDV strain, demonstrating its potential value for wildlife conservation efforts. Together, these studies suggest that recombinant RV expressing H protein from CDV stimulated high levels of adaptive immune responses (VNA), and protected all dogs challenge infection. Copyright © 2014 Elsevier B.V. All rights reserved.

Production of polyclonal antibody against Tehran strain influenza virus (A/H1N1/2009 hemagglutinin conserved domain (HA2: brief report

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Somayeh Zamani

2015-10-01

Full Text Available Background: The influenza virus is one of the most important factors for higher morbidity and mortality in the world. Recently, researchers have been focused on influenza conserved antigenic proteins such as hemagglutinin stalk domain (HA2 for vaccine production and serological studies. The HA2 plays a major role in the fusion of the virus with host cells membrane. The immunity system enables to produce antibody against HA2. The aim of this study is polyclonal antibody production against influenza HA2. Methods: This study was done in the Influenza Research Lab, Pasteur Institute of Iran, Tehran for one year from September 2013 to October 2014. In the present study, recombinant HA2 protein was produced in prokaryotic system and purified using Nickel affinity chromatography. The purified HA2 was mixed with Freund’s adjuvant (complete and incomplete and injected into two New Zealand white rabbits by intramuscularly and subcutaneously routes. Immunization was continued for several months with two weeks interval. Before each immunization, blood was drawn by venous puncture from the rabbit ear. Function of rabbit's sera was evaluated using radial immunodiffusion (RID in both forms, Single RID (SRID and Double RID (DRID. Finally, antiserum activity against HA2 was evaluated using western blotting as serological assay. Results: Sedimentary line and zone was observed in RID assays (SRID and DRID represent interaction between HA2 protein and anti- HA2 antibody. As well as, western blotting results was positive for HA2 protein. Therefore, these results showed that polyclonal antibody produced against HA2 protein can identify HA2 protein antigenic sites. Conclusion: These findings show that humoral immune responses have properly been stimulated in rabbits and these antibodies can identify HA2 protein and may be suitable for other serological methods.

Genetic characterization of canine distemper virus involved in outbreaks in farmed mink in Denmark 2012

DEFF Research Database (Denmark)

Trebbien, Ramona; Struve, T.; Hjulsager, Charlotte Kristiane

Danish farmed mink herds experienced a large outbreak of canine distemper virus in 2012. Full-length sequence analysis (1824 nucleotides) of the variable hemagglutinin (H) gene were performed on 27 viruses collected from mink and on 7 viruses collected from wild foxes. Results of the study showed...... with other European canine distemper viruses and showed the highest level of similarity (99.3 - 99.6 %) to viruses isolated from wild foxes in Germany. The fox should therefore be considered as an important wild life reservoir of canine distemper virus and may also contribute to the transmission of the virus...

Infection and transmission of live recombinant Newcastle disease virus vaccines in Rock Pigeons, European House Sparrows, and Japanese Quail

Science.gov (United States)

In China and Mexico, engineered recombinant Newcastle disease virus (rNDV) strains are used as live vaccines for the control of Newcastle disease and as vectors to express the avian influenza virus hemagglutinin (HA) gene to control avian influenza in poultry. In this study, non-target species wer...

Biological characteristics of genetic variants of Urabe AM9 mumps vaccine virus.

Science.gov (United States)

Wright, K E; Dimock, K; Brown, E G

2000-03-01

The Urabe AM9 mumps vaccine is composed of a mixture of variants distinguishable by a difference at nucleotide (nt) 1081 of the hemagglutinin-neuraminidase (HN) gene (Brown, E.G., Dimock, K., Wright, K.E., 1996. The Urabe AM9 mumps vaccine is a mixture of viruses differing at amino acid (aa) 335 of the hemagglutinin-neuraminidase gene with one form associated with disease. J. Infect. Dis. 174, 619-622.). Further genetic and biological variation was detected in plaque purified viruses from the Urabe AM9 vaccine by examining the HN gene sequence, plaque morphology, cytopathic effects and growth in Vero cells, and temperature sensitivity (ts). Infection of Vero cells with plaque purified viruses with a G at nt 1081 of the HN gene produced large, clear plaques, caused significant CPE early after infection but yielded lower titres of virus than other purified viruses. None of these viruses were ts. In contrast, half of the plaque purified viruses with an A at nt 1081 were sensitive to a temperature of 39.5 degrees C. These viruses produced small plaques, caused significant CPE and grew to low titres. Two ts viruses possessed a unique aa substitution at aa 468 of HN. The remaining A(1081) viruses were not ts, produced large plaques but little CPE, and grew to titres 10-fold higher than the G(1081) viruses. Isolates of Urabe AM9 associated with post-vaccination illness were similar to these non-ts A(1081) viruses, but could be further sub-divided into two groups on the basis of a difference at aa 464 of HN. The post-vaccination isolates may represent insufficiently attenuated components of the vaccine, while the G(1081) and ts subset of A(1081) viruses may be more fully attenuated.

Protective efficacy of Newcastle disease virus expressing soluble trimeric hemagglutinin against highly pathogenic H5N1 influenza in chickens and mice.

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Lisette A H M Cornelissen

Full Text Available BACKGROUND: Highly pathogenic avian influenza virus (HPAIV causes a highly contagious often fatal disease in poultry, resulting in significant economic losses in the poultry industry. HPAIV H5N1 also poses a major public health threat as it can be transmitted directly from infected poultry to humans. One effective way to combat avian influenza with pandemic potential is through the vaccination of poultry. Several live vaccines based on attenuated Newcastle disease virus (NDV that express influenza hemagglutinin (HA have been developed to protect chickens or mammalian species against HPAIV. However, the zoonotic potential of NDV raises safety concerns regarding the use of live NDV recombinants, as the incorporation of a heterologous attachment protein may result in the generation of NDV with altered tropism and/or pathogenicity. METHODOLOGY/PRINCIPAL FINDINGS: In the present study we generated recombinant NDVs expressing either full length, membrane-anchored HA of the H5 subtype (NDV-H5 or a soluble trimeric form thereof (NDV-sH5(3. A single intramuscular immunization with NDV-sH5(3 or NDV-H5 fully protected chickens against disease after a lethal challenge with H5N1 and reduced levels of virus shedding in tracheal and cloacal swabs. NDV-sH5(3 was less protective than NDV-H5 (50% vs 80% protection when administered via the respiratory tract. The NDV-sH5(3 was ineffective in mice, regardless of whether administered oculonasally or intramuscularly. In this species, NDV-H5 induced protective immunity against HPAIV H5N1, but only after oculonasal administration, despite the poor H5-specific serum antibody response it elicited. CONCLUSIONS/SIGNIFICANCE: Although NDV expressing membrane anchored H5 in general provided better protection than its counterpart expressing soluble H5, chickens could be fully protected against a lethal challenge with H5N1 by using the latter NDV vector. This study thus provides proof of concept for the use of recombinant

Pre-clinical evaluation of a replication-competent recombinant adenovirus serotype 4 vaccine expressing influenza H5 hemagglutinin.

Directory of Open Access Journals (Sweden)

Jeff Alexander

Full Text Available Influenza virus remains a significant health and social concern in part because of newly emerging strains, such as avian H5N1 virus. We have developed a prototype H5N1 vaccine using a recombinant, replication-competent Adenovirus serotype 4 (Ad4 vector, derived from the U.S. military Ad4 vaccine strain, to express the hemagglutinin (HA gene from A/Vietnam/1194/2004 influenza virus (Ad4-H5-Vtn. Our hypothesis is that a mucosally-delivered replicating Ad4-H5-Vtn recombinant vector will be safe and induce protective immunity against H5N1 influenza virus infection and disease pathogenesis.The Ad4-H5-Vtn vaccine was designed with a partial deletion of the E3 region of Ad4 to accommodate the influenza HA gene. Replication and growth kinetics of the vaccine virus in multiple human cell lines indicated that the vaccine virus is attenuated relative to the wild type virus. Expression of the HA transgene in infected cells was documented by flow cytometry, western blot analysis and induction of HA-specific antibody and cellular immune responses in mice. Of particular note, mice immunized intranasally with the Ad4-H5-Vtn vaccine were protected against lethal H5N1 reassortant viral challenge even in the presence of pre-existing immunity to the Ad4 wild type virus.Several non-clinical attributes of this vaccine including safety, induction of HA-specific humoral and cellular immunity, and efficacy were demonstrated using an animal model to support Phase 1 clinical trial evaluation of this new vaccine.

Human Infection with Highly Pathogenic Avian Influenza A(H7N9) Virus, China.

Science.gov (United States)

Ke, Changwen; Mok, Chris Ka Pun; Zhu, Wenfei; Zhou, Haibo; He, Jianfeng; Guan, Wenda; Wu, Jie; Song, Wenjun; Wang, Dayan; Liu, Jiexiong; Lin, Qinhan; Chu, Daniel Ka Wing; Yang, Lei; Zhong, Nanshan; Yang, Zifeng; Shu, Yuelong; Peiris, Joseph Sriyal Malik

2017-07-01

The recent increase in zoonotic avian influenza A(H7N9) disease in China is a cause of public health concern. Most of the A(H7N9) viruses previously reported have been of low pathogenicity. We report the fatal case of a patient in China who was infected with an A(H7N9) virus having a polybasic amino acid sequence at its hemagglutinin cleavage site (PEVPKRKRTAR/GL), a sequence suggestive of high pathogenicity in birds. Its neuraminidase also had R292K, an amino acid change known to be associated with neuraminidase inhibitor resistance. Both of these molecular features might have contributed to the patient's adverse clinical outcome. The patient had a history of exposure to sick and dying poultry, and his close contacts had no evidence of A(H7N9) disease, suggesting human-to-human transmission did not occur. Enhanced surveillance is needed to determine whether this highly pathogenic avian influenza A(H7N9) virus will continue to spread.

Evaluation of the Universal Viral Transport system for long-term storage of virus specimens for microbial forensics.

Science.gov (United States)

Hosokawa-Muto, Junji; Fujinami, Yoshihito; Mizuno, Natsuko

2015-08-01

Forensic microbial specimens, including bacteria and viruses, are collected at biocrime and bioterrorism scenes. Although it is preferable that the pathogens in these samples are alive and kept in a steady state, the samples may be stored for prolonged periods before analysis. Therefore, it is important to understand the effects of storage conditions on the pathogens contained within such samples. To evaluate the capacity to preserve viable virus and the viral genome, influenza virus was added to the transport medium of the Universal Viral Transport system and stored for over 3 months at various temperatures, after which virus titrations and quantitative analysis of the influenza hemagglutinin gene were performed. Although viable viruses became undetectable 29 days after the medium was stored at room temperature, viruses in the medium stored at 4°C were viable even after 99 days. A quantitative PCR analysis indicated that the hemagglutinin gene was maintained for 99 days at both 4°C and room temperature. Therefore, long-term storage at 4°C has little effect on viable virus and viral genes, so the Universal Viral Transport system can be useful for microbial forensics. This study provides important information for the handling of forensic virus specimens. Copyright © 2015 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.

Screening of random peptide library of hemagglutinin from pandemic 2009 A(H1N1 influenza virus reveals unexpected antigenically important regions.

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Wanghui Xu

Full Text Available The antigenic structure of the membrane protein hemagglutinin (HA from the 2009 A(H1N1 influenza virus was dissected with a high-throughput screening method using complex antisera. The approach involves generating yeast cell libraries displaying a pool of random peptides of controllable lengths on the cell surface, followed by one round of fluorescence-activated cell sorting (FACS against antisera from mouse, goat and human, respectively. The amino acid residue frequency appearing in the antigenic peptides at both the primary sequence and structural level was determined and used to identify "hot spots" or antigenically important regions. Unexpectedly, different antigenic structures were seen for different antisera. Moreover, five antigenic regions were identified, of which all but one are located in the conserved HA stem region that is responsible for membrane fusion. Our findings are corroborated by several recent studies on cross-neutralizing H1 subtype antibodies that recognize the HA stem region. The antigenic peptides identified may provide clues for creating peptide vaccines with better accessibility to memory B cells and better induction of cross-neutralizing antibodies than the whole HA protein. The scheme used in this study enables a direct mapping of the antigenic regions of viral proteins recognized by antisera, and may be useful for dissecting the antigenic structures of other viral proteins.

Characterization of a new dog isolate of canine distemper virus from China.

Science.gov (United States)

Qiao, J; Meng, Q; Chen, C; Xia, X; Cai, X; Ren, Y; Zhang, H

2011-01-01

Canine distemper virus (CDV) is a highly contagious pathogen of dogs. Vaccination is an effective way to protect dogs from CDV infection, but occasionally fails. In the present study, a wild type (wt) CDV, named XJ2, was isolated from a dead vaccinated dog. The hemagglutinin (H) gene of the XJ2 was amplified and analyzed for the molecular characteristics including N-glycosylation sites, phylogenesis, hydrophobicity and epitopes. The data indicated that XJ2 was a genetic variant strain of CDV. CDV-sero-negative dogs were inoculated intranasally with XJ2, developed severe clinical symptoms and died, suggesting high virulence.

Surface localization of the nuclear receptor CAR in influenza A virus-infected cells

International Nuclear Information System (INIS)

Takahashi, Tadanobu; Moriyama, Yusuke; Ikari, Akira; Sugatani, Junko; Suzuki, Takashi; Miwa, Masao

2008-01-01

Constitutive active/androstane receptor CAR is a member of the nuclear receptors which regulate transcription of xenobiotic metabolism enzymes. CAR is usually localized in the cytosol and nucleus. Here, we found that CAR was localized at the cell surface of influenza A virus (IAV)-infected cells. Additionally, we demonstrated that expression of a viral envelope glycoprotein, either hemagglutinin (HA) or neuraminidase (NA), but not viral nucleoprotein (NP), was responsible for this localization. This report is the first demonstration of CAR at the surface of tissue culture cells, and suggests that CAR may exert the IAV infection mechanism

Human monoclonal antibodies derived from a patient infected with 2009 pandemic influenza A virus broadly cross-neutralize group 1 influenza viruses

International Nuclear Information System (INIS)

Pan, Yang; Sasaki, Tadahiro; Kubota-Koketsu, Ritsuko; Inoue, Yuji; Yasugi, Mayo; Yamashita, Akifumi; Ramadhany, Ririn; Arai, Yasuha; Du, Anariwa; Boonsathorn, Naphatsawan; Ibrahim, Madiha S.

2014-01-01

Highlights: • Influenza infection can elicit heterosubtypic antibodies to group 1 influenza virus. • Three human monoclonal antibodies were generated from an H1N1-infected patient. • The antibodies predominantly recognized α-helical stem of viral hemagglutinin (HA). • The antibodies inhibited HA structural activation during the fusion process. • The antibodies are potential candidates for future antibody therapy to influenza. - Abstract: Influenza viruses are a continuous threat to human public health because of their ability to evolve rapidly through genetic drift and reassortment. Three human monoclonal antibodies (HuMAbs) were generated in this study, 1H11, 2H5 and 5G2, and they cross-neutralize a diverse range of group 1 influenza A viruses, including seasonal H1N1, 2009 pandemic H1N1 (H1N1pdm) and avian H5N1 and H9N2. The three HuMAbs were prepared by fusing peripheral blood lymphocytes from an H1N1pdm-infected patient with a newly developed fusion partner cell line, SPYMEG. All the HuMAbs had little hemagglutination inhibition activity but had strong membrane-fusion inhibition activity against influenza viruses. A protease digestion assay showed the HuMAbs targeted commonly a short α-helix region in the stalk of the hemagglutinin. Furthermore, Ile45Phe and Glu47Gly double substitutions in the α-helix region made the HA unrecognizable by the HuMAbs. These two amino acid residues are highly conserved in the HAs of H1N1, H5N1 and H9N2 viruses. The HuMAbs reported here may be potential candidates for the development of therapeutic antibodies against group 1 influenza viruses

Human monoclonal antibodies derived from a patient infected with 2009 pandemic influenza A virus broadly cross-neutralize group 1 influenza viruses

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Pan, Yang [Research Institute for Microbial Diseases, Osaka University, Suita, Osaka (Japan); Sasaki, Tadahiro [Research Institute for Microbial Diseases, Osaka University, Suita, Osaka (Japan); JST/JICA, Science and Technology Research Partnership for Sustainable Development (SATREPS), Tokyo (Japan); Kubota-Koketsu, Ritsuko [Research Institute for Microbial Diseases, Osaka University, Suita, Osaka (Japan); Kanonji Institute, The Research Foundation for Microbial Diseases of Osaka University, Kanonji, Kagawa (Japan); JST/JICA, Science and Technology Research Partnership for Sustainable Development (SATREPS), Tokyo (Japan); Inoue, Yuji [Research Institute for Microbial Diseases, Osaka University, Suita, Osaka (Japan); JST/JICA, Science and Technology Research Partnership for Sustainable Development (SATREPS), Tokyo (Japan); Yasugi, Mayo [Research Institute for Microbial Diseases, Osaka University, Suita, Osaka (Japan); Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Izumisano, Osaka (Japan); JST/JICA, Science and Technology Research Partnership for Sustainable Development (SATREPS), Tokyo (Japan); Yamashita, Akifumi; Ramadhany, Ririn; Arai, Yasuha [Research Institute for Microbial Diseases, Osaka University, Suita, Osaka (Japan); Du, Anariwa [Research Institute for Microbial Diseases, Osaka University, Suita, Osaka (Japan); JST/JICA, Science and Technology Research Partnership for Sustainable Development (SATREPS), Tokyo (Japan); Boonsathorn, Naphatsawan [Research Institute for Microbial Diseases, Osaka University, Suita, Osaka (Japan); Department of Medical Sciences, Ministry of Public Health, Muang, Nonthaburi (Thailand); JST/JICA, Science and Technology Research Partnership for Sustainable Development (SATREPS), Tokyo (Japan); Ibrahim, Madiha S. [Research Institute for Microbial Diseases, Osaka University, Suita, Osaka (Japan); Department of Microbiology and Immunology, Faculty of Veterinary Medicine, Damanhour University, Damanhour (Egypt); and others

2014-07-18

Highlights: • Influenza infection can elicit heterosubtypic antibodies to group 1 influenza virus. • Three human monoclonal antibodies were generated from an H1N1-infected patient. • The antibodies predominantly recognized α-helical stem of viral hemagglutinin (HA). • The antibodies inhibited HA structural activation during the fusion process. • The antibodies are potential candidates for future antibody therapy to influenza. - Abstract: Influenza viruses are a continuous threat to human public health because of their ability to evolve rapidly through genetic drift and reassortment. Three human monoclonal antibodies (HuMAbs) were generated in this study, 1H11, 2H5 and 5G2, and they cross-neutralize a diverse range of group 1 influenza A viruses, including seasonal H1N1, 2009 pandemic H1N1 (H1N1pdm) and avian H5N1 and H9N2. The three HuMAbs were prepared by fusing peripheral blood lymphocytes from an H1N1pdm-infected patient with a newly developed fusion partner cell line, SPYMEG. All the HuMAbs had little hemagglutination inhibition activity but had strong membrane-fusion inhibition activity against influenza viruses. A protease digestion assay showed the HuMAbs targeted commonly a short α-helix region in the stalk of the hemagglutinin. Furthermore, Ile45Phe and Glu47Gly double substitutions in the α-helix region made the HA unrecognizable by the HuMAbs. These two amino acid residues are highly conserved in the HAs of H1N1, H5N1 and H9N2 viruses. The HuMAbs reported here may be potential candidates for the development of therapeutic antibodies against group 1 influenza viruses.

Effect of priming with H1N1 influenza viruses of variable antigenic distances on challenge with 2009 pandemic H1N1 virus.

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O'Donnell, Christopher D; Wright, Amber; Vogel, Leatrice N; Wei, Chih-Jen; Nabel, Gary J; Subbarao, Kanta

2012-08-01

Compared to seasonal influenza viruses, the 2009 pandemic H1N1 (pH1N1) virus caused greater morbidity and mortality in children and young adults. People over 60 years of age showed a higher prevalence of cross-reactive pH1N1 antibodies, suggesting that they were previously exposed to an influenza virus or vaccine that was antigenically related to the pH1N1 virus. To define the basis for this cross-reactivity, ferrets were infected with H1N1 viruses of variable antigenic distance that circulated during different decades from the 1930s (Alaska/35), 1940s (Fort Monmouth/47), 1950s (Fort Warren/50), and 1990s (New Caledonia/99) and challenged with 2009 pH1N1 virus 6 weeks later. Ferrets primed with the homologous CA/09 or New Jersey/76 (NJ/76) virus served as a positive control, while the negative control was an influenza B virus that should not cross-protect against influenza A virus infection. Significant protection against challenge virus replication in the respiratory tract was observed in ferrets primed with AK/35, FM/47, and NJ/76; FW/50-primed ferrets showed reduced protection, and NC/99-primed ferrets were not protected. The hemagglutinins (HAs) of AK/35, FM/47, and FW/50 differ in the presence of glycosylation sites. We found that the loss of protective efficacy observed with FW/50 was associated with the presence of a specific glycosylation site. Our results suggest that changes in the HA occurred between 1947 and 1950, such that prior infection could no longer protect against 2009 pH1N1 infection. This provides a mechanistic understanding of the nature of serological cross-protection observed in people over 60 years of age during the 2009 H1N1 pandemic.

Development of a combined canine distemper virus specific RT-PCR protocol for the differentiation of infected and vaccinated animals (DIVA) and genetic characterization of the hemagglutinin gene of seven Chinese strains demonstrated in dogs.

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Yi, Li; Cheng, Shipeng; Xu, Hongli; Wang, Jianke; Cheng, Yuening; Yang, Shen; Luo, Bin

2012-01-01

A combined reverse-transcription polymerase chain reaction (RT-PCR) method was developed for the detection and differentiation of wild-type and vaccine strains of the canine distemper virus (CDV). A pair of primers (P1/P2) was used to detect both CDV wild-type strains and vaccines. Another pair (P3/P4) was used to detect only CDV wild-type strains. A 335bp fragment was amplified from the genomic RNA of the vaccine and wild-type strains. A 555bp fragment was amplified specifically from the genomic RNA of the wild-type strains. No amplification was achieved for the uninfected cells, cells infected with canine parvovirus, canine coronavirus, or canine adenovirus. The combined RT-PCR method detected effectively and differentiated the CDV wild-type and vaccine strains by two separate RT-PCRs. The method can be used for clinical detection and epidemiological surveillance. The phylogenetic analysis of the hemagglutinin gene of the local wild-type CDV strains revealed that the seven local isolates all belonged to the Asia-1 lineage, and were clustered closely with one another at the same location. These results suggested that the CDV genotype Asia-1 is circulating currently in domestic dogs in China. Copyright © 2011 Elsevier B.V. All rights reserved.

Whole-Genome Characterization of a Novel Human Influenza A(H1N2) Virus Variant, Brazil.

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Resende, Paola Cristina; Born, Priscila Silva; Matos, Aline Rocha; Motta, Fernando Couto; Caetano, Braulia Costa; Debur, Maria do Carmo; Riediger, Irina Nastassja; Brown, David; Siqueira, Marilda M

2017-01-01

We report the characterization of a novel reassortant influenza A(H1N2) virus not previously reported in humans. Recovered from a a pig farm worker in southeast Brazil who had influenza-like illness, this virus is a triple reassortant containing gene segments from subtypes H1N2 (hemagglutinin), H3N2 (neuraminidase), and pandemic H1N1 (remaining genes).

Fowl adenovirus serotype 9 vectored vaccine for protection of avian influenza virus

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A fowl adenovirus serotype 9, a non-pathogenic large double stranded DNA virus, was developed as a viral vector to express influenza genes as a potential vaccine. Two separate constructs were developed that expressed either the hemagglutinin gene of A/Chicken/Jalisco/2012 (H7) or A/ Chicken/Iowa/20...

Molecular phylogeography of canine distemper virus: Geographic origin and global spreading.

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Panzera, Yanina; Sarute, Nicolás; Iraola, Gregorio; Hernández, Martín; Pérez, Ruben

2015-11-01

Canine distemper virus (CDV) (Paramyxoviridae-Morbillivirus) is a worldwide spread virus causing a fatal systemic disease in a broad range of carnivore hosts. In this study we performed Bayesian inferences using 208 full-length hemagglutinin gene nucleotide sequences isolated in 16 countries during 37 years (1975-2011). The estimated time to the most recent common ancestor suggested that current CDV strains emerged in the United States in the 1880s. This ancestor diversified through time into two ancestral clades, the current America 1 lineage that recently spread to Asia, and other ancestral clade that diversified and spread worldwide to originate the remaining eight lineages characterized to date. The spreading of CDV was characterized by several migratory events with posterior local differentiation, and expansion of the virus host range. A significant genetic flow between domestic and wildlife hosts is displayed; being domestic hosts the main viral reservoirs worldwide. This study is an extensive and integrative description of spatio/temporal population dynamics of CDV lineages that provides a novel evolutionary paradigm about the origin and dissemination of the current strains of the virus. Copyright © 2015 Elsevier Inc. All rights reserved.

Avian influenza virus ecology in Iceland shorebirds: intercontinental reassortment and movement

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Hall, Jeffrey S.; Hallgrimsson, Gunnar Thor; Suwannanarn, Kamol; Sreevatsen, Srinand; Ip, Hon S.; TeSlaa, Joshua L.; Nashold, Sean W.; Dusek, Robert J.

2014-01-01

Shorebirds are a primary reservoir of avian influenza viruses (AIV). We conducted surveillance studies in Iceland shorebird populations for 3 years, documenting high serological evidence of AIV exposure in shorebirds, primarily in Ruddy Turnstones (Arenaria interpres; seroprevalence = 75%). However, little evidence of virus infection was found in these shorebird populations and only two turnstone AIVs (H2N7; H5N1) were able to be phylogenetically examined. These analyses showed that viruses from Iceland shorebirds were primarily derived from Eurasian lineage viruses, yet the H2 hemagglutinin gene segment was from a North American lineage previously detected in a gull from Iceland the previous year. The H5N1 virus was determined to be low pathogenic, however the PB2 gene was closely related to the PB2 from highly pathogenic H5N1 isolates from China. Multiple lines of evidence suggest that the turnstones were infected with at least one of these AIV while in Iceland and confirm Iceland as an important location where AIV from different continents interact and reassort, creating new virus genomes. Mounting data warrant continued surveillance for AIV in wild birds in the North Atlantic, including Canada, Greenland, and the northeast USA to determine the risks of new AI viruses and their intercontinental movement in this region.

Avian influenza virus ecology in Iceland shorebirds: intercontinental reassortment and movement.

Science.gov (United States)

Hall, Jeffrey S; Hallgrimsson, Gunnar Thor; Suwannanarn, Kamol; Sreevatsen, Srinand; Ip, Hon S; Magnusdottir, Ellen; TeSlaa, Joshua L; Nashold, Sean W; Dusek, Robert J

2014-12-01

Shorebirds are a primary reservoir of avian influenza viruses (AIV). We conducted surveillance studies in Iceland shorebird populations for 3 years, documenting high serological evidence of AIV exposure in shorebirds, primarily in Ruddy Turnstones (Arenaria interpres; seroprevalence=75%). However, little evidence of virus infection was found in these shorebird populations and only two turnstone AIVs (H2N7; H5N1) were able to be phylogenetically examined. These analyses showed that viruses from Iceland shorebirds were primarily derived from Eurasian lineage viruses, yet the H2 hemagglutinin gene segment was from a North American lineage previously detected in a gull from Iceland the previous year. The H5N1 virus was determined to be low pathogenic, however the PB2 gene was closely related to the PB2 from highly pathogenic H5N1 isolates from China. Multiple lines of evidence suggest that the turnstones were infected with at least one of these AIV while in Iceland and confirm Iceland as an important location where AIV from different continents interact and reassort, creating new virus genomes. Mounting data warrant continued surveillance for AIV in wild birds in the North Atlantic, including Canada, Greenland, and the northeast USA to determine the risks of new AI viruses and their intercontinental movement in this region. Published by Elsevier B.V.

Vaccine-induced anti-HA2 antibodies promote virus fusion and enhance influenza virus respiratory disease.

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Khurana, Surender; Loving, Crystal L; Manischewitz, Jody; King, Lisa R; Gauger, Phillip C; Henningson, Jamie; Vincent, Amy L; Golding, Hana

2013-08-28

Vaccine-induced disease enhancement has been described in connection with several viral vaccines in animal models and in humans. We investigated a swine model to evaluate mismatched influenza vaccine-associated enhanced respiratory disease (VAERD) after pH1N1 infection. Vaccinating pigs with whole inactivated H1N2 (human-like) virus vaccine (WIV-H1N2) resulted in enhanced pneumonia and disease after pH1N1 infection. WIV-H1N2 immune sera contained high titers of cross-reactive anti-pH1N1 hemagglutinin (HA) antibodies that bound exclusively to the HA2 domain but not to the HA1 globular head. No hemagglutination inhibition titers against pH1N1 (challenge virus) were measured. Epitope mapping using phage display library identified the immunodominant epitope recognized by WIV-H1N2 immune sera as amino acids 32 to 77 of pH1N1-HA2 domain, close to the fusion peptide. These cross-reactive anti-HA2 antibodies enhanced pH1N1 infection of Madin-Darby canine kidney cells by promoting virus membrane fusion activity. The enhanced fusion activity correlated with lung pathology in pigs. This study suggests a role for fusion-enhancing anti-HA2 antibodies in VAERD, in the absence of receptor-blocking virus-neutralizing antibodies. These findings should be considered during the evaluation of universal influenza vaccines designed to elicit HA2 stem-targeting antibodies.

Molecular characterization of influenza viruses collected from young children in Uberlandia, Brazil - from 2001 to 2010.

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de Mattos Silva Oliveira, Thelma Fátima; Yokosawa, Jonny; Motta, Fernando Couto; Siqueira, Marilda Mendonça; da Silveira, Hélio Lopes; Queiróz, Divina Aparecida Oliveira

2015-02-18

Influenza remains a major health problem due to the seasonal epidemics that occur every year caused by the emergence of new influenza virus strains. Hemagglutinin (HA) and neuraminidase (NA) glycoproteins are under selective pressure and subjected to frequent changes by antigenic drift. Therefore, our main objective was to investigate the influenza cases in Uberlândia city, Midwestern Brazil, in order to monitor the appearance of new viral strains, despite the availability of a prophylactic vaccine. Nasopharyngeal samples were collected from 605 children less than five years of age presenting with acute respiratory disease and tested by immunofluorescence assay (IFA) for detection of adenovirus, respiratory syncytial virus, parainfluenza virus types 1, 2, and 3 and influenza virus types A and B. A reverse transcription-PCR (RT-PCR) for influenza viruses A and B was carried out to amplify partial segments of the HA and NA genes. The nucleotide sequences were analyzed and compared with sequences of the virus strains of the vaccine available in the same year of sample collection. Forty samples (6.6%) were tested positive for influenza virus by IFA and RT-PCR, with 39 samples containing virus of type A and one of type B. By RT-PCR, the type A viruses were further characterized in subtypes H3N2, H1N2 and H1N1 (41.0%, 17.9%, and 2.6%, respectively). Deduced amino acid sequence analysis of the partial hemagglutinin sequence compared to sequences from vaccine strains, revealed that all strains found in Uberlândia had variations in the antigenic sites. The sequences of the receptor binding sites were preserved, although substitutions with similar amino acids were observed in few cases. The neuraminidase sequences did not show significant changes. All the H3 isolates detected in the 2001-2003 period had drifted from vaccine strain, unlike the isolates of the 2004-2007 period. These results suggest that the seasonal influenza vaccine effectiveness could be reduced because

Identification of Xylella fastidiosa antivirulence genes: hemagglutinin adhesins contribute a biofilm maturation to X. fastidios and colonization and attenuate virulence.

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Guilhabert, Magalie R; Kirkpatrick, Bruce C

2005-08-01

Xylella fastidosa, a gram-negative, xylem-limited bacterium, is the causal agent of several economically important plant diseases, including Pierce's disease (PD) and citrus variegated chlorosis (CVC). Until recently, the inability to transform or produce transposon mutants of X. fastidosa had been a major impediment to identifying X. fastidosa genes that mediate pathogen and plant interactions. A random transposon (Tn5) library of X. fastidosa was constructed and screened for mutants showing more severe symptoms and earlier grapevine death (hypervirulence) than did vines infected with the wild type. Seven hypervirulent mutants identified in this screen moved faster and reached higher populations than the wild type in grapevines. These results suggest that X. fastidosa attenuates its virulence in planta and that movement is important in X. fastidosa virulence. The mutated genes were sequenced and none had been described previously as antivirulence genes, although six of them showed similarity with genes of known functions in other organisms. One transposon insertion inactivated a hemagglutinin adhesin gene (PD2118), which we named HxfA. Another mutant in a second putative X. fastidosa hemagglutinin gene, PD1792 (HxfB), was constructed, and further characterization of these hxf mutants suggests that X. fastidosa hemagglutinins mediate contact between X. fastidosa cells, which results in colony formation and biofilm maturation within the xylem vessels.

Avian Influenza virus glycoproteins restrict virus replication and spread through human airway epithelium at temperatures of the proximal airways.

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Margaret A Scull

2009-05-01

Full Text Available Transmission of avian influenza viruses from bird to human is a rare event even though avian influenza viruses infect the ciliated epithelium of human airways in vitro and ex vivo. Using an in vitro model of human ciliated airway epithelium (HAE, we demonstrate that while human and avian influenza viruses efficiently infect at temperatures of the human distal airways (37 degrees C, avian, but not human, influenza viruses are restricted for infection at the cooler temperatures of the human proximal airways (32 degrees C. These data support the hypothesis that avian influenza viruses, ordinarily adapted to the temperature of the avian enteric tract (40 degrees C, rarely infect humans, in part due to differences in host airway regional temperatures. Previously, a critical residue at position 627 in the avian influenza virus polymerase subunit, PB2, was identified as conferring temperature-dependency in mammalian cells. Here, we use reverse genetics to show that avianization of residue 627 attenuates a human virus, but does not account for the different infection between 32 degrees C and 37 degrees C. To determine the mechanism of temperature restriction of avian influenza viruses in HAE at 32 degrees C, we generated recombinant human influenza viruses in either the A/Victoria/3/75 (H3N2 or A/PR/8/34 (H1N1 genetic background that contained avian or avian-like glycoproteins. Two of these viruses, A/Victoria/3/75 with L226Q and S228G mutations in hemagglutinin (HA and neuraminidase (NA from A/Chick/Italy/1347/99 and A/PR/8/34 containing the H7 and N1 from A/Chick/Italy/1347/99, exhibited temperature restriction approaching that of wholly avian influenza viruses. These data suggest that influenza viruses bearing avian or avian-like surface glycoproteins have a reduced capacity to establish productive infection at the temperature of the human proximal airways. This temperature restriction may limit zoonotic transmission of avian influenza viruses and

Multisegment one-step RT-PCR fluorescent labeling of influenza A virus genome for use in diagnostic microarray applications

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Vasin, A V; Plotnikova, M A; Klotchenko, S A; Elpaeva, E A; Komissarov, A B; Egorov, V V; Kiselev, O I [Research Institute of Influenza of the Ministry of Health and Social Development of the Russian Federation, 15/17 Prof. Popova St., St. Petersburg (Russian Federation); Sandybaev, N T; Chervyakova, O V; Strochkov, V M; Taylakova, E T; Koshemetov, J K; Mamadaliev, S M, E-mail: vasin@influenza.spb.ru [Research Institute for Biological Safety Problems of the RK NBC/SC ME and S RK, Gvardeiskiy (Kazakhstan)

2011-04-01

Microarray technology is one of the most challenging methods of influenza A virus subtyping, which is based on the antigenic properties of viral surface glycoproteins - hemagglutinin and neuraminidase. On the example of biochip for detection of influenza A/H5N1 virus we showed the possibility of using multisegment RTPCR method for amplification of fluorescently labeled cDNA of all possible influenza A virus subtypes with a single pair of primers in influenza diagnostic microarrays.

Airway protease/antiprotease imbalance in atopic asthmatics contributes to increased influenza A virus cleavage and replication

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Asthmatics are more susceptible to influenza infections, yet mechanisms mediating this enhanced susceptibility are unknown. Influenza virus hemagglutinin (HA) protein binds to sialic add residues on the host cells. HA requires cleavage to allow fusion of the viral HA with host ce...

Different evolutionary trends of swine H1N2 influenza viruses in Italy compared to European viruses.

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Moreno, Ana; Gabanelli, Elena; Sozzi, Enrica; Lelli, Davide; Chiapponi, Chiara; Ciccozzi, Massimo; Zehender, Gianguglielmo; Cordioli, Paolo

2013-12-01

European H1N2 swine influenza viruses (EU H1N2SIVs) arose from multiple reassortment events among human H1N1, human H3N2, and avian influenza viruses. We investigated the evolutionary dynamics of 53 Italian H1N2 strains by comparing them with EU H1N2 SIVs. Hemagglutinin (HA) phylogeny revealed Italian strains fell into four groups: Group A and B (41 strains) had a human H1 similar to EU H1N2SIVs, which probably originated in 1986. However Group B (38 strains) formed a subgroup that had a two-amino acid deletion at positions 146/147 in HA. Group C (11 strains) contained an avian H1 that probably originated in 1996, and Group D (1 strain) had an H1 characteristic of the 2009 pandemic strain. Neuraminidase (NA) phylogeny suggested a series of genomic reassortments had occurred. Group A had an N2 that originated from human H3N2 in the late 1970s. Group B had different human N2 that most likely arose from a reassortment with the more recent human H3N2 virus, which probably occurred in 2000. Group C had an avian-like H1 combined with an N2 gene from one of EU H1N2SIVs, EU H3N2SIVs or Human H3N2. Group D was part of the EU H3N2SIVs clade. Although selection pressure for HA and NA was low, several positively selected sites were identified in both proteins, some of which were antigenic, suggesting selection influenced the evolution of SIV. The data highlight different evolutionary trends between European viruses and currently circulating Italian B strains and show the establishment of reassortant strains involving human viruses in Italian pigs.

Phylogenetic analysis of influenza A viruses (H3N2 circulating in Zhytomyr region during 2013–2014 epidemic season

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Boyalska O. G.

2015-06-01

Full Text Available Aim. To perform phylogenetic analysis of the hemagglutinin (HA and neuraminidase (NA genes of influenza A(H3N2 viruses circulating in the Zhytomyr region during 2013–2014 epidemic season. To make comparison of the HA and NA genes sequences of the Zhytomyr region isolates with the HA and NA genes sequences of influenza viruses circulating in the world. Methods. Laboratory diagnosis was conducted by real-time polymerase chain reaction (RT-PCR. In this study the sequencing and phylogenetic analysis were carried out. Results. For the first time the genes of influenza A(H3N2 viruses isolated in the Zhytomyr region during 2013–2014 epidemic season, coding hemagglutinin and neuraminidase were compared with their orthologs. According to the results of this comparison the phylogenetic tree was constructed. Additionally, the amino acid substitutions of the influenza viruses circulating in Ukraine and worldwide were analyzed. Conclusions. The nucleotide sequences of the influenza A(H3N2 viruses genes HA and NA isolated in the Zhytomyr region were identified. Based on the nucleotide sequences of HA and NA we constructed the influenza virus phylogenetic tree demonstrating that the virus isolated in the Zhytomyr region was closely related to the Ukrainian isolate from Kharkov and in the world to the isolates from Germany, Romania, Italy.

Hemagglutinin-based polyanhydride nanovaccines against H5N1 influenza elicit protective virus neutralizing titers and cell-mediated immunity

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Ross KA

2014-12-01

Full Text Available Kathleen A Ross,1 Hyelee Loyd,2 Wuwei Wu,2 Lucas Huntimer,3 Shaheen Ahmed,4 Anthony Sambol,5 Scott Broderick,6 Zachary Flickinger,2 Krishna Rajan,6 Tatiana Bronich,4 Surya Mallapragada,1 Michael J Wannemuehler,3 Susan Carpenter,2 Balaji Narasimhan1 1Chemical and Biological Engineering, Iowa State University, Ames, IA, USA; 2Animal Science, Iowa State University, Ames, IA, USA; 3Veterinary Microbiology and Preventive Medicine, Iowa State University, Ames, IA, USA; 4Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE, USA; 5Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE, USA; 6Materials Science and Engineering, Iowa State University, Ames, IA, USA Abstract: H5N1 avian influenza is a significant global concern with the potential to become the next pandemic threat. Recombinant subunit vaccines are an attractive alternative for pandemic vaccines compared to traditional vaccine technologies. In particular, polyanhydride nanoparticles encapsulating subunit proteins have been shown to enhance humoral and cell-mediated immunity and provide protection upon lethal challenge. In this work, a recombinant H5 hemagglutinin trimer (H53 was produced and encapsulated into polyanhydride nanoparticles. The studies performed indicated that the recombinant H53 antigen was a robust immunogen. Immunizing mice with H53 encapsulated into polyanhydride nanoparticles induced high neutralizing antibody titers and enhanced CD4+ T cell recall responses in mice. Finally, the H53-based polyanhydride nanovaccine induced protective immunity against a low-pathogenic H5N1 viral challenge. Informatics analyses indicated that mice receiving the nanovaccine formulations and subsequently challenged with virus were similar to naïve mice that were not challenged. The current studies provide a basis to further exploit the advantages of polyanhydride nanovaccines in pandemic scenarios. Keywords: polymer, nanoparticle, vaccine, subunit

Enhanced genetic characterization of influenza A(H3N2) viruses and vaccine effectiveness by genetic group, 2014–2015

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Flannery, Brendan; Zimmerman, Richard K.; Gubareva, Larisa V.; Garten, Rebecca J.; Chung, Jessie R.; Nowalk, Mary Patricia; Jackson, Michael L.; Jackson, Lisa A.; Monto, Arnold S.; Ohmit, Suzanne E.; Belongia, Edward A.; McLean, Huong Q.; Gaglani, Manjusha; Piedra, Pedro A.; Mishin, Vasiliy P.; Chesnokov, Anton P.; Spencer, Sarah; Thaker, Swathi N.; Barnes, John R.; Foust, Angie; Sessions, Wendy; Xu, Xiyan; Katz, Jacqueline; Fry, Alicia M.

2018-01-01

Background During the 2014–15 US influenza season, expanded genetic characterization of circulating influenza A(H3N2) viruses was used to assess the impact of genetic variability of influenza A(H3N2) viruses on influenza vaccine effectiveness (VE). Methods A novel pyrosequencing assay was used to determine genetic group based on hemagglutinin (HA) gene sequences of influenza A(H3N2) viruses from patients enrolled US Flu Vaccine Effectiveness network sites. Vaccine effectiveness was estimated using a test-negative design comparing vaccination among patients infected with influenza A(H3N2) viruses and uninfected patients. Results Among 9710 enrollees, 1868 (19%) tested positive for influenza A(H3N2); genetic characterization of 1397 viruses showed 1134 (81%) belonged to one HA genetic group (3C.2a) of antigenically drifted H3N2 viruses. Effectiveness of 2014–15 influenza vaccination varied by A(H3N2) genetic group from 1% (95% confidence interval [CI], −14% to 14%) against illness caused by antigenically drifted A(H3N2) group 3C.2a viruses versus 44% (95% CI, 16% to 63%) against illness caused by vaccine-like A(H3N2) group 3C.3b viruses. Conclusion Effectiveness of 2014–15 influenza vaccination varied by genetic group of influenza A(H3N2) virus. Changes in hemagglutinin genes related to antigenic drift were associated with reduced vaccine effectiveness. PMID:27190176

Measles Virus Hemagglutinin epitopes immunogenic in natural infection and vaccination are targeted by broad or genotype-specific neutralizing monoclonal antibodies.

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Muñoz-Alía, Miguel Angel; Casasnovas, José M; Celma, María Luisa; Carabaña, Juan; Liton, Paloma B; Fernandez-Muñoz, Rafael

2017-05-15

Measles virus (MV) remains a leading cause of vaccine-preventable deaths in children. Protection against MV is associated with neutralizing antibodies that preferentially recognize the viral hemagglutinin (MV-H), and to a lesser extent, the fusion protein (MV-F). Although MV is serologically monotypic, 24 genotypes have been identified. Here we report three neutralization epitopes conserved in the more prevalent circulating MV genotypes, two located in the MV-H receptor binding site (RBS) (antigenic site III) and a third in MV-H/MV-F interphase (antigenic site Ia) which are essential for MV multiplication. In contrast, two MV-H neutralization epitopes, showed a genotype-specific neutralization escape due to a single amino acid change, that we mapped in the "noose" antigenic site, or an enhanced neutralization epitope (antigenic site IIa). The monoclonal antibody (mAb) neutralization potency correlated with its binding affinity and was mainly driven by kinetic dissociation rate (k off ). We developed an immunoassay for mAb binding to MV-H in its native hetero-oligomeric structure with MV-F on the surface of a MV productive steady-state persistently infected (p.i.) human cell lines, and a competitive-binding assay with serum from individuals with past infection by different MV genotypes. Binding assays revealed that a broad neutralization epitope, in RBS antigenic site, a genotype specific neutralization epitopes, in noose and IIa sites, were immunogenic in natural infection and vaccination and may elicit long-lasting humoral immunity that might contribute to explain MV immunogenic stability. These results support the design of improved measles vaccines, broad-spectrum prophylactic or therapeutic antibodies and MV-used in oncolytic therapies. Copyright © 2017 Elsevier B.V. All rights reserved.

SYBR green-based real-time reverse transcription-PCR for typing and subtyping of all hemagglutinin and neuraminidase genes of avian influenza viruses and comparison to standard serological subtyping tests

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Tsukamoto, K.; Javier, P.C.; Shishido, M.; Noguchi, D.; Pearce, J.; Kang, H.-M.; Jeong, O.M.; Lee, Y.-J.; Nakanishi, K.; Ashizawa, T.

2012-01-01

Continuing outbreaks of H5N1 highly pathogenic (HP) avian influenza virus (AIV) infections of wild birds and poultry worldwide emphasize the need for global surveillance of wild birds. To support the future surveillance activities, we developed a SYBR green-based, real-time reverse transcriptase PCR (rRT-PCR) for detecting nucleoprotein (NP) genes and subtyping 16 hemagglutinin (HA) and 9 neuraminidase (NA) genes simultaneously. Primers were improved by focusing on Eurasian or North American lineage genes; the number of mixed-base positions per primer was set to five or fewer, and the concentration of each primer set was optimized empirically. Also, 30 cycles of amplification of 1:10 dilutions of cDNAs from cultured viruses effectively reduced minor cross- or nonspecific reactions. Under these conditions, 346 HA and 345 NA genes of 349 AIVs were detected, with average sensitivities of NP, HA, and NA genes of 10 1.5, 10 2.3, and 10 3.1 50% egg infective doses, respectively. Utility of rRT-PCR for subtyping AIVs was compared with that of current standard serological tests by using 104 recent migratory duck virus isolates. As a result, all HA genes and 99% of the NA genes were genetically subtyped, while only 45% of HA genes and 74% of NA genes were serologically subtyped. Additionally, direct subtyping of AIVs in fecal samples was possible by 40 cycles of amplification: approximately 70% of HA and NA genes of NP gene-positive samples were successfully subtyped. This validation study indicates that rRT-PCR with optimized primers and reaction conditions is a powerful tool for subtyping varied AIVs in clinical and cultured samples. Copyright ?? 2012, American Society for Microbiology. All Rights Reserved.

Humoral and cell-mediated immune responses in DNA immunized mink challenged with wild-type canine distemper virus

DEFF Research Database (Denmark)

Nielsen, Line; Søgaard, Mette; Karlskov-Mortensen, Peter

2009-01-01

The aim of the study was to investigate the different phases of the immune response after DNA immunization with the hemagglutinin and nucleoprotein genes from canine distemper virus (CDV). Although attenuated live CDV vaccines have effectively reduced the incidence of disease, canine distemper...

Imported parakeets harbor H9N2 influenza A viruses that are genetically closely related to those transmitted to humans in Hong Kong.

Science.gov (United States)

Mase, M; Imada, T; Sanada, Y; Etoh, M; Sanada, N; Tsukamoto, K; Kawaoka, Y; Yamaguchi, S

2001-04-01

In 1997 and 1998, H9N2 influenza A viruses were isolated from the respiratory organs of Indian ring-necked parakeets (Psittacula Krameri manillensis) that had been imported from Pakistan to Japan. The two isolates were closely related to each other (>99% as determined by nucleotide analysis of eight RNA segments), indicating that H9N2 viruses of the same lineage were maintained in these birds for at least 1 year. The hemagglutinins and neuraminidases of both isolates showed >97% nucleotide identity with those of H9N2 viruses isolated from humans in Hong Kong in 1999, while the six genes encoding internal proteins were >99% identical to the corresponding genes of H5N1 viruses recovered during the 1997 outbreak in Hong Kong. These results suggest that the H9N2 parakeet viruses originating in Pakistan share an immediate ancestor with the H9N2 human viruses. Thus, influenza A viruses with the potential to be transmitted directly to humans may be circulating in captive birds worldwide.

H7N9 influenza A virus in turkeys in Minnesota

Science.gov (United States)

Lebarbenchon, Camille; Pedersen, J.C.; Sreevatsan, Srinand; Ramey, Andy M.; Dugan, Vivien G.; Halpin, R.A.; Ferro, Paul A.; Lupiani, B.; Enomoto, Shinichiro; Poulson, Rebecca L.; Smeltzer, M.; Cardona, Carol J.; Tompkins, S.; Wentworth, D.E.; Stallknecht, D.E.; Brown, J.

2015-01-01

Introductions of H7 Influenza A virus (IAV) from wild birds into poultry have been documented worldwide, resulting in varying degrees of morbidity and mortality. H7 IAV infection in domestic poultry has served as a source of human infection and disease. We report the detection of H7N9 subtype IAV in Minnesota turkey farms during 2009 and 2011. The full-genome was sequenced from eight isolates as well as the hemagglutinin (HA) and neuraminidase (NA) gene segments of H7 and N9 virus subtypes for 108 isolates from North American wild birds between 1986 and 2012. Through maximum likelihood and coalescent phylogenetic analyses, we identified the recent H7 and N9 IAV ancestors of the turkey-origin H7N9 IAV, estimated the time and geographic origin of the ancestral viruses, and determined the relatedness between the 2009 and the 2011 turkey-origin H7N9 IAV. Analyses supported that the 2009 and the 2011 viruses were distantly related genetically, suggesting that the two outbreaks arose from independent introduction events from wild birds. Our findings further support that the 2011 MN turkey-origin H7N9 virus was closely related to H7N9 IAV isolated in poultry in Nebraska during the same year. Although the precise origin of the wild-bird donor of the turkey-origin H7N9 IAV could not be determined, our findings suggest that, for both the NA and HA gene segments, the MN turkey-origin H7N9 viruses were related to viruses circulating in wild birds between 2006 and 2011 in the Mississippi flyway.

Shallow Boomerang-shaped Influenza Hemagglutinin G13A Mutant Structure Promotes Leaky Membrane Fusion*

Science.gov (United States)

Lai, Alex L.; Tamm, Lukas K.

2010-01-01

Our previous studies showed that an angled boomerang-shaped structure of the influenza hemagglutinin (HA) fusion domain is critical for virus entry into host cells by membrane fusion. Because the acute angle of ∼105° of the wild-type fusion domain promotes efficient non-leaky membrane fusion, we asked whether different angles would still support fusion and thus facilitate virus entry. Here, we show that the G13A fusion domain mutant produces a new leaky fusion phenotype. The mutant fusion domain structure was solved by NMR spectroscopy in a lipid environment at fusion pH. The mutant adopted a boomerang structure similar to that of wild type but with a shallower kink angle of ∼150°. G13A perturbed the structure of model membranes to a lesser degree than wild type but to a greater degree than non-fusogenic fusion domain mutants. The strength of G13A binding to lipid bilayers was also intermediate between that of wild type and non-fusogenic mutants. These membrane interactions provide a clear link between structure and function of influenza fusion domains: an acute angle is required to promote clean non-leaky fusion suitable for virus entry presumably by interaction of the fusion domain with the transmembrane domain deep in the lipid bilayer. A shallower angle perturbs the bilayer of the target membrane so that it becomes leaky and unable to form a clean fusion pore. Mutants with no fixed boomerang angle interacted with bilayers weakly and did not promote any fusion or membrane perturbation. PMID:20826788

A live attenuated H7N7 candidate vaccine virus induces neutralizing antibody that confers protection from challenge in mice, ferrets and monkeys

Science.gov (United States)

A live attenuated H7N7 candidate vaccine virus was generated by reverse genetics using the modified hemagglutinin (HA) and neuraminidase (NA) genes of HP A/Netherlands/219/03 (NL/03) (H7N7) wild-type (wt) virus and the six internal protein genes of the cold-adapted (ca) A/Ann Arbor/6/60 ca (AA ca) (...

The low-pH stability discovered in neuraminidase of 1918 pandemic influenza A virus enhances virus replication.

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Tadanobu Takahashi

Full Text Available The "Spanish" pandemic influenza A virus, which killed more than 20 million worldwide in 1918-19, is one of the serious pathogens in recorded history. Characterization of the 1918 pandemic virus reconstructed by reverse genetics showed that PB1, hemagglutinin (HA, and neuraminidase (NA genes contributed to the viral replication and virulence of the 1918 pandemic influenza virus. However, the function of the NA gene has remained unknown. Here we show that the avian-like low-pH stability of sialidase activity discovered in the 1918 pandemic virus NA contributes to the viral replication efficiency. We found that deletion of Thr at position 435 or deletion of Gly at position 455 in the 1918 pandemic virus NA was related to the low-pH stability of the sialidase activity in the 1918 pandemic virus NA by comparison with the sequences of other human N1 NAs and sialidase activity of chimeric constructs. Both amino acids were located in or near the amino acid resides that were important for stabilization of the native tetramer structure in a low-pH condition like the N2 NAs of pandemic viruses that emerged in 1957 and 1968. Two reverse-genetic viruses were generated from a genetic background of A/WSN/33 (H1N1 that included low-pH-unstable N1 NA from A/USSR/92/77 (H1N1 and its counterpart N1 NA in which sialidase activity was converted to a low-pH-stable property by a deletion and substitutions of two amino acid residues at position 435 and 455 related to the low-pH stability of the sialidase activity in 1918 NA. The mutant virus that included "Spanish Flu"-like low-pH-stable NA showed remarkable replication in comparison with the mutant virus that included low-pH-unstable N1 NA. Our results suggest that the avian-like low-pH stability of sialidase activity in the 1918 pandemic virus NA contributes to the viral replication efficiency.

Full-Genome Analysis of Avian Influenza A(H5N1) Virus from a Human, North America, 2013

Science.gov (United States)

Pabbaraju, Kanti; Tellier, Raymond; Wong, Sallene; Li, Yan; Bastien, Nathalie; Tang, Julian W.; Drews, Steven J.; Jang, Yunho; Davis, C. Todd; Tipples, Graham A.

2014-01-01

Full-genome analysis was conducted on the first isolate of a highly pathogenic avian influenza A(H5N1) virus from a human in North America. The virus has a hemagglutinin gene of clade 2.3.2.1c and is a reassortant with an H9N2 subtype lineage polymerase basic 2 gene. No mutations conferring resistance to adamantanes or neuraminidase inhibitors were found. PMID:24755439

Canine distemper virus (CDV) infection of ferrets as a model for testing Morbillivirus vaccine strategies: NYVAC- and ALVAC-based CDV recombinants protect against symptomatic infection.

OpenAIRE

Stephensen, C B; Welter, J; Thaker, S R; Taylor, J; Tartaglia, J; Paoletti, E

1997-01-01

Canine distemper virus (CDV) infection of ferrets causes an acute systemic disease involving multiple organ systems, including the respiratory tract, lymphoid system, and central nervous system (CNS). We have tested candidate CDV vaccines incorporating the fusion (F) and hemagglutinin (HA) proteins in the highly attenuated NYVAC strain of vaccinia virus and in the ALVAC strain of canarypox virus, which does not productively replicate in mammalian hosts. Juvenile ferrets were vaccinated twice ...

Experience in applying 60Co γ-rays for careful production of inactivated influenza virus vaccines

International Nuclear Information System (INIS)

Nordheim, W.; Braeuniger, S.; Schulze, P.; Dittmann, S.; Petzold, G.; Teupel, D.; Luther, P.; Tischner, H.; Baer, M.; Akademie der Wissenschaften der DDR, Leipzig. Zentralinstitut fuer Isotopen- und Strahlenforschung)

1987-01-01

Radiation doses between 12 and 13 kGy at 15-20 0 C were sufficient for mild inactivation of influenza viruses. Under these conditions the decisive surface antigens hemagglutinin and neuraminidase were treated with care, and the preparations of influenza viruses revealed good immunogenicity in the animal experiment. Morphologic alterations after application of 20 kGy could not be demonstrated in electron microscopic investigations. Doses of 9.5-9.9 kGy in combination with a very low quantity of HCHO (1:15000) is sufficient for inactivation. Reactivation of influenza viruses after treatment could not be demonstrated. (author)

Bacterially produced recombinant influenza vaccines based on virus-like particles.

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Andrea Jegerlehner

Full Text Available Although current influenza vaccines are effective in general, there is an urgent need for the development of new technologies to improve vaccine production timelines, capacities and immunogenicity. Herein, we describe the development of an influenza vaccine technology which enables recombinant production of highly efficient influenza vaccines in bacterial expression systems. The globular head domain of influenza hemagglutinin, comprising most of the protein's neutralizing epitopes, was expressed in E. coli and covalently conjugated to bacteriophage-derived virus-like particles produced independently in E.coli. Conjugate influenza vaccines produced this way were used to immunize mice and found to elicit immune sera with high antibody titers specific for the native influenza hemagglutinin protein and high hemagglutination-inhibition titers. Moreover vaccination with these vaccines induced full protection against lethal challenges with homologous and highly drifted influenza strains.

Highly pathogenic avian influenza virus (H5N1) isolated from whooper swans, Japan.

Science.gov (United States)

Uchida, Yuko; Mase, Masaji; Yoneda, Kumiko; Kimura, Atsumu; Obara, Tsuyoshi; Kumagai, Seikou; Saito, Takehiko; Yamamoto, Yu; Nakamura, Kikuyasu; Tsukamoto, Kenji; Yamaguchi, Shigeo

2008-09-01

On April 21, 2008, four whooper swans were found dead at Lake Towada, Akita prefecture, Japan. Highly pathogenic avian influenza virus of the H5N1 subtype was isolated from specimens of the affected birds. The hemagglutinin (HA) gene of the isolate belongs to clade 2.3.2 in the HA phylogenetic tree.

Seasonal Dynamics of Haptophytes and dsDNA Algal Viruses Suggest Complex Virus-Host Relationship.

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Johannessen, Torill Vik; Larsen, Aud; Bratbak, Gunnar; Pagarete, António; Edvardsen, Bente; Egge, Elianne D; Sandaa, Ruth-Anne

2017-04-20

Viruses influence the ecology and diversity of phytoplankton in the ocean. Most studies of phytoplankton host-virus interactions have focused on bloom-forming species like Emiliania huxleyi or Phaeocystis spp. The role of viruses infecting phytoplankton that do not form conspicuous blooms have received less attention. Here we explore the dynamics of phytoplankton and algal viruses over several sequential seasons, with a focus on the ubiquitous and diverse phytoplankton division Haptophyta, and their double-stranded DNA viruses, potentially with the capacity to infect the haptophytes. Viral and phytoplankton abundance and diversity showed recurrent seasonal changes, mainly explained by hydrographic conditions. By 454 tag-sequencing we revealed 93 unique haptophyte operational taxonomic units (OTUs), with seasonal changes in abundance. Sixty-one unique viral OTUs, representing Megaviridae and Phycodnaviridae , showed only distant relationship with currently isolated algal viruses. Haptophyte and virus community composition and diversity varied substantially throughout the year, but in an uncoordinated manner. A minority of the viral OTUs were highly abundant at specific time-points, indicating a boom-bust relationship with their host. Most of the viral OTUs were very persistent, which may represent viruses that coexist with their hosts, or able to exploit several host species.

Chimeric Hemagglutinin Constructs Induce Broad Protection against Influenza B Virus Challenge in the Mouse Model

OpenAIRE

Ermler, Megan E.; Kirkpatrick, Ericka; Sun, Weina; Hai, Rong; Amanat, Fatima; Chromikova, Veronika; Palese, Peter; Krammer, Florian

2017-01-01

Seasonal influenza virus epidemics represent a significant public health burden. Approximately 25% of all influenza virus infections are caused by type B viruses, and these infections can be severe, especially in children. Current influenza virus vaccines are an effective prophylaxis against infection but are impacted by rapid antigenic drift, which can lead to mismatches between vaccine strains and circulating strains. Here, we describe a broadly protective vaccine candidate based on chimeri...

Virus-like particles displaying H5, H7, H9 hemagglutinins and N1 neuraminidase elicit protective immunity to heterologous avian influenza viruses in chickens

International Nuclear Information System (INIS)

Pushko, Peter; Tretyakova, Irina; Hidajat, Rachmat; Zsak, Aniko; Chrzastek, Klaudia; Tumpey, Terrence M.; Kapczynski, Darrell R.

2017-01-01

Avian influenza (AI) viruses circulating in wild birds pose a serious threat to public health. Human and veterinary vaccines against AI subtypes are needed. Here we prepared triple-subtype VLPs that co-localized H5, H7 and H9 antigens derived from H5N1, H7N3 and H9N2 viruses. VLPs also contained influenza N1 neuraminidase and retroviral gag protein. The H5/H7/H9/N1/gag VLPs were prepared using baculovirus expression. Biochemical, functional and antigenic characteristics were determined including hemagglutination and neuraminidase enzyme activities. VLPs were further evaluated in a chicken AI challenge model for safety, immunogenicity and protective efficacy against heterologous AI viruses including H5N2, H7N3 and H9N2 subtypes. All vaccinated birds survived challenges with H5N2 and H7N3 highly pathogenic AI (HPAI) viruses, while all controls died. Immune response was also detectable after challenge with low pathogenicity AI (LPAI) H9N2 virus suggesting that H5/H7/H9/N1/gag VLPs represent a promising approach for the development of broadly protective AI vaccine. - Highlights: •VLPs were prepared that co-localized H5, H7 and H9 subtypes in a VLP envelope. •VLPs were characterized including electron microscopy, HA assay and NA enzyme activity. •Experimental VLP vaccine was evaluated in an avian influenza challenge model. •VLPs induced immune responses against heterologous H5, H7 and H9 virus challenges.

Virus-like particles displaying H5, H7, H9 hemagglutinins and N1 neuraminidase elicit protective immunity to heterologous avian influenza viruses in chickens

Energy Technology Data Exchange (ETDEWEB)

Pushko, Peter, E-mail: ppushko@medigen-usa.com [Medigen, Inc., 8420 Gas House Pike, Suite S, Frederick, MD 21701 (United States); Tretyakova, Irina; Hidajat, Rachmat [Medigen, Inc., 8420 Gas House Pike, Suite S, Frederick, MD 21701 (United States); Zsak, Aniko; Chrzastek, Klaudia [USDA SEPRL, 934 College Station Rd, Athens, GA (United States); Tumpey, Terrence M. [Influenza Division, CDC,1600 Clifton Road N.E., Atlanta, GA (United States); Kapczynski, Darrell R. [USDA SEPRL, 934 College Station Rd, Athens, GA (United States)

2017-01-15

Avian influenza (AI) viruses circulating in wild birds pose a serious threat to public health. Human and veterinary vaccines against AI subtypes are needed. Here we prepared triple-subtype VLPs that co-localized H5, H7 and H9 antigens derived from H5N1, H7N3 and H9N2 viruses. VLPs also contained influenza N1 neuraminidase and retroviral gag protein. The H5/H7/H9/N1/gag VLPs were prepared using baculovirus expression. Biochemical, functional and antigenic characteristics were determined including hemagglutination and neuraminidase enzyme activities. VLPs were further evaluated in a chicken AI challenge model for safety, immunogenicity and protective efficacy against heterologous AI viruses including H5N2, H7N3 and H9N2 subtypes. All vaccinated birds survived challenges with H5N2 and H7N3 highly pathogenic AI (HPAI) viruses, while all controls died. Immune response was also detectable after challenge with low pathogenicity AI (LPAI) H9N2 virus suggesting that H5/H7/H9/N1/gag VLPs represent a promising approach for the development of broadly protective AI vaccine. - Highlights: •VLPs were prepared that co-localized H5, H7 and H9 subtypes in a VLP envelope. •VLPs were characterized including electron microscopy, HA assay and NA enzyme activity. •Experimental VLP vaccine was evaluated in an avian influenza challenge model. •VLPs induced immune responses against heterologous H5, H7 and H9 virus challenges.

Serum amyloid P component inhibits influenza A virus infections: in vitro and in vivo studies

DEFF Research Database (Denmark)

Horvath, A; Andersen, I; Junker, K

2001-01-01

. These studies were extended to comprise five mouse-adapted influenza A strains, two swine influenza A strains, a mink influenza A virus, a ferret influenza A reassortant virus, a influenza B virus and a parainfluenza 3 virus. The HA activity of all these viruses was inhibited by SAP. Western blotting showed......Serum amyloid P component (SAP) binds in vitro Ca(2+)-dependently to several ligands including oligosaccharides with terminal mannose and galactose. We have earlier reported that SAP binds to human influenza A virus strains, inhibiting hemagglutinin (HA) activity and virus infectivity in vitro...... that SAP bound to HA trimers, monomers and HA1 and HA2 subunits of influenza A virus. Binding studies indicated that galactose, mannose and fucose moieties contributed to the SAP reacting site(s). Intranasal administration of human SAP to mice induced no demonstrable toxic reactions, and circulating...

Molecular signatures of hemagglutinin stem-directed heterosubtypic human neutralizing antibodies against influenza A viruses.

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Yuval Avnir

2014-05-01

Full Text Available Recent studies have shown high usage of the IGHV1-69 germline immunoglobulin gene for influenza hemagglutinin stem-directed broadly-neutralizing antibodies (HV1-69-sBnAbs. Here we show that a major structural solution for these HV1-69-sBnAbs is achieved through a critical triad comprising two CDR-H2 loop anchor residues (a hydrophobic residue at position 53 (Ile or Met and Phe54, and CDR-H3-Tyr at positions 98±1; together with distinctive V-segment CDR amino acid substitutions that occur in positions sparse in AID/polymerase-η recognition motifs. A semi-synthetic IGHV1-69 phage-display library screen designed to investigate AID/polη restrictions resulted in the isolation of HV1-69-sBnAbs that featured a distinctive Ile52Ser mutation in the CDR-H2 loop, a universal CDR-H3 Tyr at position 98 or 99, and required as little as two additional substitutions for heterosubtypic neutralizing activity. The functional importance of the Ile52Ser mutation was confirmed by mutagenesis and by BCR studies. Structural modeling suggests that substitution of a small amino acid at position 52 (or 52a facilitates the insertion of CDR-H2 Phe54 and CDR-H3-Tyr into adjacent pockets on the stem. These results support the concept that activation and expansion of a defined subset of IGHV1-69-encoded B cells to produce potent HV1-69-sBnAbs does not necessarily require a heavily diversified V-segment acquired through recycling/reentry into the germinal center; rather, the incorporation of distinctive amino acid substitutions by Phase 2 long-patch error-prone repair of AID-induced mutations or by random non-AID SHM events may be sufficient. We propose that these routes of B cell maturation should be further investigated and exploited as a pathway for HV1-69-sBnAb elicitation by vaccination.

Attenuated Salmonella enterica serovar Typhi and Shigella flexneri 2a strains mucosally deliver DNA vaccines encoding measles virus hemagglutinin, inducing specific immune responses and protection in cotton rats.

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Pasetti, Marcela F; Barry, Eileen M; Losonsky, Genevieve; Singh, Mahender; Medina-Moreno, Sandra M; Polo, John M; Ulmer, Jeffrey; Robinson, Harriet; Sztein, Marcelo B; Levine, Myron M

2003-05-01

Measles remains a leading cause of child mortality in developing countries. Residual maternal measles antibodies and immunologic immaturity dampen immunogenicity of the current vaccine in young infants. Because cotton rat respiratory tract is susceptible to measles virus (MV) replication after intranasal (i.n.) challenge, this model can be used to assess the efficacy of MV vaccines. Pursuing a new measles vaccine strategy that might be effective in young infants, we used attenuated Salmonella enterica serovar Typhi CVD 908-htrA and Shigella flexneri 2a CVD 1208 vaccines to deliver mucosally to cotton rats eukaryotic expression plasmid pGA3-mH and Sindbis virus-based DNA replicon pMSIN-H encoding MV hemagglutinin (H). The initial i.n. dose-response with bacterial vectors alone identified a well-tolerated dosage (1 x 10(9) to 7 x 10(9) CFU) and a volume (20 micro l) that elicited strong antivector immune responses. Animals immunized i.n. on days 0, 28, and 76 with bacterial vectors carrying DNA plasmids encoding MV H or immunized parenterally with these naked DNA vaccine plasmids developed MV plaque reduction neutralizing antibodies and proliferative responses against MV antigens. In a subsequent experiment of identical design, cotton rats were challenged with wild-type MV 1 month after the third dose of vaccine or placebo. MV titers were significantly reduced in lung tissue of animals immunized with MV DNA vaccines delivered either via bacterial live vectors or parenterally. Since attenuated serovar Typhi and S. flexneri can deliver measles DNA vaccines mucosally in cotton rats, inducing measles immune responses (including neutralizing antibodies) and protection, boosting strategies can now be evaluated in animals primed with MV DNA vaccines.

Molecular characterization of AI viruses from poultry and wild bird surveillance in Denmark

DEFF Research Database (Denmark)

Larsen, Lars Erik; Krog, Jesper Schak; Madsen, Jesper J.

Infection with avian influenza virus (AIV) in poultry may cause devastating disease although the same virus may not cause disease in wild birds. Since AI viruses can be exchanged between poultry and wild birds, surveillance in wild birds provides important knowledge for control of disease...... in poultry. AIV’s from the Danish wild bird active surveillance were characterized, focusing on viruses from 2012, and from outbreaks of AI in poultry in Denmark. The matrix (M) gene from more than 50 viruses of different subtypes and the hemagglutinin (HA) gene from more than 30 subtype H5 low pathogenic...... viruses were sequenced and compared by alignment and phylogenetic analyses. The aim was to evaluate: the origin of viruses from outbreaks of AI in Danish poultry, the design of active surveillance in Denmark, and the suitability of the molecular diagnostic RT-PCR tests employed. All M-genes from Danish...

Emergent lineages of mumps virus suggest the need for a polyvalent vaccine

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Meghan May

2018-01-01

Full Text Available Mumps outbreaks among vaccinated patients have become increasingly common in recent years. While there are multiple conditions driving this re-emergence, convention has suggested that these outbreaks are associated with waning immunity rather than vaccine escape. Molecular evidence from both the ongoing American and Dutch outbreaks in conjunction with recent structural biology studies challenge this convention, and suggest that emergent lineages of mumps virus exhibit key differences in antigenic epitopes from the vaccine strain employed: Jeryl-Lynn 5. The American and Dutch 2016–2017 outbreak lineages were examined using computational biology through the lens of diversity in immunogenic epitopes. Findings are discussed and the laboratory evidence indicating neutralization of heterologous mumps strains by serum from vaccinated individuals is reviewed. Taken together, it is concluded that the number of heterologous epitopes occurring in mumps virus in conjunction with waning immunity is facilitating small outbreaks in vaccinated patients, and that consideration of a polyvalent mumps vaccine is warranted.

In silico analysis suggests interaction between Ebola virus and the extracellular matrix

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Veljko eVeljkovic

2015-02-01

Full Text Available The worst Ebola virus (EV outbreak in history has hit Liberia, Sierra Leone and Guinea hardest and the trendlines in this crisis are grave, and now represents global public health threat concern. Limited therapeutic and/or prophylactic options which are available for humans suffering from Ebola virus disease (EVD further complicate situation. Previous studies suggested that the EV glycoprotein (GP is the main determinant causing structural damage of endothelial cells that triggers the hemorrhagic diathesis, but molecular mechanisms underlying this phenomenon remains elusive. Using the informational spectrum method (ISM, a virtual spectroscopy method for analysis of the protein-protein interactions, the interaction of GP with endothelial extracellular matrix (ECM was investigated. Presented results of this in silico study suggest that Elastin Microfibril Interface Located Proteins (EMILINs are involved in interaction between GP and ECM. This finding could contribute to better understanding of EV/endothelium interaction and its role in pathogenesis, prevention and therapy of EVD.

A novel hemagglutinin with antiproliferative activity against tumor cells from the hallucinogenic mushroom Boletus speciosus.

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Sun, Jian; Ng, Tzi-Bun; Wang, Hexiang; Zhang, Guoqing

2014-01-01

Little was known about bioactive compounds from the hallucinogenic mushroom Boletus speciosus. In the present study, a hemagglutinin (BSH, B. speciosus hemagglutinin) was isolated from its fruiting bodies and enzymatic properties were also tested. The chromatographic procedure utilized comprised anion exchange chromatography on Q-Sepharose, cation exchange chromatography on CM-Cellulose, cation exchange chromatography on SP-Sepharose, and gel filtration by FPLC on Superdex 75. The hemagglutinin was a homodimer which was estimated to be approximately 31 kDa in size. The activity of BSH was stable up to 60°C, while there was a precipitous drop in activity when the temperature was elevated to 70°C. BSH retained 25% hemagglutinating activity when exposed to 100 mM NaOH and 25 mM HCl. The activity was potently inhibited by 1.25 mM Hg(2+) and slightly inhibited by Fe(2+), Ca(2+), and Pb(2+). None of the sugars tested showed inhibition towards BSH. Its hemagglutinating activity towards human erythrocytes type A, type B, and type AB was higher than type O. The hemagglutinin showed antiproliferative activity towards hepatoma Hep G2 cells and mouse lymphocytic leukemia cells (L1210) in vitro, with IC50 of 4.7 μ M and 7.0 μ M, respectively. It also exhibited HIV-1 reverse transcriptase inhibitory activity with an IC50 of 7.1 μ M.

Antibody neutralization of retargeted measles viruses

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Lech, Patrycja J.; Pappoe, Roland; Nakamura, Takafumi; Tobin, Gregory J.; Nara, Peter L.; Russell, Stephen J.

2014-01-01

The measles virus (MV) vaccine lineage is a promising oncolytic but prior exposure to the measles vaccine or wild-type MV strains limits treatment utility due to the presence of anti-measles antibodies. MV entry can be redirected by displaying a polypeptide ligand on the Hemagglutinin (H) C-terminus. We hypothesized that retargeted MV would escape neutralization by monoclonal antibodies (mAbs) recognizing the H receptor-binding surface and be less susceptible to neutralization by human antisera. Using chimeric H proteins, with and without mutations that ablate MV receptor binding, we show that retargeted MVs escape mAbs that target the H receptor-binding surface by virtue of mutations that ablate infection via SLAM and CD46. However, C-terminally displayed domains do not mediate virus entry in the presence of human antibodies that bind to the underlying H domain. In conclusion, utility of retargeted oncolytic measles viruses does not extend to evasion of human serum neutralization. PMID:24725950

Influenza vaccines: from whole virus preparations to recombinant protein technology.

Science.gov (United States)

Huber, Victor C

2014-01-01

Vaccination against influenza represents our most effective form of prevention. Historical approaches toward vaccine creation and production have yielded highly effective vaccines that are safe and immunogenic. Despite their effectiveness, these historical approaches do not allow for the incorporation of changes into the vaccine in a timely manner. In 2013, a recombinant protein-based vaccine that induces immunity toward the influenza virus hemagglutinin was approved for use in the USA. This vaccine represents the first approved vaccine formulation that does not require an influenza virus intermediate for production. This review presents a brief history of influenza vaccines, with insight into the potential future application of vaccines generated using recombinant technology.

Recombinant Newcastle disease viral vector expressing hemagglutinin or fusion of canine distemper virus is safe and immunogenic in minks.

Science.gov (United States)

Ge, Jinying; Wang, Xijun; Tian, Meijie; Gao, Yuwei; Wen, Zhiyuan; Yu, Guimei; Zhou, Weiwei; Zu, Shulong; Bu, Zhigao

2015-05-15

Canine Distemper Virus (CDV) infects many carnivores and cause several high-mortality disease outbreaks. The current CDV live vaccine cannot be safely used in some exotic species, such as mink and ferret. Here, we generated recombinant lentogenic Newcastle disease virus (NDV) LaSota expressing either envelope glycoproyein, heamagglutinine (H) or fusion protein (F), named as rLa-CDVH and rLa-CDVF, respectively. The feasibility of these recombinant NDVs to serve as live virus-vectored CD vaccine was evaluated in minks. rLa-CDVH induced significant neutralization antibodies (NA) to CDV and provided solid protection against virulent CDV challenge. On the contrast, rLa-CDVF induced much lower NA to CDV and fail to protected mink from virulent CDV challenge. Results suggest that recombinant NDV expressing CDV H is safe and efficient candidate vaccine against CDV in mink, and maybe other host species. Copyright © 2015 Elsevier Ltd. All rights reserved.

Rapid and highly informative diagnostic assay for H5N1 influenza viruses.

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Nader Pourmand

Full Text Available A highly discriminative and information-rich diagnostic assay for H5N1 avian influenza would meet immediate patient care needs and provide valuable information for public health interventions, e.g., tracking of new and more dangerous variants by geographic area as well as avian-to-human or human-to-human transmission. In the present study, we have designed a rapid assay based on multilocus nucleic acid sequencing that focuses on the biologically significant regions of the H5N1 hemagglutinin gene. This allows the prediction of viral strain, clade, receptor binding properties, low- or high-pathogenicity cleavage site and glycosylation status. H5 HA genes were selected from nine known high-pathogenicity avian influenza subtype H5N1 viruses, based on their diversity in biologically significant regions of hemagglutinin and/or their ability to cause infection in humans. We devised a consensus pre-programmed pyrosequencing strategy, which may be used as a faster, more accurate alternative to de novo sequencing. The available data suggest that the assay described here is a reliable, rapid, information-rich and cost-effective approach for definitive diagnosis of H5N1 avian influenza. Knowledge of the predicted functional sequences of the HA will enhance H5N1 avian influenza surveillance efforts.

[Molecular analyses of human influenza viruses. Circulation of new variants since 1995/96].

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Biere, B; Schweiger, B

2008-09-01

The evolution of influenza viruses is increasingly pursued by molecular analyses that complement classical methods. The analyses focus on the two surface proteins hemagglutinin (HA) and neuraminidase (NA) which determine the viral antigenic profile. Influenza A(H3N2) viruses are exceptionally variable, so that usually at least two virus variants cocirculate at the same time. Together with influenza B viruses they caused approximately 90% of influenza virus infections in Germany during the last 12 seasons, while influenza A(H1N1) viruses only played a subordinate part. Unexpectedly, reassorted viruses of subtype A(H1N2) appeared during the seasons 2001/02 and 2002/03, but were isolated only rarely and gained no epidemiological significance. Furthermore, during the season 2001/02 influenza B viruses of the Victoria-lineage reappeared in Germany and other countries of the northern hemisphere after 10 years of absence. These viruses reassorted with the cocirculating Yamagata-like influenza B viruses, as could be seen by the appearance of viruses with a Victoria-like HA and a Yamagata-like NA.

Phylogenetic analysis of the haemagglutinin gene of canine distemper virus strains detected from giant panda and raccoon dogs in China

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2013-01-01

Background Canine distemper virus (CDV) infects a variety of carnivores, including wild and domestic Canidae. In this study, we sequenced and phylogenetic analyses of the hemagglutinin (H) genes from eight canine distemper virus (CDV) isolates obtained from seven raccoon dogs (Nyctereutes procyonoides) and a giant panda (Ailuropoda melanoleuca) in China. Results Phylogenetic analysis of the partial hemagglutinin gene sequences showed close clustering for geographic lineages, clearly distinct from vaccine strains and other wild-type foreign CDV strains, all the CDV strains were characterized as Asia-1 genotype and were highly similar to each other (91.5-99.8% nt and 94.4-99.8% aa). The giant panda and raccoon dogs all were 549Y on the HA protein in this study, irrespective of the host species. Conclusions These findings enhance our knowledge of the genetic characteristics of Chinese CDV isolates, and may facilitate the development of effective strategies for monitoring and controlling CDV for wild canids and non-cainds in China. PMID:23566727

An unexpected antibody response to an engineered influenza virus modifies CD8+ T cell responses.

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Thomas, Paul G; Brown, Scott A; Yue, Wen; So, Jenny; Webby, Richard J; Doherty, Peter C

2006-02-21

The ovalbumin(323-339) peptide that binds H2I-A(b) was engineered into the globular heads of hemagglutinin (H) molecules from serologically non-cross-reactive H1N1 and H3N2 influenza A viruses, the aim being to analyze recall CD4+ T cell responses in a virus-induced respiratory disease. Prime/challenge experiments with these H1ova and H3ova viruses in H2(b) mice gave the predicted, ovalbumin-specific CD4+ T cell response but showed an unexpectedly enhanced, early expansion of viral epitope-specific CD8+ T cells in spleen and a greatly diminished inflammatory process in the virus-infected respiratory tract. At the same time, the primary antibody response to the H3N2 challenge virus was significantly reduced, an effect that has been associated with preexisting neutralizing antibody in other experimental systems. Analysis of serum from the H1ova-primed mice showed low-level binding to H3ova but not to the wild-type H3N2 virus. Experiments with CD4+ T cell-depleted and Ig-/- mice indicated that this cross-reactive Ig is indeed responsible for the modified pathogenesis after respiratory challenge. Furthermore, the effect does not seem to be virus-dose related, although it does require infection. These findings suggest intriguing possibilities for vaccination and, at the same time, emphasize that engineered modifications in viruses may have unintended immunological consequences.

Vaccine protection of chickens against antigenically diverse H5 highly pathogenic avian influenza isolates with a live HVT vector vaccine expressing the influenza hemagglutinin gene derived from a clade 2.2 avian influenza virus.

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Kapczynski, Darrell R; Esaki, Motoyuki; Dorsey, Kristi M; Jiang, Haijun; Jackwood, Mark; Moraes, Mauro; Gardin, Yannick

2015-02-25

Vaccination is an important tool in the protection of poultry against avian influenza (AI). For field use, the overwhelming majority of AI vaccines produced are inactivated whole virus formulated into an oil emulsion. However, recombinant vectored vaccines are gaining use for their ability to induce protection against heterologous isolates and ability to overcome maternal antibody interference. In these studies, we compared protection of chickens provided by a turkey herpesvirus (HVT) vector vaccine expressing the hemagglutinin (HA) gene from a clade 2.2 H5N1 strain (A/swan/Hungary/4999/2006) against homologous H5N1 as well as heterologous H5N1 and H5N2 highly pathogenic (HP) AI challenge. The results demonstrated all vaccinated birds were protected from clinical signs of disease and mortality following homologous challenge. In addition, oral and cloacal swabs taken from challenged birds demonstrated that vaccinated birds had lower incidence and titers of viral shedding compared to sham-vaccinated birds. Following heterologous H5N1 or H5N2 HPAI challenge, 80-95% of birds receiving the HVT vector AI vaccine at day of age survived challenge with fewer birds shedding virus after challenge than sham vaccinated birds. In vitro cytotoxicity analysis demonstrated that splenic T lymphocytes from HVT-vector-AI vaccinated chickens recognized MHC-matched target cells infected with H5, as well as H6, H7, or H9 AI virus. Taken together, these studies provide support for the use of HVT vector vaccines expressing HA to protect poultry against multiple lineages of HPAI, and that both humoral and cellular immunity induced by live vaccines likely contributes to protection. Published by Elsevier Ltd.

A Novel Hemagglutinin with Antiproliferative Activity against Tumor Cells from the Hallucinogenic Mushroom Boletus speciosus

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Jian Sun

2014-01-01

Full Text Available Little was known about bioactive compounds from the hallucinogenic mushroom Boletus speciosus. In the present study, a hemagglutinin (BSH, B. speciosus hemagglutinin was isolated from its fruiting bodies and enzymatic properties were also tested. The chromatographic procedure utilized comprised anion exchange chromatography on Q-Sepharose, cation exchange chromatography on CM-Cellulose, cation exchange chromatography on SP-Sepharose, and gel filtration by FPLC on Superdex 75. The hemagglutinin was a homodimer which was estimated to be approximately 31 kDa in size. The activity of BSH was stable up to 60°C, while there was a precipitous drop in activity when the temperature was elevated to 70°C. BSH retained 25% hemagglutinating activity when exposed to 100 mM NaOH and 25 mM HCl. The activity was potently inhibited by 1.25 mM Hg2+ and slightly inhibited by Fe2+, Ca2+, and Pb2+. None of the sugars tested showed inhibition towards BSH. Its hemagglutinating activity towards human erythrocytes type A, type B, and type AB was higher than type O. The hemagglutinin showed antiproliferative activity towards hepatoma Hep G2 cells and mouse lymphocytic leukemia cells (L1210 in vitro, with IC50 of 4.7 μM and 7.0 μM, respectively. It also exhibited HIV-1 reverse transcriptase inhibitory activity with an IC50 of 7.1 μM.

Heterologous prime-boost immunization of Newcastle disease virus vectored vaccines protected broiler chickens against highly pathogenic avian influenza and Newcastle disease viruses.

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Kim, Shin-Hee; Samal, Siba K

2017-07-24

Avian Influenza virus (AIV) is an important pathogen for both human and animal health. There is a great need to develop a safe and effective vaccine for AI infections in the field. Live-attenuated Newcastle disease virus (NDV) vectored AI vaccines have shown to be effective, but preexisting antibodies to the vaccine vector can affect the protective efficacy of the vaccine in the field. To improve the efficacy of AI vaccine, we generated a novel vectored vaccine by using a chimeric NDV vector that is serologically distant from NDV. In this study, the protective efficacy of our vaccines was evaluated by using H5N1 highly pathogenic avian influenza virus (HPAIV) strain A/Vietnam/1203/2004, a prototype strain for vaccine development. The vaccine viruses were three chimeric NDVs expressing the hemagglutinin (HA) protein in combination with the neuraminidase (NA) protein, matrix 1 protein, or nonstructural 1 protein. Comparison of their protective efficacy between a single and prime-boost immunizations indicated that prime immunization of 1-day-old SPF chicks with our vaccine viruses followed by boosting with the conventional NDV vector strain LaSota expressing the HA protein provided complete protection of chickens against mortality, clinical signs and virus shedding. Further verification of our heterologous prime-boost immunization using commercial broiler chickens suggested that a sequential immunization of chickens with chimeric NDV vector expressing the HA and NA proteins following the boost with NDV vector expressing the HA protein can be a promising strategy for the field vaccination against HPAIVs and against highly virulent NDVs. Copyright © 2017 Elsevier Ltd. All rights reserved.

Establishment of an H6N2 Influenza Virus Lineage in Domestic Ducks in Southern China ▿ †

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Huang, K.; Bahl, J.; Fan, X. H.; Vijaykrishna, D.; Cheung, C. L.; Webby, R. J.; Webster, R. G.; Chen, H.; Smith, Gavin J. D.; Peiris, J. S. M.; Guan, Y.

2010-01-01

Multiple reassortment events between different subtypes of endemic avian influenza viruses have increased the genomic diversity of influenza viruses circulating in poultry in southern China. Gene exchange from the natural gene pool to poultry has contributed to this increase in genetic diversity. However, the role of domestic ducks as an interface between the natural gene pool and terrestrial poultry in the influenza virus ecosystem has not been fully characterized. Here we phylogenetically and antigenically analyzed 170 H6 viruses isolated from domestic ducks from 2000 to 2005 in southern China, which contains the largest population of domestic ducks in the world. Three distinct hemagglutinin lineages were identified. Group I contained the majority of isolates with a single internal gene complex and was endemic in domestic ducks in Guangdong from the late 1990s onward. Group II was derived from reassortment events in which the surface genes of group I viruses were replaced with novel H6 and N2 genes. Group III represented H6 viruses that undergo frequent reassortment with multiple virus subtypes from the natural gene pool. Surprisingly, H6 viruses endemic in domestic ducks and terrestrial poultry seldom reassort, but gene exchanges between viruses from domestic ducks and migratory ducks occurred throughout the surveillance period. These findings suggest that domestic ducks in southern China mediate the interaction of viruses between different gene pools and facilitate the generation of novel influenza virus variants circulating in poultry. PMID:20463062

Emergent lineages of mumps virus suggest the need for a polyvalent vaccine.

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May, Meghan; Rieder, Courtney A; Rowe, Rebecca J

2018-01-01

Mumps outbreaks among vaccinated patients have become increasingly common in recent years. While there are multiple conditions driving this re-emergence, convention has suggested that these outbreaks are associated with waning immunity rather than vaccine escape. Molecular evidence from both the ongoing American and Dutch outbreaks in conjunction with recent structural biology studies challenge this convention, and suggest that emergent lineages of mumps virus exhibit key differences in antigenic epitopes from the vaccine strain employed: Jeryl-Lynn 5. The American and Dutch 2016-2017 outbreak lineages were examined using computational biology through the lens of diversity in immunogenic epitopes. Findings are discussed and the laboratory evidence indicating neutralization of heterologous mumps strains by serum from vaccinated individuals is reviewed. Taken together, it is concluded that the number of heterologous epitopes occurring in mumps virus in conjunction with waning immunity is facilitating small outbreaks in vaccinated patients, and that consideration of a polyvalent mumps vaccine is warranted. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Replication and transmission of H9N2 influenza viruses in ferrets: evaluation of pandemic potential.

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Hongquan Wan

2008-08-01

Full Text Available H9N2 avian influenza A viruses are endemic in poultry of many Eurasian countries and have caused repeated human infections in Asia since 1998. To evaluate the potential threat of H9N2 viruses to humans, we investigated the replication and transmission efficiency of H9N2 viruses in the ferret model. Five wild-type (WT H9N2 viruses, isolated from different avian species from 1988 through 2003, were tested in vivo and found to replicate in ferrets. However these viruses achieved mild peak viral titers in nasal washes when compared to those observed with a human H3N2 virus. Two of these H9N2 viruses transmitted to direct contact ferrets, however no aerosol transmission was detected in the virus displaying the most efficient direct contact transmission. A leucine (Leu residue at amino acid position 226 in the hemagglutinin (HA receptor-binding site (RBS, responsible for human virus-like receptor specificity, was found to be important for the transmission of the H9N2 viruses in ferrets. In addition, an H9N2 avian-human reassortant virus, which contains the surface glycoprotein genes from an H9N2 virus and the six internal genes of a human H3N2 virus, showed enhanced replication and efficient transmission to direct contacts. Although no aerosol transmission was observed, the virus replicated in multiple respiratory tissues and induced clinical signs similar to those observed with the parental human H3N2 virus. Our results suggest that the establishment and prevalence of H9N2 viruses in poultry pose a significant threat for humans.

Reassortant H1N1 influenza virus vaccines protect pigs against pandemic H1N1 influenza virus and H1N2 swine influenza virus challenge.

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Yang, Huanliang; Chen, Yan; Shi, Jianzhong; Guo, Jing; Xin, Xiaoguang; Zhang, Jian; Wang, Dayan; Shu, Yuelong; Qiao, Chuanling; Chen, Hualan

2011-09-28

Influenza A (H1N1) virus has caused human influenza outbreaks in a worldwide pandemic since April 2009. Pigs have been found to be susceptible to this influenza virus under experimental and natural conditions, raising concern about their potential role in the pandemic spread of the virus. In this study, we generated a high-growth reassortant virus (SC/PR8) that contains the hemagglutinin (HA) and neuraminidase (NA) genes from a novel H1N1 isolate, A/Sichuan/1/2009 (SC/09), and six internal genes from A/Puerto Rico/8/34 (PR8) virus, by genetic reassortment. The immunogenicity and protective efficacy of this reassortant virus were evaluated at different doses in a challenge model using a homologous SC/09 or heterologous A/Swine/Guangdong/1/06(H1N2) virus (GD/06). Two doses of SC/PR8 virus vaccine elicited high-titer serum hemagglutination inhibiting (HI) antibodies specific for the 2009 H1N1 virus and conferred complete protection against challenge with either SC/09 or GD/06 virus, with reduced lung lesions and viral shedding in vaccine-inoculated animals compared with non-vaccinated control animals. These results indicated for the first time that a high-growth SC/PR8 reassortant H1N1 virus exhibits properties that are desirable to be a promising vaccine candidate for use in swine in the event of a pandemic H1N1 influenza. Copyright © 2011 Elsevier B.V. All rights reserved.

Serological characterization of guinea pigs infected with H3N2 human influenza or immunized with hemagglutinin protein

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Bushnell Ruth V

2010-08-01

Full Text Available Abstract Background Recent and previous studies have shown that guinea pigs can be infected with, and transmit, human influenza viruses. Therefore guinea pig may be a useful animal model for better understanding influenza infection and assessing vaccine strategies. To more fully characterize the model, antibody responses following either infection/re-infection with human influenza A/Wyoming/03/2003 H3N2 or immunization with its homologous recombinant hemagglutinin (HA protein were studied. Results Serological samples were collected and tested for anti-HA immunoglobulin by ELISA, antiviral antibodies by hemagglutination inhibition (HI, and recognition of linear epitopes by peptide scanning (PepScan. Animals inoculated with infectious virus demonstrated pronounced viral replication and subsequent serological conversion. Animals either immunized with the homologous HA antigen or infected, showed a relatively rapid rise in antibody titers to the HA glycoprotein in ELISA assays. Antiviral antibodies, measured by HI assay, were detectable after the second inoculation. PepScan data identified both previously recognized and newly defined linear epitopes. Conclusions Infection and/or recombinant HA immunization of guinea pigs with H3N2 Wyoming influenza virus resulted in a relatively rapid production of viral-specific antibody thus demonstrating the strong immunogenicity of the major viral structural proteins in this animal model for influenza infection. The sensitivity of the immune response supports the utility of the guinea pig as a useful animal model of influenza infection and immunization.

Influenza A Viruses of Human Origin in Swine, Brazil.

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Nelson, Martha I; Schaefer, Rejane; Gava, Danielle; Cantão, Maurício Egídio; Ciacci-Zanella, Janice Reis

2015-08-01

The evolutionary origins of the influenza A(H1N1)pdm09 virus that caused the first outbreak of the 2009 pandemic in Mexico remain unclear, highlighting the lack of swine surveillance in Latin American countries. Although Brazil has one of the largest swine populations in the world, influenza was not thought to be endemic in Brazil's swine until the major outbreaks of influenza A(H1N1)pdm09 in 2009. Through phylogenetic analysis of whole-genome sequences of influenza viruses of the H1N1, H1N2, and H3N2 subtypes collected in swine in Brazil during 2009-2012, we identified multiple previously uncharacterized influenza viruses of human seasonal H1N2 and H3N2 virus origin that have circulated undetected in swine for more than a decade. Viral diversity has further increased in Brazil through reassortment between co-circulating viruses, including A(H1N1)pdm09. The circulation of multiple divergent hemagglutinin lineages challenges the design of effective cross-protective vaccines and highlights the need for additional surveillance.

A low pathogenic H5N2 influenza virus isolated in Taiwan acquired high pathogenicity by consecutive passages in chickens.

OpenAIRE

Soda, Kosuke; Cheng, Ming-Chu; Yoshida, Hiromi; Endo, Mayumi; Lee, Shu-Hwae; Okamatsu, Masatoshi; Sakoda, Yoshihiro; Wang, Ching-Ho; Kida, Hiroshi

2011-01-01

H5N2 viruses were isolated from cloacal swab samples of apparently healthy chickens in Taiwan in 2003 and 2008 during surveillance of avian influenza. Each of the viruses was eradicated by stamping out. The official diagnosis report indicated that the Intravenous Pathogenicity Indexes (IVPIs) of the isolates were 0.00 and 0.89, respectively, indicating that these were low pathogenic strains, although the hemagglutinin of the strain isolated in 2008 (Taiwan08) had multibasic amino acid residue...

A single base-pair change in 2009 H1N1 hemagglutinin increases human receptor affinity and leads to efficient airborne viral transmission in ferrets.

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Akila Jayaraman

2011-03-01

Full Text Available The 2009 H1N1 influenza A virus continues to circulate among the human population as the predominant H1N1 subtype. Epidemiological studies and airborne transmission studies using the ferret model have shown that the transmission efficiency of 2009 H1N1 viruses is lower than that of previous seasonal strains and the 1918 pandemic H1N1 strain. We recently correlated this reduced transmission efficiency to the lower binding affinity of the 2009 H1N1 hemagglutinin (HA to α2→6 sialylated glycan receptors (human receptors. Here we report that a single point mutation (Ile219→Lys; a base pair change in the glycan receptor-binding site (RBS of a representative 2009 H1N1 influenza A virus, A/California/04/09 or CA04/09, quantitatively increases its human receptor-binding affinity. The increased human receptor-affinity is in the same range as that of the HA from highly transmissible seasonal and 1918 pandemic H1N1 viruses. Moreover, a 2009 H1N1 virus carrying this mutation in the RBS (generated using reverse genetics transmits efficiently in ferrets by respiratory droplets thereby reestablishing our previously observed correlation between human receptor-binding affinity and transmission efficiency. These findings are significant in the context of monitoring the evolution of the currently circulating 2009 H1N1 viruses.

Induction of cytotoxic T-cell responses by gene gun DNA vaccination with minigenes encoding influenza A virus HA and NP CTL-epitopes

DEFF Research Database (Denmark)

Fomsgaard, A; Nielsen, H V; Kirkby, N

1999-01-01

degree of controllability. We have examined the induction of murine CTL's by this approach using DNA plasmid minigene vaccines encoding known mouse K(k) minimal CTL epitopes (8 amino acids) from the influenza A virus hemagglutinin and nucleoprotein. We here report that such an approach is feasible...

Reoccurrence of H5Nx clade 2.3.4.4 highly pathogenic avian influenza viruses in wild birds during 2016

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The Asian-origin H5N1 A/goose/Guangdong/1/1996 (Gs/GD) lineage of high pathogenicity avian influenza viruses (HPAIV) has become widespread across four continents, affecting poultry, wild birds and humans. H5N1 HPAIV has evolved into multiple hemagglutinin (HA) genetic clades and reassorting with dif...

Carbohydrates of influenza virus. I. Glycopeptides derived from viral glycoproteins after labeling with radioactive sugars

International Nuclear Information System (INIS)

Schwarz, R.T.; Schmidt, M.F.G.; Anwer, U.; Klenk, H.D.

1977-01-01

The carbohydrate moiety of the influenza glycoproteins NA, HA 1 , and HA 2 were analyzed by labeling with radioactive sugars. Analysis of glycopeptides obtained after digestion with Pronase indicated that there are at least two different types of carbohydrate side chains. The side chain of type I is composed of glucosamine, mannose, galactose, and fucose. It is found on NA, HA 1 , and HA 2 . The side chain of type II contains a high amount of mannose and is found only on NA and HA 2 . The molecular weights of the corresponding glycopeptides obtained from virus grown in chicken ambryo cells are 2,600 for type I and 2,000 for type II. The glycoproteins of virus grown in MDBK cells have a higher molecular weight than those of virus grown in chicken embryo cells, and there is a corresponding difference in the molecular weights of the glycopeptides. Under conditions of partial inhibition of glycosylation, virus particles were isolated that contained hemagglutinin with reduced carbohydrate content. Glycopeptide analysis indicated that this reduction is due to the lack of whole carbohydrate side chains and not to the incorporation of incomplete ones. This observation suggests that glycosylation of the viral glycoproteins involves en bloc transfer of the core sugars to the polypeptide chains

Hemagglutinin Typing as an Aid in Identification of Biochemically Atypical Escherichia coli Strains

OpenAIRE

Crichton, Pamela B.; Ip, S. M.; Old, D. C.

1981-01-01

Tests for the presence of mannose-sensitive and mannose-resistant, eluting hemagglutinins and fimbriae were helpful in indicating whether biochemically atypical strains of the tribe Escherichieae might be escherichiae or shigellae.

Patterns of predicted T-cell epitopes associated with antigenic drift in influenza H3N2 hemagglutinin.

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E Jane Homan

Full Text Available Antigenic drift allowing escape from neutralizing antibodies is an important feature of transmission and survival of influenza viruses in host populations. Antigenic drift has been studied in particular detail for influenza A H3N2 and well defined antigenic clusters of this virus documented. We examine how host immunogenetics contributes to determination of the antibody spectrum, and hence the immune pressure bringing about antigenic drift. Using uTOPE™ bioinformatics analysis of predicted MHC binding, based on amino acid physical property principal components, we examined the binding affinity of all 9-mer and 15-mer peptides within the hemagglutinin 1 (HA1 of 447 H3N2 virus isolates to 35 MHC-I and 14 MHC-II alleles. We provide a comprehensive map of predicted MHC-I and MHC-II binding affinity for a broad array of HLA alleles for the H3N2 influenza HA1 protein. Each HLA allele exhibited a characteristic predicted binding pattern. Cluster analysis for each HLA allele shows that patterns based on predicted MHC binding mirror those described based on antibody binding. A single amino acid mutation or position displacement can result in a marked difference in MHC binding and hence potential T-helper function. We assessed the impact of individual amino acid changes in HA1 sequences between 10 virus isolates from 1968-2002, representative of antigenic clusters, to understand the changes in MHC binding over time. Gain and loss of predicted high affinity MHC-II binding sites with cluster transitions were documented. Predicted high affinity MHC-II binding sites were adjacent to antibody binding sites. We conclude that host MHC diversity may have a major determinant role in the antigenic drift of influenza A H3N2.

Full-Genome Sequence of a Reassortant H1N2 Influenza A Virus Isolated from Pigs in Brazil.

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Schmidt, Candice; Cibulski, Samuel Paulo; Muterle Varela, Ana Paula; Mengue Scheffer, Camila; Wendlant, Adrieli; Quoos Mayer, Fabiana; Lopes de Almeida, Laura; Franco, Ana Cláudia; Roehe, Paulo Michel

2014-12-18

In this study, the full-genome sequence of a reassortant H1N2 swine influenza virus is reported. The isolate has the hemagglutinin (HA) and neuraminidase (NA) genes from human lineage (H1-δ cluster and N2), and the internal genes (polymerase basic 1 [PB1], polymerase basic 2 [PB2], polymerase acidic [PA], nucleoprotein [NP], matrix [M], and nonstructural [NS]) are derived from human 2009 pandemic H1N1 (H1N1pdm09) virus. Copyright © 2014 Schmidt et al.

Microsurgeon Hirudo medicinalis as a Natural Bioshuttle for Spontaneous Mass Vaccination against Influenza A Virus

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Sajjad Khani

2011-09-01

Full Text Available Introduction: Recent report on existence of a stem region of hemagglutinin has arisen new hopes for vaccination of influenza A as it consist of a conserve fusion peptide shared across several influenza subtypes and can be targeted by human immune system. Methods: Given that traditional vaccination based on live attenuated viruses often fails to surpass such viral infection, a great deal of attention has been devoted to develop a safe yet efficient system for vaccination influenza A. We believe that a natural bioshuttle can be recruited for spontaneous mass vaccination. Results: Thus, here, we hypothesize that a bioengineered transgenic Hirudo medicinalis can be considered as an alive bioshuttle for in-situ vaccination against influenza A virus. By introducing the designated gene(s encoding the target fragment (i.e., stem region of hemagglutinin, this microsurgeon can act as a rapid microproducer of viral proteins for in-house mass vaccination through imparting the necessary proteins such as those, naturally presented in leech's saliva. Conclusion: This peculiar bioshuttle can be easily exploited as a medical modality choice at home resulting in greater patient compliance.

Integration of antibody by surface functionalization of graphite-encapsulated magnetic beads using ammonia gas plasma technology for capturing influenza A virus.

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Sakudo, Akikazu; Chou, Han; Ikuta, Kazuyoshi; Nagatsu, Masaaki

2015-05-01

Antibody-integrated magnetic beads have been functionalized for influenza A virus capture. First, ammonia plasma produced by a radio frequency power source was reacted with the surface of graphite-encapsulated magnetic beads to introduce amino groups. Anti-influenza A virus hemagglutinin antibody was then anchored by its surface sulfide groups to the amino groups on the beads via N-succinimidyl 3-(2-pyridyldithio) propionate. After incubation with influenza A virus, adsorption of the virus to the beads was confirmed by immunochromatography, polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), and inoculation of chicken embryonated eggs, indicating that virus infectivity is maintained and that the proposed method is useful for the enhanced detection and isolation of influenza A virus. Copyright © 2015 Elsevier Ltd. All rights reserved.

Chinese and global distribution of H9 subtype avian influenza viruses.

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Wenming Jiang

Full Text Available H9 subtype avian influenza viruses (AIVs are of significance in poultry and public health, but epidemiological studies about the viruses are scarce. In this study, phylogenetic relationships of the viruses were analyzed based on 1233 previously reported sequences and 745 novel sequences of the viral hemagglutinin gene. The novel sequences were obtained through large-scale surveys conducted in 2008-2011 in China. The results revealed distinct distributions of H9 subtype AIVs in different hosts, sites and regions in China and in the world: (1 the dominant lineage of H9 subtype AIVs in China in recent years is lineage h9.4.2.5 represented by A/chicken/Guangxi/55/2005; (2 the newly emerging lineage h9.4.2.6, represented by A/chicken/Guangdong/FZH/2011, has also become prevalent in China; (3 lineages h9.3.3, h9.4.1 and h9.4.2, represented by A/duck/Hokkaido/26/99, A/quail/Hong Kong/G1/97 and A/chicken/Hong Kong/G9/97, respectively, have become globally dominant in recent years; (4 lineages h9.4.1 and h9.4.2 are likely of more risk to public health than others; (5 different lineages have different transmission features and host tropisms. This study also provided novel experimental data which indicated that the Leu-234 (H9 numbering motif in the viral hemagglutinin gene is an important but not unique determinant in receptor-binding preference. This report provides a detailed and updated panoramic view of the epidemiological distributions of H9 subtype AIVs globally and in China, and sheds new insights for the prevention of infection in poultry and preparedness for a potential pandemic caused by the viruses.

Yeast expressed recombinant Hemagglutinin protein of Novel H1N1 elicits neutralising antibodies in rabbits and mice

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Athmaram TN

2011-11-01

Full Text Available Abstract Currently available vaccines for the pandemic Influenza A (H1N1 2009 produced in chicken eggs have serious impediments viz limited availability, risk of allergic reactions and the possible selection of sub-populations differing from the naturally occurring virus, whereas the cell culture derived vaccines are time consuming and may not meet the demands of rapid global vaccination required to combat the present/future pandemic. Hemagglutinin (HA based subunit vaccine for H1N1 requires the HA protein in glycosylated form, which is impossible with the commonly used bacterial expression platform. Additionally, bacterial derived protein requires extensive purification and refolding steps for vaccine applications. For these reasons an alternative heterologous system for rapid, easy and economical production of Hemagglutinin protein in its glycosylated form is required. The HA gene of novel H1N1 A/California/04/2009 was engineered for expression in Pichia pastoris as a soluble secreted protein. The full length HA- synthetic gene having α-secretory tag was integrated into P. pastoris genome through homologous recombination. The resultant Pichia clones having multiple copy integrants of the transgene expressed full length HA protein in the culture supernatant. The Recombinant yeast derived H1N1 HA protein elicited neutralising antibodies both in mice and rabbits. The sera from immunised animals also exhibited Hemagglutination Inhibition (HI activity. Considering the safety, reliability and also economic potential of Pichia expression platform, our preliminary data indicates the feasibility of using this system as an alternative for large-scale production of recombinant influenza HA protein in the face of influenza pandemic threat.

Detection of H5 Avian Influenza Viruses by Antigen-Capture Enzyme-Linked Immunosorbent Assay Using H5-Specific Monoclonal Antibody▿

OpenAIRE

He, Qigai; Velumani, Sumathy; Du, Qingyun; Lim, Chee Wee; Ng, Fook Kheong; Donis, Ruben; Kwang, Jimmy

2007-01-01

The unprecedented spread of highly pathogenic avian influenza virus subtype H5N1 in Asia and Europe is threatening animals and public health systems. Effective diagnosis and control management are needed to control the disease. To this end, we developed a panel of monoclonal antibodies (MAbs) against the H5N1 avian influenza virus (AIV) and implemented an antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) to detect the H5 viral antigen. Mice immunized with denatured hemagglutinin (H...

Structural basis for the development of avian virus capsids that display influenza virus proteins and induce protective immunity.

Science.gov (United States)

Pascual, Elena; Mata, Carlos P; Gómez-Blanco, Josué; Moreno, Noelia; Bárcena, Juan; Blanco, Esther; Rodríguez-Frandsen, Ariel; Nieto, Amelia; Carrascosa, José L; Castón, José R

2015-03-01

Bioengineering of viruses and virus-like particles (VLPs) is a well-established approach in the development of new and improved vaccines against viral and bacterial pathogens. We report here that the capsid of a major avian pathogen, infectious bursal disease virus (IBDV), can accommodate heterologous proteins to induce protective immunity. The structural units of the ~70-nm-diameter T=13 IBDV capsid are trimers of VP2, which is made as a precursor (pVP2). The pVP2 C-terminal domain has an amphipathic α helix that controls VP2 polymorphism. In the absence of the VP3 scaffolding protein, 466-residue pVP2 intermediates bearing this α helix assemble into genuine VLPs only when expressed with an N-terminal His6 tag (the HT-VP2-466 protein). HT-VP2-466 capsids are optimal for protein insertion, as they are large enough (cargo space, ~78,000 nm(3)) and are assembled from a single protein. We explored HT-VP2-466-based chimeric capsids initially using enhanced green fluorescent protein (EGFP). The VLP assembly yield was efficient when we coexpressed EGFP-HT-VP2-466 and HT-VP2-466 from two recombinant baculoviruses. The native EGFP structure (~240 copies/virion) was successfully inserted in a functional form, as VLPs were fluorescent, and three-dimensional cryo-electron microscopy showed that the EGFP molecules incorporated at the inner capsid surface. Immunization of mice with purified EGFP-VLPs elicited anti-EGFP antibodies. We also inserted hemagglutinin (HA) and matrix (M2) protein epitopes derived from the mouse-adapted A/PR/8/34 influenza virus and engineered several HA- and M2-derived chimeric capsids. Mice immunized with VLPs containing the HA stalk, an M2 fragment, or both antigens developed full protection against viral challenge. Virus-like particles (VLPs) are multimeric protein cages that mimic the infectious virus capsid and are potential candidates as nonliving vaccines that induce long-lasting protection. Chimeric VLPs can display or include foreign

Serum amyloid P component inhibits influenza A virus infections: in vitro and in vivo studies

DEFF Research Database (Denmark)

Horvath, A; Andersen, I; Junker, K

2001-01-01

Serum amyloid P component (SAP) binds in vitro Ca(2+)-dependently to several ligands including oligosaccharides with terminal mannose and galactose. We have earlier reported that SAP binds to human influenza A virus strains, inhibiting hemagglutinin (HA) activity and virus infectivity in vitro...... that SAP bound to HA trimers, monomers and HA1 and HA2 subunits of influenza A virus. Binding studies indicated that galactose, mannose and fucose moieties contributed to the SAP reacting site(s). Intranasal administration of human SAP to mice induced no demonstrable toxic reactions, and circulating...... on day 10 and these mice approached normal body weight, whereas control mice (one out of five surviving on day 10) died. The data provide evidence of the potential of intranasally administered SAP for prophylactic treatment of influenza A virus infections in humans....

Hemagglutinin Typing as an Aid in Identification of Biochemically Atypical Escherichia coli Strains

Science.gov (United States)

Crichton, Pamela B.; Ip, S. M.; Old, D. C.

1981-01-01

Tests for the presence of mannose-sensitive and mannose-resistant, eluting hemagglutinins and fimbriae were helpful in indicating whether biochemically atypical strains of the tribe Escherichieae might be escherichiae or shigellae. PMID:7334072

Acute Lung Injury Results from Innate Sensing of Viruses by an ER Stress Pathway

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Eike R. Hrincius

2015-06-01

Full Text Available Incursions of new pathogenic viruses into humans from animal reservoirs are occurring with alarming frequency. The molecular underpinnings of immune recognition, host responses, and pathogenesis in this setting are poorly understood. We studied pandemic influenza viruses to determine the mechanism by which increasing glycosylation during evolution of surface proteins facilitates diminished pathogenicity in adapted viruses. ER stress during infection with poorly glycosylated pandemic strains activated the unfolded protein response, leading to inflammation, acute lung injury, and mortality. Seasonal strains or viruses engineered to mimic adapted viruses displaying excess glycans on the hemagglutinin did not cause ER stress, allowing preservation of the lungs and survival. We propose that ER stress resulting from recognition of non-adapted viruses is utilized to discriminate “non-self” at the level of protein processing and to activate immune responses, with unintended consequences on pathogenesis. Understanding this mechanism should improve strategies for treating acute lung injury from zoonotic viral infections.

Molecular characterization of the receptor binding structure-activity relationships of influenza B virus hemagglutinin.

Science.gov (United States)

Carbone, V; Kim, H; Huang, J X; Baker, M A; Ong, C; Cooper, M A; Li, J; Rockman, S; Velkov, T

2013-01-01

Selectivity of α2,6-linked human-like receptors by B hemagglutinin (HA) is yet to be fully understood. This study integrates binding data with structure-recognition models to examine the impact of regional-specific sequence variations within the receptor-binding pocket on selectivity and structure activity relationships (SAR). The receptor-binding selectivity of influenza B HAs corresponding to either B/Victoria/2/1987 or the B/Yamagata/16/88 lineages was examined using surface plasmon resonance, solid-phase ELISA and gel-capture assays. Our SAR data showed that the presence of asialyl sugar units is the main determinant of receptor preference of α2,6 versus α2,3 receptor binding. Changes to the type of sialyl-glycan linkage present on receptors exhibit only a minor effect upon binding affinity. Homology-based structural models revealed that structural properties within the HA pocket, such as a glyco-conjugate at Asn194 on the 190-helix, sterically interfere with binding to avian receptor analogs by blocking the exit path of the asialyl sugars. Similarly, naturally occurring substitutions in the C-terminal region of the 190-helix and near the N-terminal end of the 140-loop narrows the horizontal borders of the binding pocket, which restricts access of the avian receptor analog LSTa. This study helps bridge the gap between ligand structure and receptor recognition for influenza B HA; and provides a consensus SAR model for the binding of human and avian receptor analogs to influenza B HA.

Targeting an Oncolytic Influenza A Virus to Tumor Tissue by Elastase

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Irina Kuznetsova

2017-12-01

Full Text Available Oncolytic viruses are currently established as a novel type of immunotherapy. The challenge is to safely target oncolytic viruses to tumors. Previously, we have generated influenza A viruses (IAVs containing deletions in the viral interferon antagonist. Those deletions have attenuated the virus in normal tissue but allowed replication in tumor cells. IAV entry is mediated by hemagglutinin (HA, which needs to be activated by a serine protease, for example, through trypsin. To further target the IAV to tumors, we have changed the trypsin cleavage site to an elastase cleavage site. We chose this cleavage site because elastase is expressed in the tumor microenvironment. Moreover, the exchange of the cleavage site previously has been shown to attenuate viral growth in lungs. Newly generated elastase-activated influenza viruses (AE viruses grew to similar titers in tumor cells as the trypsin-activated counterparts (AT viruses. Intratumoral injection of AE viruses into syngeneic B16f1 melanoma-derived tumors in mice reduced tumor growth similar to AT viruses and had a better therapeutic effect in heterologous human PANC-1-derived tumors. Therefore, the introduction of the attenuation marker “elastase cleavage site” in viral HA allows for safe, effective oncolytic virus therapy.

Influenza A and B Virus Intertypic Reassortment through Compatible Viral Packaging Signals

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Baker, Steven F.; Nogales, Aitor; Finch, Courtney; Tuffy, Kevin M.; Domm, William; Perez, Daniel R.; Topham, David J.

2014-01-01

ABSTRACT Influenza A and B viruses cocirculate in humans and together cause disease and seasonal epidemics. These two types of influenza viruses are evolutionarily divergent, and exchange of genetic segments inside coinfected cells occurs frequently within types but never between influenza A and B viruses. Possible mechanisms inhibiting the intertypic reassortment of genetic segments could be due to incompatible protein functions of segment homologs, a lack of processing of heterotypic segments by influenza virus RNA-dependent RNA polymerase, an inhibitory effect of viral proteins on heterotypic virus function, or an inability to specifically incorporate heterotypic segments into budding virions. Here, we demonstrate that the full-length hemagglutinin (HA) of prototype influenza B viruses can complement the function of multiple influenza A viruses. We show that viral noncoding regions were sufficient to drive gene expression for either type A or B influenza virus with its cognate or heterotypic polymerase. The native influenza B virus HA segment could not be incorporated into influenza A virus virions. However, by adding the influenza A virus packaging signals to full-length influenza B virus glycoproteins, we rescued influenza A viruses that possessed HA, NA, or both HA and NA of influenza B virus. Furthermore, we show that, similar to single-cycle infectious influenza A virus, influenza B virus cannot incorporate heterotypic transgenes due to packaging signal incompatibilities. Altogether, these results demonstrate that the lack of influenza A and B virus reassortants can be attributed at least in part to incompatibilities in the virus-specific packaging signals required for effective segment incorporation into nascent virions. IMPORTANCE Reassortment of influenza A or B viruses provides an evolutionary strategy leading to unique genotypes, which can spawn influenza A viruses with pandemic potential. However, the mechanism preventing intertypic reassortment or

Filamentous hemagglutinin of Bordetella pertussis: a key adhesin with immunomodulatory properties?

Czech Academy of Sciences Publication Activity Database

Romero, Rodrigo, Villarino; Osička, Radim; Šebo, Peter

2014-01-01

Roč. 9, č. 12 (2014), s. 1339-1360 ISSN 1746-0913 R&D Projects: GA ČR(CZ) P302/11/0580; GA ČR(CZ) GA13-14547S Institutional support: RVO:61388971 Keywords : Bordetella * adhesion * integrins * filamentous hemagglutinin Subject RIV: EE - Microbiology, Virology Impact factor: 4.275, year: 2014

Highly pathogenic avian influenza virus subtype H5N1 in mute swans (Cygnus olor) in Central Bosnia.

Science.gov (United States)

Goletić, Teufik; Gagić, Abdulah; Residbegović, Emina; Kustura, Aida; Kavazović, Aida; Savić, Vladimir; Harder, Timm; Starick, Elke; Prasović, Senad

2010-03-01

In order to determine the actual prevalence of avian influenza viruses (AIVs) in wild birds in Bosnia and Herzegovina, extensive surveillance was carried out between October 2005 and April 2006. A total of 394 samples representing 41 bird species were examined for the presence of influenza A virus using virus isolation in embryonated chicken eggs, PCR, and nucleotide sequencing. AIV subtype H5N1 was detected in two mute swans (Cygnus olor). The isolates were determined to be highly pathogenic avian influenza (HPAI) virus and the hemagglutinin sequence was closely similar to A/Cygnus olor/Astrakhan/ Ast05-2-10/2005 (H5N1). This is the first report of HPAI subtype H5N1 in Bosnia and Herzegovina.

Novel H7N2 and H5N6 Avian Influenza A Viruses in Sentinel Chickens: A Sentinel Chicken Surveillance Study

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Teng Zhao

2016-11-01

Full Text Available In 2014, surveillance of sentinel chicken for avian influenza virus was conducted in aquatic bird habitat near Wuxi City, Jiangsu Province, China. Two H7N2, one H5N6, and two H9N2 viruses were isolated. Sequence analysis revealed that the H7N2 virus is a novel reassortant of H7N9 and H9N2 viruses and H5N6 virus is a reassortant of H5N1 clade 2.3.4 and H6N6 viruses. Substitutions V186 and L226 (H3 numbering in the hemagglutinin (HA gene protein was found in two H7N2 viruses but not in the H5N6 virus. Two A138 and A160 mutations were identified in the HA gene protein of all three viruses but a P128 mutation was only in the H5N6 virus. A deletion of three and eleven amino acids in the neuraminidase stalk region was found in two H7N2 and H5N6 viruses, respectively. Moreover, a mutation of N31 in M2 protein was observed in both two H7N2 viruses. High similarity of these isolated viruses to viruses previously identified among poultry and humans, suggests that peridomestic aquatic birds may play a role in sustaining novel virus transmission. Therefore, continued surveillance is needed to monitor these avian influenza viruses in wild bird and domestic poultry that may pose a threat to poultry and human health.

pH-Controlled Two-Step Uncoating of Influenza Virus

Science.gov (United States)

Li, Sai; Sieben, Christian; Ludwig, Kai; Höfer, Chris T.; Chiantia, Salvatore; Herrmann, Andreas; Eghiaian, Frederic; Schaap, Iwan A.T.

2014-01-01

Upon endocytosis in its cellular host, influenza A virus transits via early to late endosomes. To efficiently release its genome, the composite viral shell must undergo significant structural rearrangement, but the exact sequence of events leading to viral uncoating remains largely speculative. In addition, no change in viral structure has ever been identified at the level of early endosomes, raising a question about their role. We performed AFM indentation on single viruses in conjunction with cellular assays under conditions that mimicked gradual acidification from early to late endosomes. We found that the release of the influenza genome requires sequential exposure to the pH of both early and late endosomes, with each step corresponding to changes in the virus mechanical response. Step 1 (pH 7.5–6) involves a modification of both hemagglutinin and the viral lumen and is reversible, whereas Step 2 (pH pH step or blocking the envelope proton channel M2 precludes proper genome release and efficient infection, illustrating the importance of viral lumen acidification during the early endosomal residence for influenza virus infection. PMID:24703306

Cross-Species Infectivity of H3N8 Influenza Virus in an Experimental Infection in Swine.

Science.gov (United States)

Solórzano, Alicia; Foni, Emanuela; Córdoba, Lorena; Baratelli, Massimiliano; Razzuoli, Elisabetta; Bilato, Dania; Martín del Burgo, María Ángeles; Perlin, David S; Martínez, Jorge; Martínez-Orellana, Pamela; Fraile, Lorenzo; Chiapponi, Chiara; Amadori, Massimo; del Real, Gustavo; Montoya, María

2015-11-01

Avian influenza A viruses have gained increasing attention due to their ability to cross the species barrier and cause severe disease in humans and other mammal species as pigs. H3 and particularly H3N8 viruses, are highly adaptive since they are found in multiple avian and mammal hosts. H3N8 viruses have not been isolated yet from humans; however, a recent report showed that equine influenza A viruses (IAVs) can be isolated from pigs, although an established infection has not been observed thus far in this host. To gain insight into the possibility of H3N8 avian IAVs to cross the species barrier into pigs, in vitro experiments and an experimental infection in pigs with four H3N8 viruses from different origins (equine, canine, avian, and seal) were performed. As a positive control, an H3N2 swine influenza virus A was used. Although equine and canine viruses hardly replicated in the respiratory systems of pigs, avian and seal viruses replicated substantially and caused detectable lesions in inoculated pigs without previous adaptation. Interestingly, antibodies against hemagglutinin could not be detected after infection by hemagglutination inhibition (HAI) test with avian and seal viruses. This phenomenon was observed not only in pigs but also in mice immunized with the same virus strains. Our data indicated that H3N8 IAVs from wild aquatic birds have the potential to cross the species barrier and establish successful infections in pigs that might spread unnoticed using the HAI test as diagnostic tool. Although natural infection of humans with an avian H3N8 influenza A virus has not yet been reported, this influenza A virus subtype has already crossed the species barrier. Therefore, we have examined the potential of H3N8 from canine, equine, avian, and seal origin to productively infect pigs. Our results demonstrated that avian and seal viruses replicated substantially and caused detectable lesions in inoculated pigs without previous adaptation. Surprisingly, we

Influenza A Viruses of Human Origin in Swine, Brazil

Science.gov (United States)

Schaefer, Rejane; Gava, Danielle; Cantão, Maurício Egídio; Ciacci-Zanella, Janice Reis

2015-01-01

The evolutionary origins of the influenza A(H1N1)pdm09 virus that caused the first outbreak of the 2009 pandemic in Mexico remain unclear, highlighting the lack of swine surveillance in Latin American countries. Although Brazil has one of the largest swine populations in the world, influenza was not thought to be endemic in Brazil’s swine until the major outbreaks of influenza A(H1N1)pdm09 in 2009. Through phylogenetic analysis of whole-genome sequences of influenza viruses of the H1N1, H1N2, and H3N2 subtypes collected in swine in Brazil during 2009–2012, we identified multiple previously uncharacterized influenza viruses of human seasonal H1N2 and H3N2 virus origin that have circulated undetected in swine for more than a decade. Viral diversity has further increased in Brazil through reassortment between co-circulating viruses, including A(H1N1)pdm09. The circulation of multiple divergent hemagglutinin lineages challenges the design of effective cross-protective vaccines and highlights the need for additional surveillance. PMID:26196759

[Activating effect of adrenaline, prednisolone and vincristine in the late periods of tick-borne encephalitis virus persistence].

Science.gov (United States)

Frolova, T V; Pogodina, V V

1984-01-01

The activating effect of adrenalin (A), prednisolone (P), and vincristine (V) on persistent infection caused by subcutaneous inoculation of Syrian hamsters with the Vasilchenko and B-383 strains of tick-borne encephalitis virus (TBE) was studied. The drugs were administered once, twice, or three times 250-270 days after virus inoculation. Complement-fixing antigen was found in the organs of the infected animals given no A, P, or V; in the organ explants synthesis of hemagglutinin was observed but no infectious virus could be isolated. After treatment of the infected hamsters with A, P, or V organ explants yielded TBE virus strains which showed either high or low virulence for white mice. The activated TBE virus strains were obtained from explants of hamster brains and spleens but not liver. V produced the most marked activating effect, A the least.

Antigenic and Molecular Characterization of Avian Influenza A(H9N2) Viruses, Bangladesh

Science.gov (United States)

Shanmuganatham, Karthik; Feeroz, Mohammed M.; Jones-Engel, Lisa; Smith, Gavin J.D.; Fourment, Mathieu; Walker, David; McClenaghan, Laura; Alam, S.M. Rabiul; Hasan, M. Kamrul; Seiler, Patrick; Franks, John; Danner, Angie; Barman, Subrata; McKenzie, Pamela; Krauss, Scott; Webby, Richard J.

2013-01-01

Human infection with avian influenza A(H9N2) virus was identified in Bangladesh in 2011. Surveillance for influenza viruses in apparently healthy poultry in live-bird markets in Bangladesh during 2008–2011 showed that subtype H9N2 viruses are isolated year-round, whereas highly pathogenic subtype H5N1 viruses are co-isolated with subtype H9N2 primarily during the winter months. Phylogenetic analysis of the subtype H9N2 viruses showed that they are reassortants possessing 3 gene segments related to subtype H7N3; the remaining gene segments were from the subtype H9N2 G1 clade. We detected no reassortment with subtype H5N1 viruses. Serologic analyses of subtype H9N2 viruses from chickens revealed antigenic conservation, whereas analyses of viruses from quail showed antigenic drift. Molecular analysis showed that multiple mammalian-specific mutations have become fixed in the subtype H9N2 viruses, including changes in the hemagglutinin, matrix, and polymerase proteins. Our results indicate that these viruses could mutate to be transmissible from birds to mammals, including humans. PMID:23968540

Transient expression of hemagglutinin antigen from low pathogenic avian influenza A (H7N7 in Nicotiana benthamiana.

Directory of Open Access Journals (Sweden)

Selvaraju Kanagarajan

Full Text Available The influenza A virus is of global concern for the poultry industry, especially the H5 and H7 subtypes as they have the potential to become highly pathogenic for poultry. In this study, the hemagglutinin (HA of a low pathogenic avian influenza virus of the H7N7 subtype isolated from a Swedish mallard Anas platyrhynchos was sequenced, characterized and transiently expressed in Nicotiana benthamiana. Recently, plant expression systems have gained interest as an alternative for the production of vaccine antigens. To examine the possibility of expressing the HA protein in N. benthamiana, a cDNA fragment encoding the HA gene was synthesized de novo, modified with a Kozak sequence, a PR1a signal peptide, a C-terminal hexahistidine (6×His tag, and an endoplasmic retention signal (SEKDEL. The construct was cloned into a Cowpea mosaic virus (CPMV-based vector (pEAQ-HT and the resulting pEAQ-HT-HA plasmid, along with a vector (pJL3:p19 containing the viral gene-silencing suppressor p19 from Tomato bushy stunt virus, was agro-infiltrated into N. benthamiana. The highest gene expression of recombinant plant-produced, uncleaved HA (rHA0, as measured by quantitative real-time PCR was detected at 6 days post infiltration (dpi. Guided by the gene expression profile, rHA0 protein was extracted at 6 dpi and subsequently purified utilizing the 6×His tag and immobilized metal ion adsorption chromatography. The yield was 0.2 g purified protein per kg fresh weight of leaves. Further molecular characterizations showed that the purified rHA0 protein was N-glycosylated and its identity confirmed by liquid chromatography-tandem mass spectrometry. In addition, the purified rHA0 exhibited hemagglutination and hemagglutination inhibition activity indicating that the rHA0 shares structural and functional properties with native HA protein of H7 influenza virus. Our results indicate that rHA0 maintained its native antigenicity and specificity, providing a good source of

Genetic analysis of Asian measles virus strains--new endemic genotype in Nepal.

Science.gov (United States)

Truong, A T; Mulders, M N; Gautam, D C; Ammerlaan, W; de Swart, R L; King, C C; Osterhaus, A D; Muller, C P

2001-07-01

In many parts of Asia measles virus (MV) continues to be endemic. However, little is known about the genetic characteristics of viruses circulating on this continent. This study reports the molecular epidemiological analysis based on the entire nucleocapsid (N) and hemagglutinin (H) genes of the first isolates from Nepal and Taiwan, as well as of recent MV strains from India, Indonesia, and China. Four isolates collected in various regions in Nepal during 1999 belonged to a new genotype, tentatively called D8. Another Nepalese isolate and one from India belonged to genotype D4. The diversity of the Nepalese strains indicated that measles continues to be endemic in this country. The isolate from Taiwan grouped with D3 viruses and one Chinese strain isolated in The Netherlands was assigned to the previously described clade H, known to be endemic in Mainland China. Molecular characterization emerges as an important tool for monitoring virus endemicity and vaccination efforts.

Complete Genome Sequence of a Novel Reassortant Avian Influenza H1N2 Virus Isolated from a Domestic Sparrow in 2012

OpenAIRE

Xie, Zhixun; Guo, Jie; Xie, Liji; Liu, Jiabo; Pang, Yaoshan; Deng, Xianwen; Xie, Zhiqin; Fan, Qing; Luo, Sisi

2013-01-01

We report here the complete genome sequence of a novel H1N2 avian influenza virus strain, A/Sparrow /Guangxi/GXs-1/2012 (H1N2), isolated from a sparrow in the Guangxi Province of southern China in 2012. All of the 8 gene segments (hemagglutinin [HA], nucleoprotein [NP], matrix [M], polymerase basic 2 [PB2], neuraminidase [NA], polymerase acidic [PA], polymerase basic 1 [PB1], and nonstructural [NS] genes) of this natural recombinant virus are attributed to the Eurasian lineage, and phylogenet...

Vaccination with recombinant RNA replicon particles protects chickens from H5N1 highly pathogenic avian influenza virus.

Directory of Open Access Journals (Sweden)

Stefan J Halbherr

Full Text Available Highly pathogenic avian influenza viruses (HPAIV of subtype H5N1 not only cause a devastating disease in domestic chickens and turkeys but also pose a continuous threat to public health. In some countries, H5N1 viruses continue to circulate and evolve into new clades and subclades. The rapid evolution of these viruses represents a problem for virus diagnosis and control. In this work, recombinant vesicular stomatitis virus (VSV vectors expressing HA of subtype H5 were generated. To comply with biosafety issues the G gene was deleted from the VSV genome. The resulting vaccine vector VSV*ΔG(HA was propagated on helper cells providing the VSV G protein in trans. Vaccination of chickens with a single intramuscular dose of 2×10⁸ infectious replicon particles without adjuvant conferred complete protection from lethal H5N1 infection. Subsequent application of the same vaccine strongly boosted the humoral immune response and completely prevented shedding of challenge virus and transmission to sentinel birds. The vaccine allowed serological differentiation of infected from vaccinated animals (DIVA by employing a commercially available ELISA. Immunized chickens produced antibodies with neutralizing activity against multiple H5 viruses representing clades 1, 2.2, 2.5, and low-pathogenic avian influenza viruses (classical clade. Studies using chimeric H1/H5 hemagglutinins showed that the neutralizing activity was predominantly directed against the globular head domain. In summary, these results suggest that VSV replicon particles are safe and potent DIVA vaccines that may help to control avian influenza viruses in domestic poultry.

Association of an alphasatellite with tomato yellow leaf curl virus and ageratum yellow vein virus in Japan is suggestive of a recent introduction.

Science.gov (United States)

Shahid, Muhammad Shafiq; Ikegami, Masato; Waheed, Abdul; Briddon, Rob W; Natsuaki, Keiko T

2014-01-14

Samples were collected in 2011 from tomato plants exhibiting typical tomato leaf curl disease symptoms in the vicinity of Komae, Japan. PCR mediated amplification, cloning and sequencing of all begomovirus components from two plants from different fields showed the plants to be infected by Tomato yellow leaf curl virus (TYLCV) and Ageratum yellow vein virus (AYVV). Both viruses have previously been shown to be present in Japan, although this is the first identification of AYVV on mainland Japan; the virus previously having been shown to be present on the Okinawa Islands. The plant harboring AYVV was also shown to contain the betasatellite Tomato leaf curl Java betasatellite (ToLCJaB), a satellite not previously shown to be present in Japan. No betasatellite was associated with the TYLCV infected tomato plants analyzed here, consistent with earlier findings for this virus in Japan. Surprisingly both plants were also found to harbor an alphasatellite; no alphasatellites having previously been reported from Japan. The alphasatellite associated with both viruses was shown to be Sida yellow vein China alphasatellite which has previously only been identified in the Yunnan Province of China and Nepal. The results suggest that further begomoviruses, and their associated satellites, are being introduced to Japan. The significance of these findings is discussed.

Association of an Alphasatellite with Tomato Yellow Leaf Curl Virus and Ageratum Yellow Vein Virus in Japan Is Suggestive of a Recent Introduction

Directory of Open Access Journals (Sweden)

Muhammad Shafiq Shahid

2014-01-01

Full Text Available Samples were collected in 2011 from tomato plants exhibiting typical tomato leaf curl disease symptoms in the vicinity of Komae, Japan. PCR mediated amplification, cloning and sequencing of all begomovirus components from two plants from different fields showed the plants to be infected by Tomato yellow leaf curl virus (TYLCV and Ageratum yellow vein virus (AYVV. Both viruses have previously been shown to be present in Japan, although this is the first identification of AYVV on mainland Japan; the virus previously having been shown to be present on the Okinawa Islands. The plant harboring AYVV was also shown to contain the betasatellite Tomato leaf curl Java betasatellite (ToLCJaB, a satellite not previously shown to be present in Japan. No betasatellite was associated with the TYLCV infected tomato plants analyzed here, consistent with earlier findings for this virus in Japan. Surprisingly both plants were also found to harbor an alphasatellite; no alphasatellites having previously been reported from Japan. The alphasatellite associated with both viruses was shown to be Sida yellow vein China alphasatellite which has previously only been identified in the Yunnan Province of China and Nepal. The results suggest that further begomoviruses, and their associated satellites, are being introduced to Japan. The significance of these findings is discussed.

Strategies and challenges for eliciting immunity against avian influenza virus in birds.

Science.gov (United States)

Swayne, David E; Kapczynski, Darrell

2008-10-01

Vaccines and vaccination have emerged during the past two decades as essential tools in avian influenza (AI) control for poultry, because they increase resistance to infection, prevent illness and death, reduce virus replication and shed from respiratory and alimentary tracts, and reduce virus transmission to birds and mammals, including humans. Such protection in birds is primarily mediated by homosubtypic humoral immunity against the hemagglutinin protein, but cell-mediated and innate immunity contribute to protection in some bird species. The immune response to the neuraminidase protein can contribute to protection, but immunity to the viral internal proteins is generally not protective. Although, some preliminary studies with M2e protein in chickens suggest partial protection may be achievable. Historically, the H5 subtype AI vaccines have demonstrated broad homosubtypic protection, primarily against H5 high-pathogenicity (HP) AI viruses isolated in the early stages of outbreaks. However, as H5 viruses have become endemic and outbreaks prolonged, some drift variants with resistance to earlier H5 AI vaccines have emerged in Central America, China, Egypt, and Indonesia. How widespread such drift variants are will remain unknown until more detailed genetic and antigenic analyses are conducted on field isolates. Future vaccines will utilize biotechnology to produce new AI vaccine seed strains using HA genes more closely matching circulating field viruses. In addition, newer technologies for AI vaccines will improve vaccine coverage by using mass application technologies for example by drinking water, by spray, or via injection in ovo or at the hatchery.

A chimeric measles virus with a lentiviral envelope replicates exclusively in CD4+/CCR5+ cells

International Nuclear Information System (INIS)

Mourez, Thomas; Mesel-Lemoine, Mariana; Combredet, Chantal; Najburg, Valerie; Cayet, Nadege; Tangy, Frederic

2011-01-01

We generated a replicating chimeric measles virus in which the hemagglutinin and fusion surface glycoproteins were replaced with the gp160 envelope glycoprotein of simian immunodeficiency virus (SIVmac239). Based on a previously cloned live-attenuated Schwarz vaccine strain of measles virus (MV), this chimera was rescued at high titers using reverse genetics in CD4+ target cells. Cytopathic effect consisted in the presence of large cell aggregates evolving to form syncytia, as observed during SIV infection. The morphology of the chimeric virus was identical to that of the parent MV particles. The presence of SIV gp160 as the only envelope protein on chimeric particles surface altered the cell tropism of the new virus from CD46+ to CD4+ cells. Used as an HIV candidate vaccine, this MV/SIVenv chimeric virus would mimic transient HIV-like infection, benefiting both from HIV-like tropism and the capacity of MV to replicate in dendritic cells, macrophages and lymphocytes.

A Role for the Mannose-Sensitive Hemagglutinin in Biofilm Formation by Vibrio cholerae El Tor

Science.gov (United States)

Watnick, Paula I.; Fullner, Karla Jean; Kolter, Roberto

1999-01-01

While much has been learned regarding the genetic basis of host-pathogen interactions, less is known about the molecular basis of a pathogen’s survival in the environment. Biofilm formation on abiotic surfaces represents a survival strategy utilized by many microbes. Here it is shown that Vibrio cholerae El Tor does not use the virulence-associated toxin-coregulated pilus to form biofilms on borosilicate but rather uses the mannose-sensitive hemagglutinin (MSHA) pilus, which plays no role in pathogenicity. In contrast, attachment of V. cholerae to chitin is shown to be independent of the MSHA pilus, suggesting divergent pathways for biofilm formation on nutritive and nonnutritive abiotic surfaces. PMID:10348878

In Silico Prediction and Experimental Confirmation of HA Residues Conferring Enhanced Human Receptor Specificity of H5N1 Influenza A Viruses

Science.gov (United States)

Schmier, Sonja; Mostafa, Ahmed; Haarmann, Thomas; Bannert, Norbert; Ziebuhr, John; Veljkovic, Veljko; Dietrich, Ursula; Pleschka, Stephan

2015-06-01

Newly emerging influenza A viruses (IAV) pose a major threat to human health by causing seasonal epidemics and/or pandemics, the latter often facilitated by the lack of pre-existing immunity in the general population. Early recognition of candidate pandemic influenza viruses (CPIV) is of crucial importance for restricting virus transmission and developing appropriate therapeutic and prophylactic strategies including effective vaccines. Often, the pandemic potential of newly emerging IAV is only fully recognized once the virus starts to spread efficiently causing serious disease in humans. Here, we used a novel phylogenetic algorithm based on the informational spectrum method (ISM) to identify potential CPIV by predicting mutations in the viral hemagglutinin (HA) gene that are likely to (differentially) affect critical interactions between the HA protein and target cells from bird and human origin, respectively. Predictions were subsequently validated by generating pseudotyped retrovirus particles and genetically engineered IAV containing these mutations and characterizing potential effects on virus entry and replication in cells expressing human and avian IAV receptors, respectively. Our data suggest that the ISM-based algorithm is suitable to identify CPIV among IAV strains that are circulating in animal hosts and thus may be a new tool for assessing pandemic risks associated with specific strains.

CANINE DISTEMPER VIRUS IN A WILD FAR EASTERN LEOPARD ( PANTHERA PARDUS ORIENTALIS).

Science.gov (United States)

Sulikhan, Nadezhda S; Gilbert, Martin; Blidchenko, Ekaterina Yu; Naidenko, Sergei V; Ivanchuk, Galina V; Gorpenchenko, Tatiana Yu; Alshinetskiy, Mikhail V; Shevtsova, Elena I; Goodrich, John M; Lewis, John C M; Goncharuk, Mikhail S; Uphyrkina, Olga V; Rozhnov, Vyatcheslav V; Shedko, Sergey V; McAloose, Denise; Miquelle, Dale G

2018-01-01

The critically endangered population of Far Eastern leopards ( Panthera pardus orientalis) may number as few as 60 individuals and is at risk from stochastic processes such as infectious disease. During May 2015, a case of canine distemper virus (CDV) was diagnosed in a wild leopard exhibiting severe neurologic disease in the Russian territory of Primorskii Krai. Amplified sequences of the CDV hemagglutinin gene and phosphoprotein gene aligned within the Arctic-like clade of CDV, which includes viruses from elsewhere in Russia, China, Europe, and North America. Histologic examination of cerebral tissue revealed perivascular lymphoid cuffing and demyelination of the white matter consistent with CDV infection. Neutralizing antibodies against CDV were detected in archived serum from two wild Far Eastern leopards sampled during 1993-94, confirming previous exposure in the population. This leopard population is likely too small to maintain circulation of CDV, suggesting that infections arise from spillover from more-abundant domestic or wild carnivore reservoirs. Increasing the population size and establishment of additional populations of leopards would be important steps toward securing the future of this subspecies and reducing the risk posed by future outbreaks of CDV or other infectious diseases.

Characterization of influenza A(H1N1)pdm09 viruses isolated from Nepalese and Indian outbreak patients in early 2015.

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Nakamura, Kazuya; Shirakura, Masayuki; Fujisaki, Seiichiro; Kishida, Noriko; Burke, David F; Smith, Derek J; Kuwahara, Tomoko; Takashita, Emi; Takayama, Ikuyo; Nakauchi, Mina; Chadha, Mandeep; Potdar, Varsha; Bhushan, Arvind; Upadhyay, Bishnu Prasad; Shakya, Geeta; Odagiri, Takato; Kageyama, Tsutomu; Watanabe, Shinji

2017-09-01

We characterized influenza A(H1N1)pdm09 isolates from large-scale outbreaks that occurred in Nepal and India in early 2015. Although no specific viral features, which may have caused the outbreaks, were identified, an S84N substitution in hemagglutinin was frequently observed. Chronological phylogenetic analysis revealed that these Nepalese and Indian viruses possessing the S84N substitution constitute potential ancestors of the novel genetic subclade 6B.1 virus that spread globally in the following (2015/16) influenza season. Thus, active surveillance of circulating influenza viruses in the Southern Asia region, including Nepal and India, would be beneficial for detecting novel variant viruses prior to their worldwide spread. © 2017 The Authors. Influenza and Other Respiratory Viruses Published by John Wiley & Sons Ltd.

Evolution of an Eurasian avian-like influenza virus in naïve and vaccinated pigs.

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Pablo R Murcia

Full Text Available Influenza viruses are characterized by an ability to cross species boundaries and evade host immunity, sometimes with devastating consequences. The 2009 pandemic of H1N1 influenza A virus highlights the importance of pigs in influenza emergence, particularly as intermediate hosts by which avian viruses adapt to mammals before emerging in humans. Although segment reassortment has commonly been associated with influenza emergence, an expanded host-range is also likely to be associated with the accumulation of specific beneficial point mutations. To better understand the mechanisms that shape the genetic diversity of avian-like viruses in pigs, we studied the evolutionary dynamics of an Eurasian Avian-like swine influenza virus (EA-SIV in naïve and vaccinated pigs linked by natural transmission. We analyzed multiple clones of the hemagglutinin 1 (HA1 gene derived from consecutive daily viral populations. Strikingly, we observed both transient and fixed changes in the consensus sequence along the transmission chain. Hence, the mutational spectrum of intra-host EA-SIV populations is highly dynamic and allele fixation can occur with extreme rapidity. In addition, mutations that could potentially alter host-range and antigenicity were transmitted between animals and mixed infections were commonplace, even in vaccinated pigs. Finally, we repeatedly detected distinct stop codons in virus samples from co-housed pigs, suggesting that they persisted within hosts and were transmitted among them. This implies that mutations that reduce viral fitness in one host, but which could lead to fitness benefits in a novel host, can circulate at low frequencies.

Heterosubtypic immunity to influenza A virus infections in mallards may explain existence of multiple virus subtypes.

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Neus Latorre-Margalef

Full Text Available Wild birds, particularly duck species, are the main reservoir of influenza A virus (IAV in nature. However, knowledge of IAV infection dynamics in the wild bird reservoir, and the development of immune responses, are essentially absent. Importantly, a detailed understanding of how subtype diversity is generated and maintained is lacking. To address this, 18,679 samples from 7728 Mallard ducks captured between 2002 and 2009 at a single stopover site in Sweden were screened for IAV infections, and the resulting 1081 virus isolates were analyzed for patterns of immunity. We found support for development of homosubtypic hemagglutinin (HA immunity during the peak of IAV infections in the fall. Moreover, re-infections with the same HA subtype and related prevalent HA subtypes were uncommon, suggesting the development of natural homosubtypic and heterosubtypic immunity (p-value = 0.02. Heterosubtypic immunity followed phylogenetic relatedness of HA subtypes, both at the level of HA clades (p-value = 0.04 and the level of HA groups (p-value = 0.05. In contrast, infection patterns did not support specific immunity for neuraminidase (NA subtypes. For the H1 and H3 Clades, heterosubtypic immunity showed a clear temporal pattern and we estimated within-clade immunity to last at least 30 days. The strength and duration of heterosubtypic immunity has important implications for transmission dynamics of IAV in the natural reservoir, where immune escape and disruptive selection may increase HA antigenic variation and explain IAV subtype diversity.

Baculovirus display of fusion protein of Peste des petits ruminants virus and hemagglutination protein of Rinderpest virus and immunogenicity of the displayed proteins in mouse model

International Nuclear Information System (INIS)

Masmudur Rahman, Md.; Shaila, M.S.; Gopinathan, Karumathil P.

2003-01-01

Recombinant Bombyx mori nucleopolyhedroviruses (BmNPV) displaying the immunodominant ectodomains of fusion glycoprotein (F) of Peste des petitis ruminants virus (PPRV) and the hemagglutinin protein (H) of Rinderpest virus (RPV), on the budded virions as well as the surface of the infected host cells have been constructed. The F and H protein sequences were inserted in-frame within the amino-terminal region of BmNPV envelope glycoprotein GP64 expressing under the strong viral polyhedrin (polh) promoter. We improved the recombinant virus selection in BmNPV by incorporating the green fluorescent protein gene (gfp) as selection marker under a separate promoter within the transfer cassette harboring the desired genes. Following infection of the insect larvae or the host-derived BmN cells with these recombinant BmNPVs, the expressed GP64 fusion proteins were displayed on the host cell surface and the budded virions. The antigenic epitopes of the recombinant proteins were properly displayed and the recombinant virus particles induced immune response in mice against PPRV or RPV

Mallard or chicken? Comparing the isolation of avian influenza A viruses in embryonated Mallard and chicken eggs

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Josef D. Järhult

2015-09-01

Full Text Available Background: To date, the most efficient and robust method for isolating avian influenza A viruses (IAVs is using embryonated chicken eggs (ECEs. It is known that low-pathogenic avian IAVs undergo rapid genetic changes when introduced to poultry holdings, but the factors driving mutagenesis are not well understood. Despite this, there is limited data on the effects of the standard method of virus isolation of avian-derived viruses, that is, whether isolation in ECEs causes adaptive changes in avian IAVs. Eggs from a homologous species could potentially offer an isolation vessel less prone to induce adaptive changes. Methods: We performed eight serial passages of two avian IAVs isolated from fecal samples of wild Mallards in both ECEs and embryonated Mallard eggs, and hemagglutination assay titers and hemagglutinin sequences were compared. Results: There was no obvious difference in titers between ECEs and embryonated Mallard eggs. Sequence analyses of the isolates showed no apparent difference in the rate of introduction of amino acid substitutions in the hemagglutinin gene (three substitutions in total in embryonated Mallard eggs and two substitutions in ECEs. Conclusion: Embryonated Mallard eggs seem to be good isolation vessels for avian IAVs but carry some practical problems such as limited availability and short egg-laying season of Mallards. Our study finds isolation of Mallard-derived avian IAVs in ECEs non-inferior to isolation in embryonated Mallard eggs, but more research in the area may be warranted as this is a small-scale study.

Outbreak and genotyping of canine distemper virus in captive Siberian tigers and red pandas

OpenAIRE

Zhang, He; Shan, Fen; Zhou, Xia; Li, Bing; Zhai, Jun-Qiong; Zou, Shu-Zhan; Wu, Meng-Fan; Chen, Wu; Zhai, Shao-Lun; Luo, Man-Lin

2017-01-01

In this study, four canine distemper virus (CDV) strains were isolated from captive Siberian tigers (Panthera tigris altaica) and red pandas (Ailurus fulgens) during two separate CDV outbreaks in a zoo in Guangdong province, China. Sequence alignment and phylogenetic analyses based on the full-length hemagglutinin (H) and fusion (F) genes showed that they were closely identical to genotype Asia-1. Prior to confirmation of CDV in Siberian tigers, to control spread of the disease, a live attenu...

The evolutionary genetics and emergence of avian influenza viruses in wild birds.

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Vivien G Dugan

2008-05-01

Full Text Available We surveyed the genetic diversity among avian influenza virus (AIV in wild birds, comprising 167 complete viral genomes from 14 bird species sampled in four locations across the United States. These isolates represented 29 type A influenza virus hemagglutinin (HA and neuraminidase (NA subtype combinations, with up to 26% of isolates showing evidence of mixed subtype infection. Through a phylogenetic analysis of the largest data set of AIV genomes compiled to date, we were able to document a remarkably high rate of genome reassortment, with no clear pattern of gene segment association and occasional inter-hemisphere gene segment migration and reassortment. From this, we propose that AIV in wild birds forms transient "genome constellations," continually reshuffled by reassortment, in contrast to the spread of a limited number of stable genome constellations that characterizes the evolution of mammalian-adapted influenza A viruses.

Characterization of Avian H9N2 Influenza Viruses from United Arab Emirates 2000 to 2003

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Aamir, U. B.; Wernery, Ulrich; Ilyushina, N.; Webster, R. G.

2009-01-01

Our aim was to establish the phylogenetic relation of H9N2 avian viruses in the Middle East to other Asian H9N2 lineages by characterization of 7 viruses isolated from United Arab Emirates (2000-2003). All these viruses had an additional basic amino acid at the hemagglutinin-connecting peptide; 6 contained a mutation associated with increased affinity toward human-like sialic acid substrates. The viruses' surface glycoproteins and most internal genes were >90% similar to those of A/Quail/Hong Kong/G1/97 (H9N2) lineage. The hemadsorbing site of neuraminidase had up to 4 amino acid substitutions, as do human pandemic viruses. M2 sequence analysis revealed amino acid changes at 2 positions, with increasing resistance to amantadine in cell culture. They replicated efficiently in inoculated chickens and were successfully transmitted to contacts. They continue to maintain H5N1-like genes and may augment the spread of H5N1 viruses through regional co-circulation and inapparent infection. These viruses may present as potential pandemic candidates themselves. PMID:17157891

Synthetic generation of influenza vaccine viruses for rapid response to pandemics.

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Dormitzer, Philip R; Suphaphiphat, Pirada; Gibson, Daniel G; Wentworth, David E; Stockwell, Timothy B; Algire, Mikkel A; Alperovich, Nina; Barro, Mario; Brown, David M; Craig, Stewart; Dattilo, Brian M; Denisova, Evgeniya A; De Souza, Ivna; Eickmann, Markus; Dugan, Vivien G; Ferrari, Annette; Gomila, Raul C; Han, Liqun; Judge, Casey; Mane, Sarthak; Matrosovich, Mikhail; Merryman, Chuck; Palladino, Giuseppe; Palmer, Gene A; Spencer, Terika; Strecker, Thomas; Trusheim, Heidi; Uhlendorff, Jennifer; Wen, Yingxia; Yee, Anthony C; Zaveri, Jayshree; Zhou, Bin; Becker, Stephan; Donabedian, Armen; Mason, Peter W; Glass, John I; Rappuoli, Rino; Venter, J Craig

2013-05-15

During the 2009 H1N1 influenza pandemic, vaccines for the virus became available in large quantities only after human infections peaked. To accelerate vaccine availability for future pandemics, we developed a synthetic approach that very rapidly generated vaccine viruses from sequence data. Beginning with hemagglutinin (HA) and neuraminidase (NA) gene sequences, we combined an enzymatic, cell-free gene assembly technique with enzymatic error correction to allow rapid, accurate gene synthesis. We then used these synthetic HA and NA genes to transfect Madin-Darby canine kidney (MDCK) cells that were qualified for vaccine manufacture with viral RNA expression constructs encoding HA and NA and plasmid DNAs encoding viral backbone genes. Viruses for use in vaccines were rescued from these MDCK cells. We performed this rescue with improved vaccine virus backbones, increasing the yield of the essential vaccine antigen, HA. Generation of synthetic vaccine seeds, together with more efficient vaccine release assays, would accelerate responses to influenza pandemics through a system of instantaneous electronic data exchange followed by real-time, geographically dispersed vaccine production.

In vitro assessment of attachment pattern and replication efficiency of H5N1 influenza A viruses with altered receptor specificity.

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Chutinimitkul, Salin; van Riel, Debby; Munster, Vincent J; van den Brand, Judith M A; Rimmelzwaan, Guus F; Kuiken, Thijs; Osterhaus, Albert D M E; Fouchier, Ron A M; de Wit, Emmie

2010-07-01

The continuous circulation of the highly pathogenic avian influenza (HPAI) H5N1 virus has been a cause of great concern. The possibility of this virus acquiring specificity for the human influenza A virus receptor, alpha2,6-linked sialic acids (SA), and being able to transmit efficiently among humans is a constant threat to human health. Different studies have described amino acid substitutions in hemagglutinin (HA) of clinical HPAI H5N1 isolates or that were introduced experimentally that resulted in an increased, but not exclusive, binding of these virus strains to alpha2,6-linked SA. We introduced all previously described amino acid substitutions and combinations thereof into a single genetic background, influenza virus A/Indonesia/5/05 HA, and tested the receptor specificity of these 27 mutant viruses. The attachment pattern to ferret and human tissues of the upper and lower respiratory tract of viruses with alpha2,6-linked SA receptor preference was then determined and compared to the attachment pattern of a human influenza A virus (H3N2). At least three mutant viruses showed an attachment pattern to the human respiratory tract similar to that of the human H3N2 virus. Next, the replication efficiencies of these mutant viruses and the effects of three different neuraminidases on virus replication were determined. These data show that influenza virus A/Indonesia/5/05 potentially requires only a single amino acid substitution to acquire human receptor specificity, while at the same time remaining replication competent, thus suggesting that the pandemic threat posed by HPAI H5N1 is far from diminished.

In Vitro Assessment of Attachment Pattern and Replication Efficiency of H5N1 Influenza A Viruses with Altered Receptor Specificity▿

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Chutinimitkul, Salin; van Riel, Debby; Munster, Vincent J.; van den Brand, Judith M. A.; Rimmelzwaan, Guus F.; Kuiken, Thijs; Osterhaus, Albert D. M. E.; Fouchier, Ron A. M.; de Wit, Emmie

2010-01-01

The continuous circulation of the highly pathogenic avian influenza (HPAI) H5N1 virus has been a cause of great concern. The possibility of this virus acquiring specificity for the human influenza A virus receptor, α2,6-linked sialic acids (SA), and being able to transmit efficiently among humans is a constant threat to human health. Different studies have described amino acid substitutions in hemagglutinin (HA) of clinical HPAI H5N1 isolates or that were introduced experimentally that resulted in an increased, but not exclusive, binding of these virus strains to α2,6-linked SA. We introduced all previously described amino acid substitutions and combinations thereof into a single genetic background, influenza virus A/Indonesia/5/05 HA, and tested the receptor specificity of these 27 mutant viruses. The attachment pattern to ferret and human tissues of the upper and lower respiratory tract of viruses with α2,6-linked SA receptor preference was then determined and compared to the attachment pattern of a human influenza A virus (H3N2). At least three mutant viruses showed an attachment pattern to the human respiratory tract similar to that of the human H3N2 virus. Next, the replication efficiencies of these mutant viruses and the effects of three different neuraminidases on virus replication were determined. These data show that influenza virus A/Indonesia/5/05 potentially requires only a single amino acid substitution to acquire human receptor specificity, while at the same time remaining replication competent, thus suggesting that the pandemic threat posed by HPAI H5N1 is far from diminished. PMID:20392847

Molecular characterization of influenza viruses circulating in Northern Italy during two seasons (2005/2006 and 2006/2007) of low influenza activity.

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Pariani, Elena; Amendola, Antonella; Zappa, Alessandra; Bianchi, Silvia; Colzani, Daniela; Anselmi, Giovanni; Zanetti, Alessandro; Tanzi, Elisabetta

2008-11-01

The influenza activity and circulation of influenza viruses in Lombardy (the most populous Italian region) were observed during two consecutive seasons (2005/2006 and 2006/2007) characterized by low influenza activity by the Italian Influenza Surveillance Network. The molecular characteristics of circulating viruses were analyzed to evaluate the introduction of new variants and emergence of vaccine-escape viruses. In both seasons, the epidemic in Lombardy was sustained almost exclusively by influenza A viruses, accounting for 80.5% and 93.6% of total detections, respectively, and the co-circulation of A/H3 viruses belonging to distinct phylogenetic groups was observed. The A/H1N1 viruses isolated during the 2005/2006 season were closely related to A/New Caledonia/20/99, while the hemagglutinin (HA) sequences of the A/H1N1 viruses from the 2006/2007 season exhibited a greater diversity. These viruses were A/Solomon Islands/3/2006-like and showed several variants. All B isolates were similar to B/Malaysia/2506/2004 belonging to the B/Victoria/2/87-lineage. Influenza B virus was the dominant virus in Europe in the 2005/2006 season and accounted for the 20% of total detections in Lombardy. Overall, the viruses studied presented heterogeneity in their HA sequences suggesting the circulation of a miscellaneous set of variants during the two seasons notwithstanding the medium-low activity of influenza. The importance of virological surveillance of influenza viruses is recognized widely and the molecular characterization of the viruses, especially in vaccinated subjects, is of particular importance to evaluate the introduction and circulation of new variants. 2008 Wiley-Liss, Inc.

Genetic makeup of amantadine-resistant and oseltamivir-resistant human influenza A/H1N1 viruses.

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Zaraket, Hassan; Saito, Reiko; Suzuki, Yasushi; Baranovich, Tatiana; Dapat, Clyde; Caperig-Dapat, Isolde; Suzuki, Hiroshi

2010-04-01

The emergence and widespread occurrence of antiviral drug-resistant seasonal human influenza A viruses, especially oseltamivir-resistant A/H1N1 virus, are major concerns. To understand the genetic background of antiviral drug-resistant A/H1N1 viruses, we performed full genome sequencing of prepandemic A/H1N1 strains. Seasonal influenza A/H1N1 viruses, including antiviral-susceptible viruses, amantadine-resistant viruses, and oseltamivir-resistant viruses, obtained from several areas in Japan during the 2007-2008 and 2008-2009 influenza seasons were analyzed. Sequencing of the full genomes of these viruses was performed, and the phylogenetic relationships among the sequences of each individual genome segment were inferred. Reference genome sequences from the Influenza Virus Resource database were included to determine the closest ancestor for each segment. Phylogenetic analysis revealed that the oseltamivir-resistant strain evolved from a reassortant oseltamivir-susceptible strain (clade 2B) which circulated in the 2007-2008 season by acquiring the H275Y resistance-conferring mutation in the NA gene. The oseltamivir-resistant lineage (corresponding to the Northern European resistant lineage) represented 100% of the H1N1 isolates from the 2008-2009 season and further acquired at least one mutation in each of the polymerase basic protein 2 (PB2), polymerase basic protein 1 (PB1), hemagglutinin (HA), and neuraminidase (NA) genes. Therefore, a reassortment event involving two distinct oseltamivir-susceptible lineages, followed by the H275Y substitution in the NA gene and other mutations elsewhere in the genome, contributed to the emergence of the oseltamivir-resistant lineage. In contrast, amantadine-resistant viruses from the 2007-2008 season distinctly clustered in clade 2C and were characterized by extensive amino acid substitutions across their genomes, suggesting that a fitness gap among its genetic components might have driven these mutations to maintain it in the

Recombinant IgA Is Sufficient To Prevent Influenza Virus Transmission in Guinea Pigs

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Seibert, Christopher W.; Rahmat, Saad; Krause, Jens C.; Eggink, Dirk; Albrecht, Randy A.; Goff, Peter H.; Krammer, Florian; Duty, J. Andrew; Bouvier, Nicole M.; García-Sastre, Adolfo

2013-01-01

A serum hemagglutination inhibition (HAI) titer of 40 or greater is thought to be associated with reduced influenza virus pathogenesis in humans and is often used as a correlate of protection in influenza vaccine studies. We have previously demonstrated that intramuscular vaccination of guinea pigs with inactivated influenza virus generates HAI titers greater than 300 but does not protect vaccinated animals from becoming infected with influenza virus by transmission from an infected cage mate. Only guinea pigs intranasally inoculated with a live influenza virus or a live attenuated virus vaccine, prior to challenge, were protected from transmission (A. C. Lowen et al., J. Virol. 83:2803–2818, 2009.). Because the serum HAI titer is mostly determined by IgG content, these results led us to speculate that prevention of viral transmission may require IgA antibodies or cellular immune responses. To evaluate this hypothesis, guinea pigs and ferrets were administered a potent, neutralizing mouse IgG monoclonal antibody, 30D1 (Ms 30D1 IgG), against the A/California/04/2009 (H1N1) virus hemagglutinin and exposed to respiratory droplets from animals infected with this virus. Even though HAI titers were greater than 160 1 day postadministration, Ms 30D1 IgG did not prevent airborne transmission to passively immunized recipient animals. In contrast, intramuscular administration of recombinant 30D1 IgA (Ms 30D1 IgA) prevented transmission to 88% of recipient guinea pigs, and Ms 30D1 IgA was detected in animal nasal washes. Ms 30D1 IgG administered intranasally also prevented transmission, suggesting the importance of mucosal immunity in preventing influenza virus transmission. Collectively, our data indicate that IgG antibodies may prevent pathogenesis associated with influenza virus infection but do not protect from virus infection by airborne transmission, while IgA antibodies are more important for preventing transmission of influenza viruses. PMID:23698296

Structural and antigenic variation among diverse clade 2 H5N1 viruses.

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David A Shore

Full Text Available Antigenic variation among circulating H5N1 highly pathogenic avian influenza A viruses mandates the continuous production of strain-specific pre-pandemic vaccine candidates and represents a significant challenge for pandemic preparedness. Here we assessed the structural, antigenic and receptor-binding properties of three H5N1 HPAI virus hemagglutinins, which were recently selected by the WHO as vaccine candidates [A/Egypt/N03072/2010 (Egypt10, clade 2.2.1, A/Hubei/1/2010 (Hubei10, clade 2.3.2.1 and A/Anhui/1/2005 (Anhui05, clade 2.3.4]. These analyses revealed that antigenic diversity among these three isolates was restricted to changes in the size and charge of amino acid side chains at a handful of positions, spatially equivalent to the antigenic sites identified in H1 subtype viruses circulating among humans. All three of the H5N1 viruses analyzed in this study were responsible for fatal human infections, with the most recently-isolated strains, Hubei10 and Egypt10, containing multiple residues in the receptor-binding site of the HA, which were suspected to enhance mammalian transmission. However, glycan-binding analyses demonstrated a lack of binding to human α2-6-linked sialic acid receptor analogs for all three HAs, reinforcing the notion that receptor-binding specificity contributes only partially to transmissibility and pathogenesis of HPAI viruses and suggesting that changes in host specificity must be interpreted in the context of the host and environmental factors, as well as the virus as a whole. Together, our data reveal structural linkages with phylogenetic and antigenic analyses of recently emerged H5N1 virus clades and should assist in interpreting the significance of future changes in antigenic and receptor-binding properties.

Isolation and genetic characterization of avian influenza viruses and a Newcastle disease virus from wild birds in Barbados: 2003-2004.

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Douglas, Kirk O; Lavoie, Marc C; Kim, L Mia; Afonso, Claudio L; Suarez, David L

2007-09-01

Zoonotic transmission of an H5N1 avian influenza A virus to humans in 2003-present has generated increased public health and scientific interest in the prevalence and variability of influenza A viruses in wild birds and their potential threat to human health. Migratory waterfowl and shorebirds are regarded as the primordial reservoir of all influenza A viral subtypes and have been repeatedly implicated in avian influenza outbreaks in domestic poultry and swine. All of the 16 hemagglutinin and nine neuraminidase influenza subtypes have been isolated from wild birds, but waterfowl of the order Anseriformes are the most commonly infected. Using 9-to-11-day-old embryonating chicken egg culture, virus isolation attempts were conducted on 168 cloacal swabs from various resident, imported, and migratory bird species in Barbados during the months of July to October of 2003 and 2004. Hemagglutination assay and reverse transcription-polymerase chain reaction were used to screen all allantoic fluids for the presence of hemagglutinating agents and influenza A virus. Hemagglutination positive-influenza negative samples were also tested for Newcastle disease virus (NDV), which is also found in waterfowl. Two influenza A viruses and one NDV were isolated from Anseriformes (40/168), with isolation rates of 5.0% (2/40) and 2.5% (1/40), respectively, for influenza A and NDV. Sequence analysis of the influenza A virus isolates showed them to be H4N3 viruses that clustered with other North American avian influenza viruses. This is the first report of the presence of influenza A virus and NDV in wild birds in the English-speaking Caribbean.

Transmission of Hemagglutinin D222G Mutant Strain of Pandemic (H1N1) 2009 Virus

Science.gov (United States)

Facchini, Marzia; Spagnolo, Domenico; De Marco, Maria A.; Calzoletti, Laura; Zanetti, Alessandro; Fumagalli, Roberto; Tanzi, Maria L.; Cassone, Antonio; Rezza, Giovanni; Donatelli, Isabella

2010-01-01

A pandemic (H1N1) 2009 virus strain carrying the D222G mutation was identified in a severely ill man and was transmitted to a household contact. Only mild illness developed in the contact, despite his obesity and diabetes. The isolated virus reacted fully with an antiserum against the pandemic vaccine strain. PMID:20409386

Divergent H7 immunogens offer protection from H7N9 virus challenge.

Science.gov (United States)

Krammer, Florian; Albrecht, Randy A; Tan, Gene S; Margine, Irina; Hai, Rong; Schmolke, Mirco; Runstadler, Jonathan; Andrews, Sarah F; Wilson, Patrick C; Cox, Rebecca J; Treanor, John J; García-Sastre, Adolfo; Palese, Peter

2014-04-01

The emergence of avian H7N9 viruses in humans in China has renewed concerns about influenza pandemics emerging from Asia. Vaccines are still the best countermeasure against emerging influenza virus infections, but the process from the identification of vaccine seed strains to the distribution of the final product can take several months. In the case of the 2009 H1N1 pandemic, a vaccine was not available before the first pandemic wave hit and therefore came too late to reduce influenza morbidity. H7 vaccines based on divergent isolates of the Eurasian and North American lineages have been tested in clinical trials, and seed strains and reagents are already available and can potentially be used initially to curtail influenza-induced disease until a more appropriately matched H7N9 vaccine is ready. In a challenge experiment in the mouse model, we assessed the efficacy of both inactivated virus and recombinant hemagglutinin vaccines made from seed strains that are divergent from H7N9 from each of the two major H7 lineages. Furthermore, we analyzed the cross-reactive responses of sera from human subjects vaccinated with heterologous North American and Eurasian lineage H7 vaccines to H7N9. Vaccinations with inactivated virus and recombinant hemagglutinin protein preparations from both lineages raised hemagglutination-inhibiting antibodies against H7N9 viruses and protected mice from stringent viral challenges. Similar cross-reactivity was observed in sera of human subjects from a clinical trial with a divergent H7 vaccine. Existing H7 vaccine candidates based on divergent strains could be used as a first line of defense against an H7N9 pandemic. In addition, this also suggests that H7N9 vaccines that are currently under development might be stockpiled and used for divergent avian H7 strains that emerge in the future. Sporadic human infections with H7N9 viruses started being reported in China in the early spring of 2013. Despite a significant drop in the number of

Temperature-sensitive mutants of influenza A virus. XIV. Production and evaluation of influenza A/Georgia/74-ts-1[E] recombinant viruses in human adults.

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Richman, D D; Murphy, B R; Belshe, R B; Rusten, H M; Chanock, R M; Blacklow, N R; Parrino, T A; Rose, F B; Levine, M M; Caplan, E

1977-08-01

The two temperature-sensitive (ts) lesions present in influenza A/Hong Kong/68-ts-1[E] (H3N2 68) virus were transferred via genetic reassortment to influenza A/Georgia/74 (H3N2 74) wild-type virus. A recombinant clone possessing both ts lesions and the shutoff temperature of 38 C of the Hong Kong/68 ts donor and the two surface antigens of the Georgia/74 wild-type virus was administered to 32 seronegative adult volunteers. Thirty-one volunteers were infected, of whom only five experienced mild afebrile upper respiratory tract illness. The wild-type recipient virus was a cloned population that induced illness in five of six infected volunteers. Therfore, the attenuation exhibited by the Georgia/74-ts-1[E] virus could reasonably be assumed to be due to the acquisition of the two ts-1[E] lesions by the Georgia/74 wild-type virus. The serum and nasal wash antibody responses of the ts-1[E] vaccinees were equivalent to those of the volunteers who received wild-type virus. The two ts lesions present in the Hong Kong/68-ts-1[E] virus have now been transferred three times to a wild-type virus bearing a new hemagglutinin, and in each instance the new ts recombination exhibited a similar, satisfactory level of attenuation and antigenicity for adults. It seems likely that the transfer of the ts-1[E] lesions to any new influenza virus will regularly result in attenuation of a recombinat virus possessing the new surface antigens.

Acquisition of human-type receptor binding specificity by new H5N1 influenza virus sublineages during their emergence in birds in Egypt.

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Yohei Watanabe

2011-05-01

Full Text Available Highly pathogenic avian influenza A virus subtype H5N1 is currently widespread in Asia, Europe, and Africa, with 60% mortality in humans. In particular, since 2009 Egypt has unexpectedly had the highest number of human cases of H5N1 virus infection, with more than 50% of the cases worldwide, but the basis for this high incidence has not been elucidated. A change in receptor binding affinity of the viral hemagglutinin (HA from α2,3- to α2,6-linked sialic acid (SA is thought to be necessary for H5N1 virus to become pandemic. In this study, we conducted a phylogenetic analysis of H5N1 viruses isolated between 2006 and 2009 in Egypt. The phylogenetic results showed that recent human isolates clustered disproportionally into several new H5 sublineages suggesting that their HAs have changed their receptor specificity. Using reverse genetics, we found that these H5 sublineages have acquired an enhanced binding affinity for α2,6 SA in combination with residual affinity for α2,3 SA, and identified the amino acid mutations that produced this new receptor specificity. Recombinant H5N1 viruses with a single mutation at HA residue 192 or a double mutation at HA residues 129 and 151 had increased attachment to and infectivity in the human lower respiratory tract but not in the larynx. These findings correlated with enhanced virulence of the mutant viruses in mice. Interestingly, these H5 viruses, with increased affinity to α2,6 SA, emerged during viral diversification in bird populations and subsequently spread to humans. Our findings suggested that emergence of new H5 sublineages with α2,6 SA specificity caused a subsequent increase in human H5N1 influenza virus infections in Egypt, and provided data for understanding the virus's pandemic potential.

Spatiotemporal Analysis of the Genetic Diversity of Seal Influenza A(H10N7) Virus, Northwestern Europe

DEFF Research Database (Denmark)

Bodewes, Rogier; Zohari, Siamak; Krog, Jesper Schak

2016-01-01

and Denmark. Within a few months, this virus spread to seals of the coastal waters of Germany and the Netherlands, causing the death of thousands of animals. Genetic analysis of the hemagglutinin (HA) and neuraminidase (NA) genes of this seal influenza A(H10N7) virus revealed that it was most closely related...... to various avian influenza A(H10N7) viruses. The collection of samples from infected seals during the course of the outbreak provided a unique opportunity to follow the adaptation of the avian virus to its new seal host. Sequence data for samples collected from 41 different seals from four different......, various sequencing methods were used to elucidate the genetic changes that occurred after the introduction and subsequent spread of an avian influenza A(H10N7) virus among harbor seals of northwestern Europe by use of various samples collected during the outbreak. Such detailed knowledge of genetic...

Fusion protein gene nucleotide sequence similarities, shared antigenic sites and phylogenetic analysis suggest that phocid distemper virus 2 and canine distemper virus belong to the same virus entity.

NARCIS (Netherlands)

I.K.G. Visser (Ilona); R.W.J. van der Heijden (Roger); M.W.G. van de Bildt (Marco); M.J.H. Kenter (Marcel); C. Örvell; A.D.M.E. Osterhaus (Albert)

1993-01-01

textabstractNucleotide sequencing of the fusion protein (F) gene of phocid distemper virus-2 (PDV-2), recently isolated from Baikal seals (Phoca sibirica), revealed an open reading frame (nucleotides 84 to 2075) with two potential in-frame ATG translation initiation codons. We suggest that the

Virological surveillance and preliminary antigenic characterization of influenza viruses in pigs in five European countries from 2006 to 2008.

Science.gov (United States)

Kyriakis, C S; Brown, I H; Foni, E; Kuntz-Simon, G; Maldonado, J; Madec, F; Essen, S C; Chiapponi, C; Van Reeth, K

2011-03-01

This study presents the results of the virological surveillance for swine influenza viruses (SIVs) in Belgium, UK, Italy, France and Spain from 2006 to 2008. Our major aims were to clarify the occurrence of the three SIV subtypes - H1N1, H3N2 and H1N2 - at regional levels, to identify novel reassortant viruses and to antigenically compare SIVs with human H1N1 and H3N2 influenza viruses. Lung tissue and/or nasal swabs from outbreaks of acute respiratory disease in pigs were investigated by virus isolation. The hemagglutinin (HA) and neuraminidase (NA) subtypes were determined using standard methods. Of the total 169 viruses, 81 were classified as 'avian-like' H1N1, 36 as human-like H3N2 and 47 as human-like H1N2. Only five novel reassortant viruses were identified: two H1N1 viruses had a human-like HA and three H1N2 viruses an avian-like HA. All three SIV subtypes were detected in Belgium, Italy and Spain, while only H1N1 and H1N2 viruses were found in UK and Northwestern France. Cross-hemagglutination inhibition (HI) tests with hyperimmune sera against selected older and recent human influenza viruses showed a strong antigenic relationship between human H1N1 and H3N2 viruses from the 1980s and H1N2 and H3N2 human-like SIVs, confirming their common origin. However, antisera against human viruses isolated during the last decade did not react with currently circulating H1 or H3 SIVs, suggesting that especially young people may be, to some degree, susceptible to SIV infections. © 2009 Blackwell Verlag GmbH.

H3N2 influenza infection elicits more cross-reactive and less clonally expanded anti-hemagglutinin antibodies than influenza vaccination.

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M Anthony Moody

Full Text Available BACKGROUND: During the recent H1N1 influenza pandemic, excess morbidity and mortality was seen in young but not older adults suggesting that prior infection with influenza strains may have protected older subjects. In contrast, a history of recent seasonal trivalent vaccine in younger adults was not associated with protection. METHODS AND FINDINGS: To study hemagglutinin (HA antibody responses in influenza immunization and infection, we have studied the day 7 plasma cell repertoires of subjects immunized with seasonal trivalent inactivated influenza vaccine (TIV and compared them to the plasma cell repertoires of subjects experimentally infected (EI with influenza H3N2 A/Wisconsin/67/2005. The majority of circulating plasma cells after TIV produced influenza-specific antibodies, while most plasma cells after EI produced antibodies that did not react with influenza HA. While anti-HA antibodies from TIV subjects were primarily reactive with single or few HA strains, anti-HA antibodies from EI subjects were isolated that reacted with multiple HA strains. Plasma cell-derived anti-HA antibodies from TIV subjects showed more evidence of clonal expansion compared with antibodies from EI subjects. From an H3N2-infected subject, we isolated a 4-member clonal lineage of broadly cross-reactive antibodies that bound to multiple HA subtypes and neutralized both H1N1 and H3N2 viruses. This broad reactivity was not detected in post-infection plasma suggesting this broadly reactive clonal lineage was not immunodominant in this subject. CONCLUSION: The presence of broadly reactive subdominant antibody responses in some EI subjects suggests that improved vaccine designs that make broadly reactive antibody responses immunodominant could protect against novel influenza strains.

Histopathological evaluation of the diversity of cells susceptible to H5N1 virulent avian influenza virus.

Science.gov (United States)

Ogiwara, Haru; Yasui, Fumihiko; Munekata, Keisuke; Takagi-Kamiya, Asako; Munakata, Tsubasa; Nomura, Namiko; Shibasaki, Futoshi; Kuwahara, Kazuhiko; Sakaguchi, Nobuo; Sakoda, Yoshihiro; Kida, Hiroshi; Kohara, Michinori

2014-01-01

Patients infected with highly pathogenic avian influenza A H5N1 viruses (H5N1 HPAIV) show diffuse alveolar damage. However, the temporal progression of tissue damage and repair after viral infection remains poorly defined. Therefore, we assessed the sequential histopathological characteristics of mouse lung after intranasal infection with H5N1 HPAIV or H1N1 2009 pandemic influenza virus (H1N1 pdm). We determined the amount and localization of virus in the lung through IHC staining and in situ hybridization. IHC used antibodies raised against the virus protein and antibodies specific for macrophages, type II pneumocytes, or proliferating cell nuclear antigen. In situ hybridization used RNA probes against both viral RNA and mRNA encoding the nucleoprotein and the hemagglutinin protein. H5N1 HPAIV infection and replication were observed in multiple lung cell types and might result in rapid progression of lung injury. Both type II pneumocytes and macrophages proliferated after H5N1 HPAIV infection. However, the abundant macrophages failed to block the viral attack, and proliferation of type II pneumocytes failed to restore the damaged alveoli. In contrast, mice infected with H1N1 pdm exhibited modest proliferation of type II pneumocytes and macrophages and slight alveolar damage. These results suggest that the virulence of H5N1 HPAIV results from the wide range of cell tropism of the virus, excessive virus replication, and rapid development of diffuse alveolar damage. Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Epizootic canine distemper virus infection among wild mammals.

Science.gov (United States)

Kameo, Yuki; Nagao, Yumiko; Nishio, Yohei; Shimoda, Hiroshi; Nakano, Hitoshi; Suzuki, Kazuo; Une, Yumi; Sato, Hiroshi; Shimojima, Masayuki; Maeda, Ken

2012-01-27

In the spring of 2007, seven raccoon dogs and a weasel were captured near the city of Tanabe in Wakayama prefecture, Japan. The causative agent of the animals' death 1-2 days after capture was identified as canine distemper virus (CDV) by virus isolation, immunostaining with an anti-CDV polyclonal antibody, and a commercially available CDV antigen-detection kit. Sequence analysis of hemagglutinin genes indicated the isolated viruses belong to genotype Asia-1 and possess the substitution from tyrosine (Y) to histidine (H) at position 549 that is associated with the spread of CDV to non-canine hosts. A serosurvey for CDV was then conducted among wild animals in the region. The animals assayed consisted of 104 raccoons, 41 wild boars, 19 raccoon dogs, five Sika deer, two badgers, one weasel, one marten, one Siberian weasel and one fox. Virus-neutralization (VN) tests showed that, except for fox and weasel, all of the species assayed had VN antibodies to CDV. Interestingly, 11 of the 41 wild boars (27%) and two of the five Sika deer assayed possessed VN antibodies to CDV. These findings indicate that CDV infection was widespread among wild mammals during this epizootic. Copyright © 2011 Elsevier B.V. All rights reserved.

A novel single virus infection system reveals that influenza virus preferentially infects cells in g1 phase.

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Ryuta Ueda

Full Text Available BACKGROUND: Influenza virus attaches to sialic acid residues on the surface of host cells via the hemagglutinin (HA, a glycoprotein expressed on the viral envelope, and enters into the cytoplasm by receptor-mediated endocytosis. The viral genome is released and transported in to the nucleus, where transcription and replication take place. However, cellular factors affecting the influenza virus infection such as the cell cycle remain uncharacterized. METHODS/RESULTS: To resolve the influence of cell cycle on influenza virus infection, we performed a single-virus infection analysis using optical tweezers. Using this newly developed single-virus infection system, the fluorescence-labeled influenza virus was trapped on a microchip using a laser (1064 nm at 0.6 W, transported, and released onto individual H292 human lung epithelial cells. Interestingly, the influenza virus attached selectively to cells in the G1-phase. To clarify the molecular differences between cells in G1- and S/G2/M-phase, we performed several physical and chemical assays. Results indicated that: 1 the membranes of cells in G1-phase contained greater amounts of sialic acids (glycoproteins than the membranes of cells in S/G2/M-phase; 2 the membrane stiffness of cells in S/G2/M-phase is more rigid than those in G1-phase by measurement using optical tweezers; and 3 S/G2/M-phase cells contained higher content of Gb3, Gb4 and GlcCer than G1-phase cells by an assay for lipid composition. CONCLUSIONS: A novel single-virus infection system was developed to characterize the difference in influenza virus susceptibility between G1- and S/G2/M-phase cells. Differences in virus binding specificity were associated with alterations in the lipid composition, sialic acid content, and membrane stiffness. This single-virus infection system will be useful for studying the infection mechanisms of other viruses.

Unexpected Genome Variability at Multiple Loci Suggests Cacao Swollen Shoot Virus Comprises Multiple, Divergent Molecular Variants.

Science.gov (United States)

Cacao swollen shoot virus (CSSV) [Badnavirus, Caulimoviridae] causes swollen shoot disease of Theobroma cacao L. in West Africa. Since ~2000, various diagnostic tests have failed to detect CSSV in ~50-70% of symptomatic cacao plants, suggesting the possible emergence of new, previously uncharacteriz...

Influenza A Virus Hemagglutinin is Required for the Assembly of Viral Components Including Bundled vRNPs at the Lipid Raft

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Naoki Takizawa

2016-09-01

Full Text Available The influenza glycoproteins, hemagglutinin (HA and neuraminidase (NA, which are associated with the lipid raft, have the potential to initiate virion budding. However, the role of these viral proteins in infectious virion assembly is still unclear. In addition, it is not known how the viral ribonucleoprotein complex (vRNP is tethered to the budding site. Here, we show that HA is necessary for the efficient progeny virion production and vRNP packaging in the virion. We also found that the level of HA does not affect the bundling of the eight vRNP segments, despite reduced virion production. Detergent solubilization and a subsequent membrane flotation analysis indicated that the accumulation of nucleoprotein, viral polymerases, NA, and matrix protein 1 (M1 in the lipid raft fraction was delayed without HA. Based on our results, we inferred that HA plays a role in the accumulation of viral components, including bundled vRNPs, at the lipid raft.

Influenza A Virus Hemagglutinin is Required for the Assembly of Viral Components Including Bundled vRNPs at the Lipid Raft.

Science.gov (United States)

Takizawa, Naoki; Momose, Fumitaka; Morikawa, Yuko; Nomoto, Akio

2016-09-10

The influenza glycoproteins, hemagglutinin (HA) and neuraminidase (NA), which are associated with the lipid raft, have the potential to initiate virion budding. However, the role of these viral proteins in infectious virion assembly is still unclear. In addition, it is not known how the viral ribonucleoprotein complex (vRNP) is tethered to the budding site. Here, we show that HA is necessary for the efficient progeny virion production and vRNP packaging in the virion. We also found that the level of HA does not affect the bundling of the eight vRNP segments, despite reduced virion production. Detergent solubilization and a subsequent membrane flotation analysis indicated that the accumulation of nucleoprotein, viral polymerases, NA, and matrix protein 1 (M1) in the lipid raft fraction was delayed without HA. Based on our results, we inferred that HA plays a role in the accumulation of viral components, including bundled vRNPs, at the lipid raft.

Virulence and transmissibility of H1N2 influenza virus in ferrets imply the continuing threat of triple-reassortant swine viruses.

Science.gov (United States)

Pascua, Philippe Noriel Q; Song, Min-Suk; Lee, Jun Han; Baek, Yun Hee; Kwon, Hyeok-il; Park, Su-Jin; Choi, Eun Hye; Lim, Gyo-Jin; Lee, Ok-Jun; Kim, Si-Wook; Kim, Chul-Joong; Sung, Moon Hee; Kim, Myung Hee; Yoon, Sun-Woo; Govorkova, Elena A; Webby, Richard J; Webster, Robert G; Choi, Young-Ki

2012-09-25

Efficient worldwide swine surveillance for influenza A viruses is urgently needed; the emergence of a novel reassortant pandemic H1N1 (pH1N1) virus in 2009 demonstrated that swine can be the direct source of pandemic influenza and that the pandemic potential of viruses prevalent in swine populations must be monitored. We used the ferret model to assess the pathogenicity and transmissibility of predominant Korean triple-reassortant swine (TRSw) H1N2 and H3N2 influenza viruses genetically related to North American strains. Although most of the TRSw viruses were moderately pathogenic, one [A/Swine/Korea/1204/2009; Sw/1204 (H1N2)] was virulent in ferrets, causing death within 10 d of inoculation, and was efficiently transmitted to naive contact ferrets via respiratory droplets. Although molecular analysis did not reveal known virulence markers, the Sw/1204 virus acquired mutations in hemagglutinin (HA) (Asp-225-Gly) and neuraminidase (NA) (Ser-315-Asn) proteins during the single ferret passage. The contact-Sw/1204 virus became more virulent in mice, replicated efficiently in vitro, extensively infected human lung tissues ex vivo, and maintained its ability to replicate and transmit in swine. Reverse-genetics studies further indicated that the HA(225G) and NA(315N) substitutions contributed substantially in altering virulence and transmissibility. These findings support the continuing threat of some field TRSw viruses to human and animal health, reviving concerns on the capacity of pigs to create future pandemic viruses. Apart from warranting continued and enhanced global surveillance, this study also provides evidence on the emerging roles of HA(225G) and NA(315N) as potential virulence markers in mammals.

Antigenic Variation in H5N1 clade 2.1 Viruses in Indonesia from 2005 to 2011

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Vivi Setiawaty

2013-01-01

Full Text Available Influenza A (H5N1 virus, has spread to several countries in the world and has a high mortality rate. Meanwhile, the virus has evolved into several clades. The human influenza A (H5N1 virus circulating in Indonesia is a member of clade 2.1, which is different in antigenicity from other clades of influenza A (H5N1. An analysis of the antigenic variation in the H5 hemagglutinin gene (HA of the influenza A (H5N1 virus strains circulating in Indonesia has been undertaken. Several position of amino acid mutations, including mutations at positions 35, 53, 141, 145, 163, 174, 183, 184, 189, and 231, have been identified. The mutation Val-174-Iso appears to play an important role in immunogenicity and cross-reactivity with rabbit antisera. This study shows that the evolution of the H5HA antigenic variation of the influenza A (H5N1 virus circulating in Indonesia from 2005 to 2011 may affect the immunogenicity of the virus.

Dual function of the hemagglutinin H5 fused to chicken CD154 in a ...

African Journals Online (AJOL)

Dual function of the hemagglutinin H5 fused to chicken CD154 in a potential strategy of DIVA against avian influenza disease: preliminary study. AG Pose, ES Rodriguez, AC Mendez, JN Gomez, AV Redondo, ER Rodriguez, EMG Ramos, AA Gutierrez, MPR Molto, DG Roche, YS Ugalde, AM Lopez ...

Tropism and Infectivity of Influenza Virus, Including Highly Pathogenic Avian H5N1 Virus, in Ferret Tracheal Differentiated Primary Epithelial Cell Cultures

Science.gov (United States)

Zeng, Hui; Goldsmith, Cynthia S.; Maines, Taronna R.; Belser, Jessica A.; Gustin, Kortney M.; Pekosz, Andrew; Zaki, Sherif R.; Katz, Jacqueline M.

2013-01-01

Tropism and adaptation of influenza viruses to new hosts is partly dependent on the distribution of the sialic acid (SA) receptors to which the viral hemagglutinin (HA) binds. Ferrets have been established as a valuable in vivo model of influenza virus pathogenesis and transmission because of similarities to humans in the distribution of HA receptors and in clinical signs of infection. In this study, we developed a ferret tracheal differentiated primary epithelial cell culture model that consisted of a layered epithelium structure with ciliated and nonciliated cells on its apical surface. We found that human-like (α2,6-linked) receptors predominated on ciliated cells, whereas avian-like (α2,3-linked) receptors, which were less abundant, were presented on nonciliated cells. When we compared the tropism and infectivity of three human (H1 and H3) and two avian (H1 and H5) influenza viruses, we observed that the human influenza viruses primarily infected ciliated cells and replicated efficiently, whereas a highly pathogenic avian H5N1 virus (A/Vietnam/1203/2004) replicated efficiently within nonciliated cells despite a low initial infection rate. Furthermore, compared to other influenza viruses tested, VN/1203 virus replicated more efficiently in cells isolated from the lower trachea and at a higher temperature (37°C) compared to a lower temperature (33°C). VN/1203 virus infection also induced higher levels of immune mediator genes and cell death, and virus was recovered from the basolateral side of the cell monolayer. This ferret tracheal differentiated primary epithelial cell culture system provides a valuable in vitro model for studying cellular tropism, infectivity, and the pathogenesis of influenza viruses. PMID:23255802

Involvement of UL24 in herpes-simplex-virus-1-induced dispersal of nucleolin

International Nuclear Information System (INIS)

Lymberopoulos, Maria H.; Pearson, Angela

2007-01-01

UL24 of herpes simplex virus 1 is important for efficient viral replication, but its function is unknown. We generated a recombinant virus, vHA-UL24, encoding UL24 with an N-terminal hemagglutinin tag. By indirect immunofluorescence at 9 h post-infection (hpi), we detected HA-UL24 in nuclear foci and in cytoplasmic speckles. HA-UL24 partially co-localized with nucleolin, but not with ICP8 or coilin, markers for nucleoli, viral replication compartments, and Cajal bodies respectively. HA-UL24 staining was often juxtaposed to that of another nucleolar protein, fibrillarin. Analysis of HSV-1-induced nucleolar modifications revealed that by 18 hpi, nucleolin staining had dispersed, and fibrillarin staining went from clusters of small spots to a few separate but prominent spots. Fibrillarin redistribution appeared to be independent of UL24. In contrast, cells infected with a UL24-deficient virus retained foci of nucleolin staining. Our results demonstrate involvement of UL24 in dispersal of nucleolin during infection

In vitro infection of pupae with Israeli acute paralysis virus suggests disturbance of transcriptional homeostasis in honey bees (Apis mellifera.

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Humberto F Boncristiani

Full Text Available The ongoing decline of honey bee health worldwide is a serious economic and ecological concern. One major contributor to the decline are pathogens, including several honey bee viruses. However, information is limited on the biology of bee viruses and molecular interactions with their hosts. An experimental protocol to test these systems was developed, using injections of Israeli Acute Paralysis Virus (IAPV into honey bee pupae reared ex-situ under laboratory conditions. The infected pupae developed pronounced but variable patterns of disease. Symptoms varied from complete cessation of development with no visual evidence of disease to rapid darkening of a part or the entire body. Considerable differences in IAPV titer dynamics were observed, suggesting significant variation in resistance to IAPV among and possibly within honey bee colonies. Thus, selective breeding for virus resistance should be possible. Gene expression analyses of three separate experiments suggest IAPV disruption of transcriptional homeostasis of several fundamental cellular functions, including an up-regulation of the ribosomal biogenesis pathway. These results provide first insights into the mechanisms of IAPV pathogenicity. They mirror a transcriptional survey of honey bees afflicted with Colony Collapse Disorder and thus support the hypothesis that viruses play a critical role in declining honey bee health.

Consecutive natural influenza a virus infections in sentinel mallards in the evident absence of subtype-specific hemagglutination inhibiting antibodies.

Science.gov (United States)

Globig, A; Fereidouni, S R; Harder, T C; Grund, C; Beer, M; Mettenleiter, T C; Starick, E

2013-10-01

Dabbling ducks, particularly Mallards (Anas platyrhynchos) have been frequently and consistently reported to play a pivotal role as a reservoir of low pathogenic avian influenza viruses (AIV). From October 2006 to November 2008, hand-raised Mallard ducks kept at a pond in an avifaunistically rich area of Southern Germany served as sentinel birds in the AIV surveillance programme in Germany. The pond was regularly visited by several species of dabbling ducks. A flock of sentinel birds, consisting of the same 16 individual birds during the whole study period, was regularly tested virologically and serologically for AIV infections. Swab samples were screened by RT-qPCR and, if positive, virus was isolated in embryonated chicken eggs. Serum samples were tested by the use of competitive ELISA and hemagglutinin inhibition (HI) assay. Sequences of full-length hemagglutinin (HA) and neuraminidase (NA) genes were phylogenetically analysed. Four episodes of infections with Eurasian-type AIV occurred in August (H6N8), October/November (H3N2, H2N3) 2007, in January (H3N2) and September (H3N8) 2008. The HA and NA genes of the H3N2 viruses of October 2007 and January 2008 were almost identical rendering the possibility of a re-introduction of that virus from the environment of the sentinel flock highly likely. The HA of the H3N8 virus of September 2008 belonged to a different cluster. As a correlate of the humoral immune response, titres of nucleocapsid protein-specific antibodies fluctuated in correlation with the course of AIV infection episodes. However, no specific systemic response of hemagglutination inhibiting antibodies could be demonstrated even if homologous viral antigens were used. Besides being useful as early indicators for the circulation of influenza viruses in a specific region, the sentinel ducks also contributed to gaining insights into the ecobiology of AIV infection in aquatic wild birds. © 2012 Blackwell Verlag GmbH.

X-ray structure of the hemagglutinin of a potential H3 avian progenitor of the 1968 Hong Kong pandemic influenza virus

International Nuclear Information System (INIS)

Ha Ya; Stevens, David J.; Skehel, John J.; Wiley, Don C.

2003-01-01

We have determined the structure of the HA of an avian influenza virus, A/duck/Ukraine/63, a member of the same antigenic subtype, H3, as the virus that caused the 1968 Hong Kong influenza pandemic, and a possible progenitor of the pandemic virus. We find that structurally significant differences between the avian and the human HAs are restricted to the receptor-binding site particularly the substitutions Q226L and G228S that cause the site to open and residues within it to rearrange, including the conserved residues Y98, W153, and H183. We have also analyzed complexes formed by the HA with sialopentasaccharides in which the terminal sialic acid is in either α2,3- or α2,6-linkage to galactose. Comparing the structures of complexes in which an α2,3-linked receptor analog is bound to the H3 avian HA or to an H5 avian HA leads to the suggestion that all avian influenza HAs bind to their preferred α2,3-linked receptors similarly, with the analog in a trans conformation about the glycosidic linkage. We find that α2,6-linked analogs are bound by both human and avian HAs in a cis conformation, and that the incompatibility of an α2,6-linked receptor with the α2,3-linkage-specific H3 avian HA-binding site is partially resolved by a small change in the position and orientation of the sialic acid. We discuss our results in relation to the mechanism of transfer of influenza viruses between species

Genetic drift of HA and NA in Danish swine influenza virus from the period 2003-2012

DEFF Research Database (Denmark)

Fobian, Kristina; Breum, Solvej Østergaard; Hjulsager, Charlotte Kristiane

2012-01-01

. Currently at least three influenza A subtypes (H1N1, H1N2 and H3N2) are endemic in the Danish swine population, and since 2010 the pandemic virus (H1N1pdm09) have also frequently been detected. The focus in this study will be on H1N1 and H1N2, since the prevalence of H3N2 have declined over the past years...... will provide a more complete picture of the molecular epidemiology of the H1N1 and H1N2 swine influenza viruses in Denmark. A thorough knowledge of the antigenic drift in surface genes is very important concerning evaluation of the zoonotic potential of existing and future swine influenza virus strains......The aim of this study is to analyze; the genetic drift in hemagglutinin (HA) and neuraminidase (NA) genes from influenza viruses isolated from Danish swine over the past decade; the antigenic evolution and relatedness between swine influenza virus strains of the H1 subtype by antigenic cartography...

Replication and adaptive mutations of low pathogenic avian influenza viruses in tracheal organ cultures of different avian species.

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Henning Petersen

Full Text Available Transmission of avian influenza viruses (AIV between different avian species may require genome mutations that allow efficient virus replication in a new species and could increase virulence. To study the role of domestic poultry in the evolution of AIV we compared replication of low pathogenic (LP AIV of subtypes H9N2, H7N7 and H6N8 in tracheal organ cultures (TOC and primary embryo fibroblast cultures of chicken, turkey, Pekin duck and homing pigeon. Virus strain-dependent and avian species-related differences between LPAIV were observed in growth kinetics and induction of ciliostasis in TOC. In particular, our data demonstrate high susceptibility to LPAIV of turkey TOC contrasted with low susceptibility of homing pigeon TOC. Serial virus passages in the cells of heterologous host species resulted in adaptive mutations in the AIV genome, especially in the receptor-binding site and protease cleavage site of the hemagglutinin. Our data highlight differences in susceptibility of different birds to AIV viruses and emphasizes potential role of poultry in the emergence of new virus variants.

Molecular epidemiology of circulating highly pathogenic avian influenza (H5N1) virus in chickens, in Bangladesh, 2007-2010

DEFF Research Database (Denmark)

Ahmed, Syed Sayeem Uddin; Themudo, Goncalo Espregueira Cruz; Christensen, Jens Peter

2012-01-01

Bangladesh has been severely hit by highly pathogenic avian influenza H5N1 (HPAI-H5N1). However, little is known about the genetic diversity and the evolution of the circulating viruses in Bangladesh. In the present study, we analyzed the hemagglutinin gene of 30 Bangladeshi chicken isolates from...... several amino acid substitutions, but they are not indicative of adaptation toward human infection. The Mantel correlation test confirmed significant correlation between genetic distances and temporal distances between the viruses. The Bayesian tree shows that isolates from waves 3 and 4 derived from...... virus in Bangladesh. Furthermore, the formation of a subclade capable of transmission to humans cannot be ruled out. The findings of this study might provide valuable information for future surveillance, prevention and control programme....

Evaluation of multiplex assay platforms for detection of influenza hemagglutinin subtype specific antibody responses.

Science.gov (United States)

Li, Zhu-Nan; Weber, Kimberly M; Limmer, Rebecca A; Horne, Bobbi J; Stevens, James; Schwerzmann, Joy; Wrammert, Jens; McCausland, Megan; Phipps, Andrew J; Hancock, Kathy; Jernigan, Daniel B; Levine, Min; Katz, Jacqueline M; Miller, Joseph D

2017-05-01

Influenza hemagglutination inhibition (HI) and virus microneutralization assays (MN) are widely used for seroprevalence studies. However, these assays have limited field portability and are difficult to fully automate for high throughput laboratory testing. To address these issues, three multiplex influenza subtype-specific antibody detection assays were developed using recombinant hemagglutinin antigens in combination with Chembio, Luminex ® , and ForteBio ® platforms. Assay sensitivity, specificity, and subtype cross-reactivity were evaluated using a panel of well characterized human sera. Compared to the traditional HI, assay sensitivity ranged from 87% to 92% and assay specificity in sera collected from unexposed persons ranged from 65% to 100% across the platforms. High assay specificity (86-100%) for A(H5N1) rHA was achieved for sera from exposed or unexposed to hetorosubtype influenza HAs. In contrast, assay specificity for A(H1N1)pdm09 rHA using sera collected from A/Vietnam/1204/2004 (H5N1) vaccinees in 2008 was low (22-30%) in all platforms. Although cross-reactivity against rHA subtype proteins was observed in each assay platform, the correct subtype specific responses were identified 78%-94% of the time when paired samples were available for analysis. These results show that high throughput and portable multiplex assays that incorporate rHA can be used to identify influenza subtype specific infections. Published by Elsevier B.V.

Molecular epidemiology of H9N2 influenza viruses in Northern Europe.

Science.gov (United States)

Lindh, Erika; Ek-Kommonen, Christine; Väänänen, Veli-Matti; Vaheri, Antti; Vapalahti, Olli; Huovilainen, Anita

2014-08-27

Low pathogenic avian influenza viruses are maintained in wild bird populations throughout the world. Avian influenza viruses are characterized by their efficient ability to reassort and adapt, which enables them to cross the species barrier and enhances their zoonotic potential. Influenza viruses of the H9N2 subtype appear endemic among poultry in Eurasia. They usually exist as low-pathogenic strains and circulate between wild bird populations, poultry and birds sold at live bird markets. Direct transmission of H9N2 viruses, with receptor specificities similar to human influenza strains, to pigs and humans has been reported on several occasions. H9N2 virus was first encountered in Finland in 2009, during routine screening of hunted wild waterfowl. The next year, H9N2 influenza viruses were isolated from wild birds on four occasions, including once from a farmed mallard. We have investigated the relationship between the reared and wild bird isolates by sequencing the hemagglutinin and the neuraminidase genes of the Finnish H9N2 viruses. Nucleotide sequence comparison and phylogenetic analyses indicate that H9N2 was transmitted from wild birds to reared birds in 2010, and that highly identical strains have been circulating in Europe during the last few years. Copyright © 2014 Elsevier B.V. All rights reserved.

Fully human broadly neutralizing monoclonal antibodies against influenza A viruses generated from the memory B cells of a 2009 pandemic H1N1 influenza vaccine recipient

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Hu, Weibin [Molecular Virus Unit, Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200025 (China); Chen, Aizhong [Key Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China); Miao, Yi [Shanghai Xuhui Central Hospital, Shanghai 200031 (China); Xia, Shengli [Center for Disease Control and Prevention of Henan Province, Zhengzhou 450016 (China); Ling, Zhiyang; Xu, Ke; Wang, Tongyan [Molecular Virus Unit, Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200025 (China); Xu, Ying; Cui, Jun; Wu, Hongqiang; Hu, Guiyu; Tian, Lin; Wang, Lingling [Key Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China); Shu, Yuelong [Chinese Center for Disease Control and Prevention, Beijing 102206 (China); Ma, Xiaowei [Hualan Biological Bacterin Company, Xinxiang 453003 (China); Xu, Bianli; Zhang, Jin [Center for Disease Control and Prevention of Henan Province, Zhengzhou 450016 (China); Lin, Xiaojun, E-mail: linxiaojun@hualan.com [Hualan Biological Bacterin Company, Xinxiang 453003 (China); Bian, Chao, E-mail: cbian@sibs.ac.cn [Key Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China); Sun, Bing, E-mail: bsun@sibs.ac.cn [Molecular Virus Unit, Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200025 (China); Key Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China)

2013-01-20

Whether the 2009 pandemic H1N1 influenza vaccine can induce heterosubtypic cross-protective anti-hemagglutinin (HA) neutralizing antibodies is an important issue. We obtained a panel of fully human monoclonal antibodies from the memory B cells of a 2009 pandemic H1N1 influenza vaccine recipient. Most of the monoclonal antibodies targeted the HA protein but not the HA1 fragment. Among the analyzed antibodies, seven mAbs exhibited neutralizing activity against several influenza A viruses of different subtypes. The conserved linear epitope targeted by the neutralizing mAbs (FIEGGWTGMVDGWYGYHH) is part of the fusion peptide on HA2. Our work suggests that a heterosubtypic neutralizing antibody response primarily targeting the HA stem region exists in recipients of the 2009 pandemic H1N1 influenza vaccine. The HA stem region contains various conserved neutralizing epitopes with the fusion peptide as an important one. This work may aid in the design of a universal influenza A virus vaccine.

Fully human broadly neutralizing monoclonal antibodies against influenza A viruses generated from the memory B cells of a 2009 pandemic H1N1 influenza vaccine recipient

International Nuclear Information System (INIS)

Hu, Weibin; Chen, Aizhong; Miao, Yi; Xia, Shengli; Ling, Zhiyang; Xu, Ke; Wang, Tongyan; Xu, Ying; Cui, Jun; Wu, Hongqiang; Hu, Guiyu; Tian, Lin; Wang, Lingling; Shu, Yuelong; Ma, Xiaowei; Xu, Bianli; Zhang, Jin; Lin, Xiaojun; Bian, Chao; Sun, Bing

2013-01-01

Whether the 2009 pandemic H1N1 influenza vaccine can induce heterosubtypic cross-protective anti-hemagglutinin (HA) neutralizing antibodies is an important issue. We obtained a panel of fully human monoclonal antibodies from the memory B cells of a 2009 pandemic H1N1 influenza vaccine recipient. Most of the monoclonal antibodies targeted the HA protein but not the HA1 fragment. Among the analyzed antibodies, seven mAbs exhibited neutralizing activity against several influenza A viruses of different subtypes. The conserved linear epitope targeted by the neutralizing mAbs (FIEGGWTGMVDGWYGYHH) is part of the fusion peptide on HA2. Our work suggests that a heterosubtypic neutralizing antibody response primarily targeting the HA stem region exists in recipients of the 2009 pandemic H1N1 influenza vaccine. The HA stem region contains various conserved neutralizing epitopes with the fusion peptide as an important one. This work may aid in the design of a universal influenza A virus vaccine.

Evidences Suggesting Involvement of Viruses in Oral Squamous Cell Carcinoma

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Gupta, Kanupriya; Metgud, Rashmi

2013-01-01

Oral cancer is one of the most common cancers and it constitutes a major health problem particularly in developing countries. Oral squamous cell carcinoma (OSCC) represents the most frequent of all oral neoplasms. Several risk factors have been well characterized to be associated with OSCC with substantial evidences. The etiology of OSCC is complex and involves many factors. The most clearly defined potential factors are smoking and alcohol, which substantially increase the risk of OSCC. However, despite this clear association, a substantial proportion of patients develop OSCC without exposure to them, emphasizing the role of other risk factors such as genetic susceptibility and oncogenic viruses. Some viruses are strongly associated with OSCC while the association of others is less frequent and may depend on cofactors for their carcinogenic effects. Therefore, the exact role of viruses must be evaluated with care in order to improve the diagnosis and treatment of OSCC. Although a viral association within a subset of OSCC has been shown, the molecular and histopathological characteristics of these tumors have yet to be clearly defined. PMID:24455418

Evidences Suggesting Involvement of Viruses in Oral Squamous Cell Carcinoma

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Kanupriya Gupta

2013-01-01

Full Text Available Oral cancer is one of the most common cancers and it constitutes a major health problem particularly in developing countries. Oral squamous cell carcinoma (OSCC represents the most frequent of all oral neoplasms. Several risk factors have been well characterized to be associated with OSCC with substantial evidences. The etiology of OSCC is complex and involves many factors. The most clearly defined potential factors are smoking and alcohol, which substantially increase the risk of OSCC. However, despite this clear association, a substantial proportion of patients develop OSCC without exposure to them, emphasizing the role of other risk factors such as genetic susceptibility and oncogenic viruses. Some viruses are strongly associated with OSCC while the association of others is less frequent and may depend on cofactors for their carcinogenic effects. Therefore, the exact role of viruses must be evaluated with care in order to improve the diagnosis and treatment of OSCC. Although a viral association within a subset of OSCC has been shown, the molecular and histopathological characteristics of these tumors have yet to be clearly defined.

Application and evaluation of RT-PCR-ELISA for the nucleoprotein and RT-PCR for detection of low-pathogenic H5 and H7 subtypes of avian influenza virus

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Dybkær, Karen; Munch, Mette; Handberg, Kurt J.

2004-01-01

Three 1-tube Reverse Transcriptase Polymerase Chain Reactions (RT-PCR) directed against the genes encoding the nucleoprotein (NP) and the H5 and H7 hemagglutinin (HA) gene, respectively, were used for detection of avian influenza virus (AIV) in various specimens. A total of 1,040 samples...... originating from chickens experimentally infected with 2 different low pathogenic avian influenza viruses, from domestic ducks and from wild aquatic birds were examined. The outcome of 1) the universal AIV RT-PCR including a PCR-enzyme-linked immunosorbent assay (ELISA) procedure directed against NP (NP RT...

Analysis of human infectious avian influenza virus: hemagglutinin genetic characteristics in Asia and Africa from 2004 to 2009.

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Zhang, Jirong; Lei, Fumin

2010-09-01

In the present study, we used nucleotide and protein sequences of avian influenza virus H5N1, which were obtained in Asia and Africa, analyzed HA proteins using ClustalX1.83 and MEGA4.0, and built a genetic evolutionary tree of HA nucleotides. The analysis revealed that the receptor specificity amino acid of A/HK/213/2003, A/Turkey/65596/2006 and etc mutated into QNG, which could bind with á-2, 3 galactose and á-2, 6 galactose. A mutation might thus take place and lead to an outbreak of human infections of avian influenza virus. The mutations of HA protein amino acids from 2004 to 2009 coincided with human infections provided by the World Health Organization, indicating a "low-high-highest-high-low" pattern. We also found out that virus strains in Asia are from different origins: strains from Southeast Asia and East Asia are of the same origin, whereas those from West Asia, South Asia and Africa descend from one ancestor. The composition of the phylogenetic tree and mutations of key site amino acids in HA proteins reflected the fact that the majority of strains are regional and long term, and virus diffusions exist between China, Laos, Malaysia, Indonesia, Azerbaijan, Turkey and Iraq. We would advise that pertinent vaccines be developed and due attention be paid to the spread of viruses between neighboring countries and the dangers of virus mutation and evolution. © 2010 ISZS, Blackwell Publishing and IOZ/CAS.

[Evolution of the receptor specificity of influenza viruses hemagglutinin in its transfer from duck to pig and man].

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Marinina, V P; Gambarian, A S; Tuzikov, A B; Pozynina, G V; Bovin, N V; Fediakina, I T; Iamnikova, S S; L'vov, D K; Matrosevich, M N

2004-01-01

The receptor properties of H1 and H2 influenza viruses (IV), isolated from duck, pig and man were studied by using the natural and synthetic sialoglycoconjugates. It was shown that viruses, isolated from different hosts, adapt themselves to the host cell receptors. The IV affinity was increasing to 6'sialy(N-acetyllactosamine) in proportion as amino acids (in positions 138, 190, 194 and 225), which are for avian IV, were increasingly replacing. Some of the porcine viruses display adaptation to the human receptor, i.e. 6'sialy(N-acetyllactosamine), however, all tested porcine influenza viruses, belonging to different evolution branches, acquired even more affinity to sulphated and fucozyled derivatives of 3'sialy(N-acetyliactosamine)-(Neu5AC alpha 2-3 g AL beta 1-4(fUC alpha 1-3)(6-sulfo)GlcNAc beta).

Adaptation of Pandemic H1N1 Influenza Viruses in Mice▿

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Ilyushina, Natalia A.; Khalenkov, Alexey M.; Seiler, Jon P.; Forrest, Heather L.; Bovin, Nicolai V.; Marjuki, Henju; Barman, Subrata; Webster, Robert G.; Webby, Richard J.

2010-01-01

The molecular mechanism by which pandemic 2009 influenza A viruses were able to sufficiently adapt to humans is largely unknown. Subsequent human infections with novel H1N1 influenza viruses prompted an investigation of the molecular determinants of the host range and pathogenicity of pandemic influenza viruses in mammals. To address this problem, we assessed the genetic basis for increased virulence of A/CA/04/09 (H1N1) and A/TN/1-560/09 (H1N1) isolates, which are not lethal for mice, in a new mammalian host by promoting their mouse adaptation. The resulting mouse lung-adapted variants showed significantly enhanced growth characteristics in eggs, extended extrapulmonary tissue tropism, and pathogenicity in mice. All mouse-adapted viruses except A/TN/1-560/09-MA2 grew faster and to higher titers in cells than the original strains. We found that 10 amino acid changes in the ribonucleoprotein (RNP) complex (PB2 E158G/A, PA L295P, NP D101G, and NP H289Y) and hemagglutinin (HA) glycoprotein (K119N, G155E, S183P, R221K, and D222G) controlled enhanced mouse virulence of pandemic isolates. HA mutations acquired during adaptation affected viral receptor specificity by enhancing binding to α2,3 together with decreasing binding to α2,6 sialyl receptors. PB2 E158G/A and PA L295P amino acid substitutions were responsible for the significant enhancement of transcription and replication activity of the mouse-adapted H1N1 variants. Taken together, our findings suggest that changes optimizing receptor specificity and interaction of viral polymerase components with host cellular factors are the major mechanisms that contribute to the optimal competitive advantage of pandemic influenza viruses in mice. These modulators of virulence, therefore, may have been the driving components of early evolution, which paved the way for novel 2009 viruses in mammals. PMID:20592084

Protection of White Leghorn chickens by U.S. emergency H5 vaccination against clade 2.3.4.4 H5N2 high pathogenicity avian influenza virus.

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Bertran, Kateri; Balzli, Charles; Lee, Dong-Hun; Suarez, David L; Kapczynski, Darrell R; Swayne, David E

2017-11-01

During December 2014-June 2015, the U.S. experienced a high pathogenicity avian influenza (HPAI) outbreak caused by clade 2.3.4.4 H5Nx Goose/Guangdong lineage viruses with devastating consequences for the poultry industry. Three vaccines, developed based on updating existing registered vaccines or currently licensed technologies, were evaluated for possible use: an inactivated reverse genetics H5N1 vaccine (rgH5N1) and an RNA particle vaccine (RP-H5), both containing the hemagglutinin gene of clade 2.3.4.4 strain, and a recombinant herpesvirus turkey vectored vaccine (rHVT-H5) containing the hemagglutinin gene of clade 2.2 strain. The efficacy of the three vaccines, alone or in combination, was assessed in White Leghorn chickens against clade 2.3.4.4 H5N2 HPAI virus challenge. In Study 1, single (rHVT-H5) and prime-boost (rHVT-H5+rgH5N1 or rHVT-H5+RP-H5) vaccination strategies protected chickens with high levels of protective immunity and significantly reduced virus shedding. In Study 2, single vaccination with either rgH5N1 or RP-H5 vaccines provided clinical protection in adult chickens and significantly reduced virus shedding. In Study 3, double rgH5N1 vaccination protected adult chickens from clinical signs and mortality when challenged 20weeks post-boost, with high levels of long-lasting protective immunity and significantly reduced virus shedding. These studies support the use of genetically related vaccines, possibly in combination with a broad protective priming vaccine, for emergency vaccination programs against clade 2.3.4.4 H5Nx HPAI virus in young and adult layer chickens. Published by Elsevier Ltd.

Deep sequencing of H7N8 avian influenza viruses from surveillance zone supports H7N8 high pathogenicity avian influenza was limited to a single outbreak farm in Indiana during 2016.

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Lee, Dong-Hun; Torchetti, Mia Kim; Killian, Mary Lea; Swayne, David E

2017-07-01

In mid-January 2016, an outbreak of H7N8 high-pathogenicity avian influenza virus (HPAIV) in commercial turkeys occurred in Indiana. Surveillance within the 10km control zone identified H7N8 low-pathogenicity avian influenza virus (LPAIV) in nine surrounding turkey flocks but no other HPAIV-affected premises. We sequenced four of the H7N8 HPAIV isolated from the single farm and nine LPAIV identified during control zone surveillance. Evaluation included phylogenetic network analysis indicating close relatedness across the HPAIV and LPAIV, and that the progenitor H7N8 LPAIV spread among the affected turkey farms in Indiana, followed by spontaneous mutation to HPAIV on a single premise through acquisition of three basic amino acids at the hemagglutinin cleavage site. Deep sequencing of the available viruses failed to identify subpopulations in either the HPAIV or LPAIV suggesting mutation to HPAIV likely occurred on a single farm and the HPAIV did not spread to epidemiologically linked LPAIV-affected farms. Published by Elsevier Inc.

Dual function of the hemagglutinin H5 fused to chicken CD154 in a ...

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Dual function of the hemagglutinin H5 fused to chicken CD154 in a potential strategy of DIVA against avian influenza disease: preliminary study. ... The PDF file you selected should load here if your Web browser has a PDF reader plug-in installed (for example, a recent version of Adobe Acrobat Reader). If you would like ...

Cross-protection against European swine influenza viruses in the context of infection immunity against the 2009 pandemic H1N1 virus: studies in the pig model of influenza.

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Qiu, Yu; De Hert, Karl; Van Reeth, Kristien

2015-09-24

Pigs are natural hosts for the same influenza virus subtypes as humans and are a valuable model for cross-protection studies with influenza. In this study, we have used the pig model to examine the extent of virological protection between a) the 2009 pandemic H1N1 (pH1N1) virus and three different European H1 swine influenza virus (SIV) lineages, and b) these H1 viruses and a European H3N2 SIV. Pigs were inoculated intranasally with representative strains of each virus lineage with 6- and 17-week intervals between H1 inoculations and between H1 and H3 inoculations, respectively. Virus titers in nasal swabs and/or tissues of the respiratory tract were determined after each inoculation. There was substantial though differing cross-protection between pH1N1 and other H1 viruses, which was directly correlated with the relatedness in the viral hemagglutinin (HA) and neuraminidase (NA) proteins. Cross-protection against H3N2 was almost complete in pigs with immunity against H1N2, but was weak in H1N1/pH1N1-immune pigs. In conclusion, infection with a live, wild type influenza virus may offer substantial cross-lineage protection against viruses of the same HA and/or NA subtype. True heterosubtypic protection, in contrast, appears to be minimal in natural influenza virus hosts. We discuss our findings in the light of the zoonotic and pandemic risks of SIVs.

[Molecular characterization of human influenza viruses--a look back on the last 10 years].

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Schweiger, Brunhilde

2006-01-01

Influenza A (H3N2) viruses and influenza B viruses have caused more than 90% of influenza infections in Germany during the last then years. Continuous and extensive antigenic variation was evident for both the hemagglutinin (HA) and neuraminidase (NA) surface proteins of H3N2 and influenza B viruses. Molecular characterisation revealed an ongoing genetic drift even in years when the antigenic profiles of circulating strains were indistinguishable from those of the previous season. Retrospective phylogenetic studies showed that viruses similar to vaccine strains circulated one or two years before a given strain was recommended as vaccine strain. New drift variants of H3N2 viruses with significantly changed antigenic features appeared during the seasons 1997/1998 and 2002/2003. Most influenza seasons were characterised by a co-circulation of at least two different lineages of H3N2 viruses. Genetic reassortment between H3N2 viruses belonging to separate lineages caused the different evolutionary pathways of the HA and viruses was responsible for the occurrence of H1N2 viruses during the season 2001/02. This new subtype has been detected only sporadically in Germany. The evolution of influenza B viruses was characterised by the re-emergence of B/Victoria/2/87-lineage viruses and their co-circulation with viruses of the B/Yamagata/16/88-lineage. Reassortant B viruses possessing a Victoria/87-lineage HA and a Yamagata/88-like NA were predominant in Germany during 2002/03 and 2004/05.

Identification of amino acid substitutions with compensational effects in the attachment protein of canine distemper virus.

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Sattler, Ursula; Khosravi, Mojtaba; Avila, Mislay; Pilo, Paola; Langedijk, Johannes P; Ader-Ebert, Nadine; Alves, Lisa A; Plattet, Philippe; Origgi, Francesco C

2014-07-01

The hemagglutinin (H) gene of canine distemper virus (CDV) encodes the receptor-binding protein. This protein, together with the fusion (F) protein, is pivotal for infectivity since it contributes to the fusion of the viral envelope with the host cell membrane. Of the two receptors currently known for CDV (nectin-4 and the signaling lymphocyte activation molecule [SLAM]), SLAM is considered the most relevant for host susceptibility. To investigate how evolution might have impacted the host-CDV interaction, we examined the functional properties of a series of missense single nucleotide polymorphisms (SNPs) naturally accumulating within the H-gene sequences during the transition between two distinct but related strains. The two strains, a wild-type strain and a consensus strain, were part of a single continental outbreak in European wildlife and occurred in distinct geographical areas 2 years apart. The deduced amino acid sequence of the two H genes differed at 5 residues. A panel of mutants carrying all the combinations of the SNPs was obtained by site-directed mutagenesis. The selected mutant, wild type, and consensus H proteins were functionally evaluated according to their surface expression, SLAM binding, fusion protein interaction, and cell fusion efficiencies. The results highlight that the most detrimental functional effects are associated with specific sets of SNPs. Strikingly, an efficient compensational system driven by additional SNPs appears to come into play, virtually neutralizing the negative functional effects. This system seems to contribute to the maintenance of the tightly regulated function of the H-gene-encoded attachment protein. Importance: To investigate how evolution might have impacted the host-canine distemper virus (CDV) interaction, we examined the functional properties of naturally occurring single nucleotide polymorphisms (SNPs) in the hemagglutinin gene of two related but distinct strains of CDV. The hemagglutinin gene encodes the

Evolution of highly pathogenic H5N1 avian influenza viruses in Vietnam between 2001 and 2007.

Directory of Open Access Journals (Sweden)

Xiu-Feng Wan

Full Text Available Highly pathogenic avian influenza (HPAI H5N1 viruses have caused dramatic economic losses to the poultry industry of Vietnam and continue to pose a serious threat to public health. As of June 2008, Vietnam had reported nearly one third of worldwide laboratory confirmed human H5N1 infections. To better understand the emergence, spread and evolution of H5N1 in Vietnam we studied over 300 H5N1 avian influenza viruses isolated from Vietnam since their first detection in 2001. Our phylogenetic analyses indicated that six genetically distinct H5N1 viruses were introduced into Vietnam during the past seven years. The H5N1 lineage that evolved following the introduction in 2003 of the A/duck/Hong Kong/821/2002-like viruses, with clade 1 hemagglutinin (HA, continued to predominate in southern Vietnam as of May 2007. A virus with a clade 2.3.4 HA newly introduced into northern Vietnam in 2007, reassorted with pre-existing clade 1 viruses, resulting in the emergence of novel genotypes with neuraminidase (NA and/or internal gene segments from clade 1 viruses. A total of nine distinct genotypes have been present in Vietnam since 2001, including five that were circulating in 2007. At least four of these genotypes appear to have originated in Vietnam and represent novel H5N1 viruses not reported elsewhere. Geographic and temporal analyses of H5N1 infection dynamics in poultry suggest that the majority of viruses containing new genes were first detected in northern Vietnam and subsequently spread to southern Vietnam after reassorting with pre-existing local viruses in northern Vietnam. Although the routes of entry and spread of H5N1 in Vietnam remain speculative, enhanced poultry import controls and virologic surveillance efforts may help curb the entry and spread of new HPAI viral genes.

Reassortment and evolution of current human influenza A and B viruses.

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Xu, Xiyan; Lindstrom, Stephen E; Shaw, Michael W; Smith, Catherine B; Hall, Henrietta E; Mungall, Bruce A; Subbarao, Kanta; Cox, Nancy J; Klimov, Alexander

2004-07-01

During the 2001-2002 influenza season, human influenza A (H1N2) reassortant viruses were detected globally. The hemagglutinin (HA) of these H1N2 viruses was similar to that of the A/New Caledonia/20/99 (H1N1) vaccine strain both antigenically and genetically, while their neuraminidase (NA) was antigenically and genetically related to that of recent human influenza H3N2 reference viruses such as A/Moscow/10/99. All six internal genes of the H1N2 reassortants originated from an H3N2 virus. After being detected only in eastern Asia during the past 10 years, Influenza B/Victoria/2/87 lineage viruses reappeared in many countries outside of Asia in 2001. Additionally, reassortant influenza B viruses possessing an HA similar to that of B/Shandong/7/97, a recent B/Victoria/2/87 lineage reference strain, and an NA closely related to that of B/Sichuan/379/99, a recent B/Yamagata/16/88 lineage reference strain, were isolated globally and became the predominant influenza B epidemic strain. The current influenza vaccine is expected to provide good protection against H1N2 viruses because it contains A/New Caledonia/20/99 (H1N1) and A/Panama/2007/99 (H3N2) like viruses whose H1 HA or N2 NA are antigenically similar to those of recent circulating H1N2 viruses. On the other hand, widespread circulation of influenza B Victoria lineage viruses required inclusion of a strain from this lineage in influenza vaccines for the 2002-2003 season.

Highly Pathogenic Avian Influenza A(H5N1) Viruses at the Animal-Human Interface in Vietnam, 2003-2010.

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Creanga, Adrian; Hang, Nguyen Le Khanh; Cuong, Vuong Duc; Nguyen, Ha T; Phuong, Hoang Vu Mai; Thanh, Le Thi; Thach, Nguyen Co; Hien, Pham Thi; Tung, Nguyen; Jang, Yunho; Balish, Amanda; Dang, Nguyen Hoang; Duong, Mai Thuy; Huong, Ngo Thu; Hoa, Do Ngoc; Tho, Nguyen Dang; Klimov, Alexander; Kapella, Bryan K; Gubareva, Larisa; Kile, James C; Hien, Nguyen Tran; Mai, Le Quynh; Davis, C Todd

2017-09-15

Mutation and reassortment of highly pathogenic avian influenza A(H5N1) viruses at the animal-human interface remain a major concern for emergence of viruses with pandemic potential. To understand the relationship of H5N1 viruses circulating in poultry and those isolated from humans, comprehensive phylogenetic and molecular analyses of viruses collected from both hosts in Vietnam between 2003 and 2010 were performed. We examined the temporal and spatial distribution of human cases relative to H5N1 poultry outbreaks and characterized the genetic lineages and amino acid substitutions in each gene segment identified in humans relative to closely related viruses from avian hosts. Six hemagglutinin clades and 8 genotypes were identified in humans, all of which were initially identified in poultry. Several amino acid mutations throughout the genomes of viruses isolated from humans were identified, indicating the potential for poultry viruses infecting humans to rapidly acquire molecular markers associated with mammalian adaptation and antiviral resistance. Published by Oxford University Press for the Infectious Diseases Society of America 2017. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Isolation and Characterization of Avian Influenza Viruses, Including Highly Pathogenic H5N1, from Poultry in Live Bird Markets in Hanoi, Vietnam, in 2001

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Nguyen, Doan C.; Uyeki, Timothy M.; Jadhao, Samadhan; Maines, Taronna; Shaw, Michael; Matsuoka, Yumiko; Smith, Catherine; Rowe, Thomas; Lu, Xiuhua; Hall, Henrietta; Xu, Xiyan; Balish, Amanda; Klimov, Alexander; Tumpey, Terrence M.; Swayne, David E.; Huynh, Lien P. T.; Nghiem, Ha K.; Nguyen, Hanh H. T.; Hoang, Long T.; Cox, Nancy J.; Katz, Jacqueline M.

2005-01-01

Since 1997, outbreaks of highly pathogenic (HP) H5N1 and circulation of H9N2 viruses among domestic poultry in Asia have posed a threat to public health. To better understand the extent of transmission of avian influenza viruses (AIV) to humans in Asia, we conducted a cross-sectional virologic study in live bird markets (LBM) in Hanoi, Vietnam, in October 2001. Specimens from 189 birds and 18 environmental samples were collected at 10 LBM. Four influenza A viruses of the H4N6 (n = 1), H5N2 (n = 1), and H9N3 (n = 2) subtypes were isolated from healthy ducks for an isolation frequency of over 30% from this species. Two H5N1 viruses were isolated from healthy geese. The hemagglutinin (HA) genes of these H5N1 viruses possessed multiple basic amino acid motifs at the cleavage site, were HP for experimentally infected chickens, and were thus characterized as HP AIV. These HA genes shared high amino acid identities with genes of other H5N1 viruses isolated in Asia during this period, but they were genetically distinct from those of H5N1 viruses isolated from poultry and humans in Vietnam during the early 2004 outbreaks. These viruses were not highly virulent for experimentally infected ducks, mice, or ferrets. These results establish that HP H5N1 viruses with properties similar to viruses isolated in Hong Kong and mainland China circulated in Vietnam as early as 2001, suggest a common source for H5N1 viruses circulating in these Asian countries, and provide a framework to better understand the recent widespread emergence of HP H5N1 viruses in Asia. PMID:15767421

Protection of guinea pigs by vaccination with a recombinant swinepox virus co-expressing HA1 genes of swine H1N1 and H3N2 influenza viruses.

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Xu, Jiarong; Yang, Deji; Huang, Dongyan; Xu, Jiaping; Liu, Shichao; Lin, Huixing; Zhu, Haodan; Liu, Bao; Lu, Chengping

2013-03-01

Swine influenza (SI) is an acute respiratory infectious disease of swine caused by swine influenza virus (SIV). SIV is not only an important respiratory pathogen in pigs but also a potent threat to human health. Here, we report the construction of a recombinant swinepox virus (rSPV/H3-2A-H1) co-expressing hemagglutinin (HA1) of SIV subtypes H1N1 and H3N2. Immune responses and protection efficacy of the rSPV/H3-2A-H1 were evaluated in guinea pigs. Inoculation of rSPV/H3-2A-H1 yielded neutralizing antibodies against SIV H1N1 and H3N2. The IFN-γ and IL-4 concentrations in the supernatant of lymphocytes stimulated with purified SIV HA1 antigen were significantly higher (P guinea pigs against SIV H1N1 or H3N2 challenge was observed. No SIV shedding was detected from guinea pigs vaccinated with rSPV/H3-2A-H1 after challenge. Most importantly, the guinea pigs immunized with rSPV/H3-2A-H1 did not show gross and micrographic lung lesions. However, the control guinea pigs experienced distinct gross and micrographic lung lesions at 7 days post-challenge. Our data suggest that the recombinant swinepox virus encoding HA1 of SIV H1N1 and H3N2 might serve as a promising candidate vaccine for protection against SIV H1N1 and H3N2 infections.

Surveillance programs in Denmark has revealed the circulation of novel reassortant influenza A viruses in swine

DEFF Research Database (Denmark)

Larsen, Lars Erik; Hjulsager, Charlotte Kristiane; Trebbien, Ramona

2014-01-01

avH1N1 and H3N2 which is different from the dominating European H1N2 subtype (1). The prevalence of the H1N1pdm09 virus in swine has increased since 2009 in some countries including Denmark. Here we present the results of the national passive surveillance program on influenza in swine performed from...... by the combination of the gene segments hemagglutinin (HA) and neuraminidase (NA). In most European countries, the avian-like (av)H1N1, the 2009 pandemic variant (H1N1pdm09), H1N2 and H3N2 subtypes have constituted the dominating SIV subtypes during recent years. In Denmark, the H1N2 subtype is a reassortant between......Swine influenza is a respiratory disease caused by multiple subtypes of influenza A virus. Swine influenza virus (SIV) is enzootic in swine populations in Europe, Asia, North and South America. The influenza A virus genome consist of eight distinct gene segments and SIV subtypes are defined...

Detection of Influenza C Viruses Among Outpatients and Patients Hospitalized for Severe Acute Respiratory Infection, Minnesota, 2013-2016.

Science.gov (United States)

Thielen, Beth K; Friedlander, Hannah; Bistodeau, Sarah; Shu, Bo; Lynch, Brian; Martin, Karen; Bye, Erica; Como-Sabetti, Kathryn; Boxrud, David; Strain, Anna K; Chaves, Sandra S; Steffens, Andrea; Fowlkes, Ashley L; Lindstrom, Stephen; Lynfield, Ruth

2018-03-19

Existing literature suggests that influenza C typically causes mild respiratory tract disease. However, clinical and epidemiological data are limited. Four outpatient clinics and 3 hospitals submitted clinical data and respiratory specimens through a surveillance network for acute respiratory infection (ARI) from May 2013 through December 2016. Specimens were tested using multitarget nucleic acid amplification for 19-22 respiratory pathogens, including influenza C. Influenza C virus was detected among 59 of 10 202 (0.58%) hospitalized severe ARI cases and 11 of 2282 (0.48%) outpatients. Most detections occurred from December to March, 73% during the 2014-2015 season. Influenza C detections occurred among patients of all ages, with rates being similar between inpatients and outpatients. The highest rate of detection occurred among children aged 6-24 months (1.2%). Among hospitalized cases, 7 required intensive care. Medical comorbidities were reported in 58% of hospitalized cases and all who required intensive care. At least 1 other respiratory pathogen was detected in 40 (66%) cases, most commonly rhinovirus/enterovirus (25%) and respiratory syncytial virus (20%). The hemagglutinin-esterase-fusion gene was sequenced in 37 specimens, and both C/Kanagawa and C/Sao Paulo lineages were detected in inpatients and outpatients. We found seasonal circulation of influenza C with year-to-year variability. Detection was most frequent among young children but occurred in all ages. Some cases that were positive for influenza C, particularly those with comorbid conditions, had severe disease, suggesting a need for further study of the role of influenza C virus in the pathogenesis of respiratory disease.

Chemical Synthesis and In Vitro Evaluation of a Phage Display-Derived Peptide Active against Infectious Salmon Anemia Virus.

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Ojeda, Nicolás; Cárdenas, Constanza; Guzmán, Fanny; Marshall, Sergio H

2016-04-01

Infectious salmon anemia virus (ISAV) is the etiological agent of the disease by the same name and causes major losses in the salmon industry worldwide. Epizootic ISAV outbreaks have occurred in Norway and, to a lesser degree, in Canada. In 2007, an ISAV outbreak in Chile destroyed most of the seasonal production and endangered the entire Chilean salmon industry. None of the existing prophylactic approaches have demonstrated efficacy in providing absolute protection from or even a palliative effect on ISAV proliferation. Sanitary control measures for ISAV, based on molecular epidemiology data, have proven insufficient, mainly due to high salmon culture densities and a constant presence of a nonpathogenic strain of the virus. This report describes an alternative treatment approach based on interfering peptides selected from a phage display library. The screening of a phage display heptapeptide library resulted in the selection of a novel peptide with significant in vitro antiviral activity against ISAV. This peptide specifically interacted with the viral hemagglutinin-esterase protein, thereby impairing virus binding, with plaque reduction assays showing a significant reduction in viral yields. The identified peptide acts at micromolar concentrations against at least two different pathogenic strains of the virus, without detectable cytotoxic effects on the tested fish cells. Therefore, antiviral peptides represent a novel alternative for controlling ISAV and, potentially, other fish pathogens. Identifying novel methods for the efficient control of infectious diseases is imperative for the future of global aquaculture. The present study used a phage display heptapeptide library to identify a peptide with interfering activity against a key protein of the infectious salmon anemia virus (ISAV). A piscine orthomyxovirus, ISAV is a continuous threat to the commercial sustainability of cultured salmon production worldwide. The complex epidemiological strategy of this

Serologic evidence of influenza A (H14) virus introduction into North America

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Latorre-Margalef, Neus; Ramey, Andy M.; Fojtik, Alinde; Stallknecht, David E.

2015-01-01

Although a diverse population of influenza A viruses (IAVs) is maintained among ducks, geese, shorebirds, and gulls, not all of the 16 avian hemagglutinin (HA) subtypes are equally represented (1). The 14th HA subtype, commonly known as the H14 subtype, was historically limited to isolates from the former Soviet Union in the 1980s (2) and was not subsequently detected until 2010, when isolated in Wisconsin, USA from long-tailed ducks and a white-winged scoter (3–5). In the United States, the H14 subtype has since been isolated in California (6), Mississippi, and Texas (7); and has been reported in waterfowl in Guatemala (7). In this study, we examined whether there was serologic evidence of H14 spread among ducks in North America before (2006–2010) and after (2011–2014) the initial detection of the H14 subtype virus on this continent.

Avian influenza A virus subtype H5N2 in a red-lored Amazon parrot.

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Hawkins, Michelle G; Crossley, Beate M; Osofsky, Anna; Webby, Richard J; Lee, Chang-Won; Suarez, David L; Hietala, Sharon K

2006-01-15

A 3-month-old red-lored Amazon parrot (Amazona autumnalis autumnalis) was evaluated for severe lethargy. Avian influenza virus hemagglutinin subtype H5N2 with low pathogenicity was characterized by virus isolation, real-time reverse transcriptase PCR assay, chicken intravenous pathogenicity index, and reference sera. The virus was also determined to be closely related to a virus lineage that had been reported only in Mexico and Central America. The chick was admitted to the hospital and placed in quarantine. Supportive care treatment was administered. Although detection of H5 avian influenza virus in birds in the United States typically results in euthanasia of infected birds, an alternative strategy with strict quarantine measures and repeated diagnostic testing was used. The chick recovered from the initial clinical signs after 4 days and was released from quarantine 9 weeks after initial evaluation after 2 consecutive negative virus isolation and real-time reverse transcriptase PCR assay results. To the authors' knowledge, this is the first report of H5N2 avian influenza A virus isolated from a psittacine bird and represents the first introduction of this virus into the United States, most likely by illegal importation of psittacine birds. Avian influenza A virus should be considered as a differential diagnosis for clinical signs of gastrointestinal tract disease in psittacine birds, especially in birds with an unknown history of origin. Although infection with avian influenza virus subtype H5 is reportable, destruction of birds is not always required.

Infection and Replication of Influenza Virus at the Ocular Surface.

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Creager, Hannah M; Kumar, Amrita; Zeng, Hui; Maines, Taronna R; Tumpey, Terrence M; Belser, Jessica A

2018-04-01

Although influenza viruses typically cause respiratory tract disease, some viruses, particularly those with an H7 hemagglutinin, have been isolated from the eyes of conjunctivitis cases. Previous work has shown that isolates of multiple subtypes from both ocular and respiratory infections are capable of replication in human ex vivo ocular tissues and corneal or conjunctival cell monolayers, leaving the determinants of ocular tropism unclear. Here, we evaluated the effect of several variables on tropism for ocular cells cultured in vitro and examined the potential effect of the tear film on viral infectivity. All viruses tested were able to replicate in primary human corneal epithelial cell monolayers subjected to aerosol inoculation. The temperature at which cells were cultured postinoculation minimally affected infectivity. Replication efficiency, in contrast, was reduced at 33°C relative to that at 37°C, and this effect was slightly greater for the conjunctivitis isolates than for the respiratory ones. With the exception of a seasonal H3N2 virus, the subset of viruses studied in multilayer corneal tissue constructs also replicated productively after either aerosol or liquid inoculation. Human tears significantly inhibited the hemagglutination of both ocular and nonocular isolates, but the effect on viral infectivity was more variable, with tears reducing the infectivity of nonocular isolates more than ocular isolates. These data suggest that most influenza viruses may be capable of establishing infection if they reach the surface of ocular cells but that this is more likely for ocular-tropic viruses, as they are better able to maintain their infectivity during passage through the tear film. IMPORTANCE The potential spread of zoonotic influenza viruses to humans represents an important threat to public health. Unfortunately, despite the importance of cellular and tissue tropism to pathogenesis, determinants of influenza virus tropism have yet to be fully

Viral RNA Degradation and Diffusion Act as a Bottleneck for the Influenza A Virus Infection Efficiency.

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Max Schelker

2016-10-01

Full Text Available After endocytic uptake, influenza viruses transit early endosomal compartments and eventually reach late endosomes. There, the viral glycoprotein hemagglutinin (HA triggers fusion between endosomal and viral membrane, a critical step that leads to release of the viral segmented genome destined to reach the cell nucleus. Endosomal maturation is a complex process involving acidification of the endosomal lumen as well as endosome motility along microtubules. While the pH drop is clearly critical for the conformational change and membrane fusion activity of HA, the effect of intracellular transport dynamics on the progress of infection remains largely unclear. In this study, we developed a comprehensive mathematical model accounting for the first steps of influenza virus infection. We calibrated our model with experimental data and challenged its predictions using recombinant viruses with altered pH sensitivity of HA. We identified the time point of virus-endosome fusion and thereby the diffusion distance of the released viral genome to the nucleus as a critical bottleneck for efficient virus infection. Further, we concluded and supported experimentally that the viral RNA is subjected to cytosolic degradation strongly limiting the probability of a successful genome import into the nucleus.

Genomic and Phylogenetic Characterization of Novel, Recombinant H5N2 Avian Influenza Virus Strains Isolated from Vaccinated Chickens with Clinical Symptoms in China

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Huaiying Xu

2015-02-01

Full Text Available Infection of poultry with diverse lineages of H5N2 avian influenza viruses has been documented for over three decades in different parts of the world, with limited outbreaks caused by this highly pathogenic avian influenza virus. In the present study, three avian H5N2 influenza viruses, A/chicken/Shijiazhuang/1209/2013, A/chicken/Chiping/0321/2014, and A/chicken/Laiwu/0313/2014, were isolated from chickens with clinical symptoms of avian influenza. Complete genomic and phylogenetic analyses demonstrated that all three isolates are novel recombinant viruses with hemagglutinin (HA and matrix (M genes derived from H5N1, and remaining genes derived from H9N2-like viruses. The HA cleavage motif in all three strains (PQIEGRRRKR/GL is characteristic of a highly pathogenic avian influenza virus strain. These results indicate the occurrence of H5N2 recombination and highlight the importance of continued surveillance of the H5N2 subtype virus and reformulation of vaccine strains.

Novel virus influenza A (H1N1sw in South-Eastern France, April-August 2009.

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Antoine Nougairède

Full Text Available BACKGROUND: In April 2009, the first cases of pandemic (H1N1-2009 influenza [H1N1sw] virus were detected in France. Virological surveillance was undertaken in reference laboratories of the seven French Defence Zones. METHODOLOGY/PRINCIPAL FINDINGS: We report results of virological analyses performed in the Public Hospitals of Marseille during the first months of the outbreak. (i Nasal swabs were tested using rapid influenza diagnostic test (RIDT and two RT-PCR assays. Epidemiological characteristics of the 99 first suspected cases were analyzed, including detection of influenza virus and 18 other respiratory viruses. During three months, a total of 1,815 patients were tested (including 236 patients infected H1N1sw virus and distribution in age groups and results of RIDT were analyzed. (ii 600 sera received before April 2009 and randomly selected from in-patients were tested by a standard hemagglutination inhibition assay for antibody to the novel H1N1sw virus. (iii One early (May 2009 and one late (July 2009 viral isolates were characterized by sequencing the complete hemagglutinine and neuraminidase genes. (iiii Epidemiological characteristics of a cluster of cases that occurred in July 2009 in a summer camp were analyzed. CONCLUSIONS/SIGNIFICANCE: This study presents new virological and epidemiological data regarding infection by the pandemic A/H1N1 virus in Europe. Distribution in age groups was found to be similar to that previously reported for seasonal H1N1. The first seroprevalence data made available for a European population suggest a previous exposure of individuals over 40 years old to influenza viruses antigenically related to the pandemic (H1N1-2009 virus. Genomic analysis indicates that strains harbouring a new amino-acid pattern in the neuraminidase gene appeared secondarily and tended to supplant the first strains. Finally, in contrast with previous reports, our data support the use of RIDT for the detection of infection in

Phylogenetic evidence of a new canine distemper virus lineage among domestic dogs in Colombia, South America.

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Espinal, Maria A; Díaz, Francisco J; Ruiz-Saenz, Julian

2014-08-06

Canine distemper virus (CDV) is a highly contagious viral disease of carnivores affecting both wild and domestic populations. The hemagglutinin gene, encoding for the attachment protein that determines viral tropism, shows high heterogeneity among strains, allowing for the distinction of ten different lineages distributed worldwide according to a geographic pattern. We obtained the sequences of the full-length H gene of 15 wild-type CDV strains circulating in domestic dog populations from the Aburrá Valley, Colombia. A phylogenetic analysis of H gene nucleotide sequences from Colombian CDV viruses along with field isolates from different geographic regions and vaccine strains was performed. Colombian wild-type viruses formed a distinct monophyletic cluster clearly separated from the previously identified wild-type and vaccine lineages, suggesting that a novel genetic variant, quite different from vaccines and other lineages, is circulating among dog populations in the Aburrá Valley. We propose naming this new lineage as "South America 3". This information indicates that there are at least three different CDV lineages circulating in domestic and wild carnivore populations in South America. The first one, renamed Europe/South America 1, circulates in Brazil and Uruguay; the second, South America 2, appears to be restricted to Argentina; and the third, South America 3, which comprises all the strains characterized in this study, may also be circulating in other northern countries of South America. Copyright © 2014 Elsevier B.V. All rights reserved.

Immunogenicity of peptides of measles virus origin and influence of adjuvants.

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Halassy, Beata; Mateljak, Sanja; Bouche, Fabienne B; Pütz, Mike M; Muller, Claude P; Frkanec, Ruza; Habjanec, Lidija; Tomasić, Jelka

2006-01-12

Epitope-based peptide antigens have been under development for protection against measles virus. The immunogenicity of five peptides composed of the same B cell epitope (BCE) (H236-250 of the measles virus hemagglutinin), and different T cell epitopes of measles virus fusion protein (F421-435, F256-270, F288-302) and nucleoprotein (NP335-345) was studied in mice (subcutaneous immunisation). The adjuvant effects of peptidoglycan monomer (PGM), Montanide ISA 720 and 206 were also investigated. Results showed basic differences in peptide immunogenicity that were consistent with already described structural differences. PGM elevated peptide-specific IgG when applied together with four of five tested peptides. A strong synergistic effect was observed after co-immunisation of mice with a mixture containing all five chimeric peptides in small and equal amounts. Results revealed for the first time that immunisation with several peptides having the common BCE generated significantly higher levels of both anti-peptide and anti-BCE IgG in comparison to those obtained after immunisation with a single peptide in much higher quantity. Further improvement of immune response was obtained after incorporation of such a peptide mixture into oil-based adjuvants.

Structural basis of influenza virus fusion inhibition by the antiviral drug Arbidol

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Kadam, Rameshwar U.; Wilson, Ian A.

2016-12-21

The broad-spectrum antiviral drug Arbidol shows efficacy against influenza viruses by targeting the hemagglutinin (HA) fusion machinery. However, the structural basis of the mechanism underlying fusion inhibition by Arbidol has remained obscure, thereby hindering its further development as a specific and optimized influenza therapeutic. We determined crystal structures of Arbidol in complex with influenza virus HA from pandemic 1968 H3N2 and recent 2013 H7N9 viruses. Arbidol binds in a hydrophobic cavity in the HA trimer stem at the interface between two protomers. This cavity is distal to the conserved epitope targeted by broadly neutralizing stem antibodies and is ~16 Å from the fusion peptide. Arbidol primarily makes hydrophobic interactions with the binding site but also induces some conformational rearrangements to form a network of inter- and intraprotomer salt bridges. By functioning as molecular glue, Arbidol stabilizes the prefusion conformation of HA that inhibits the large conformational rearrangements associated with membrane fusion in the low pH of the endosome. This unique binding mode compared with the small-molecule inhibitors of other class I fusion proteins enhances our understanding of how small molecules can function as fusion inhibitors and guides the development of broad-spectrum therapeutics against influenza virus.

AcEST: BP919664 [AcEST

Lifescience Database Archive (English)

Full Text Available 5W9_9INFA Hemagglutinin OS=Influenza A virus (A/cinnamon teal/Bolivia/4537/2001(H...o BlastX Result : TrEMBL tr_hit_id Q0Q5W9 Definition tr|Q0Q5W9|Q0Q5W9_9INFA Hemagglutinin OS=Influenza A virus (A/cinnamon teal/Boliv...ia/4537/2001(H7N3)) Align length 51 Score (bit) 36.6 E-v

ANALISIS GEN HAEMAGGLUTININ PADA VIRUS CAMPAK LIAR

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Subangkit Subangkit

2015-05-01

Full Text Available AbstrakPenyakit Campak disebabkan oleh virus campak yang termasuk genus Morbilivirus dan Family Paramyxoviridae. Penyakit campak masih menjadi masalah kesehatan karena masih ditemukan Kejadian Luar Biasa (KLB di Indonesia. Salah satu penyebab terjadinya KLB tersebut diduga sebagaiakibat perbedaan antigenesitas antara strain vaksin yang digunakan dengan strain virus campak liar yang beredar di Indonesia. Penelitian ini bertujuan mendapatkan gambaran tentang karakteristik genetik gen Haemagglutinin virus campak liar yang ada di Indonesia. Spesimen yang digunakan sebanyak 27 isolat virus penyebab KLB dari 17 propinsi selama periode tahun 2003-2010. Isolat virus dilakukan pemeriksaan secara RT-PCR dan sekuensing dengan metode Sanger. Hasil sekuensing dianalisis dengan menggunakan perangkat lunak Bioedit 7.0 dan MEGA 4.0. Hasil penelitian didapatkan perbedaan 10 asam amino antara virus campak strain vaksin CAM-70 dan virus campak liar pada posisi D416N; K424T; V451M; N455T; V466I; I473T; F476L; Y481S atau Y481N; H495N; G505D. Kesimpulan penelitian ini adalah terdapat perbedaan karakteristik genetik antara virus campak liar di Indonesia berbeda dengan strain virus vaksin CAM-70.Kata kunci : Campak, Analisis Molekuler, Hemagglutinin, CD46AbstractMeasles is caused by virus belonging to the genus Morbilivirus and Family Paramyxoviridae. Measles is still a public health problem because outbreak of measles still found in Indonesia. Outbreak is suspected as a result of differences in antigenicity between vaccine strains used with wild-type measles virus strains circulating in Indonesia. This study aims to get genetic characteristics of wild-type measles virus haemagglutinin gene in Indonesia. The specimens were used 27 viral isolates from 17 provinces period 2003-2010. Viral isolates examined by RT-PCR and sequencing with Sanger method. Sequencing analysis were conducted using Bioedit 7.0 and MEGA 4.0 software. The results showed 10 amino acid differences

Rapid strategy for screening by pyrosequencing of influenza virus reassortants--candidates for live attenuated vaccines.

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Shcherbik, Svetlana V; Pearce, Nicholas C; Levine, Marnie L; Klimov, Alexander I; Villanueva, Julie M; Bousse, Tatiana L

2014-01-01

Live attenuated influenza vaccine viruses (LAIVs) can be generated by classical reassortment of gene segments between a cold adapted, temperature sensitive and attenuated Master Donor Virus (MDV) and a seasonal wild-type (wt) virus. The vaccine candidates contain hemagglutinin (HA) and neuraminidase (NA) genes derived from the circulating wt viruses and the remaining six genes derived from the MDV strains. Rapid, efficient selection of the viruses with 6∶2 genome compositions from the large number of genetically different viruses generated during reassortment is essential for the biannual production schedule of vaccine viruses. This manuscript describes a new approach for the genotypic analysis of LAIV reassortant virus clones based on pyrosequencing. LAIV candidate viruses were created by classical reassortment of seasonal influenza A (H3N2) (A/Victoria/361/2011, A/Ohio/02/2012, A/Texas/50/2012) or influenza A (H7N9) (A/Anhui/1/2013) wt viruses with the MDV A/Leningrad/134/17/57(H2N2). Using strain-specific pyrosequencing assays, mixed gene variations were detected in the allantoic progenies during the cloning procedure. The pyrosequencing analysis also allowed for estimation of the relative abundance of segment variants in mixed populations. This semi-quantitative approach was used for selecting specific clones for the subsequent cloning procedures. The present study demonstrates that pyrosequencing analysis is a useful technique for rapid and reliable genotyping of reassortants and intermediate clones during the preparation of LAIV candidates, and can expedite the selection of vaccine virus candidates.

Rapid strategy for screening by pyrosequencing of influenza virus reassortants--candidates for live attenuated vaccines.

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Svetlana V Shcherbik

Full Text Available BACKGROUND: Live attenuated influenza vaccine viruses (LAIVs can be generated by classical reassortment of gene segments between a cold adapted, temperature sensitive and attenuated Master Donor Virus (MDV and a seasonal wild-type (wt virus. The vaccine candidates contain hemagglutinin (HA and neuraminidase (NA genes derived from the circulating wt viruses and the remaining six genes derived from the MDV strains. Rapid, efficient selection of the viruses with 6∶2 genome compositions from the large number of genetically different viruses generated during reassortment is essential for the biannual production schedule of vaccine viruses. METHODOLOGY/PRINCIPAL FINDINGS: This manuscript describes a new approach for the genotypic analysis of LAIV reassortant virus clones based on pyrosequencing. LAIV candidate viruses were created by classical reassortment of seasonal influenza A (H3N2 (A/Victoria/361/2011, A/Ohio/02/2012, A/Texas/50/2012 or influenza A (H7N9 (A/Anhui/1/2013 wt viruses with the MDV A/Leningrad/134/17/57(H2N2. Using strain-specific pyrosequencing assays, mixed gene variations were detected in the allantoic progenies during the cloning procedure. The pyrosequencing analysis also allowed for estimation of the relative abundance of segment variants in mixed populations. This semi-quantitative approach was used for selecting specific clones for the subsequent cloning procedures. CONCLUSIONS/SIGNIFICANCE: The present study demonstrates that pyrosequencing analysis is a useful technique for rapid and reliable genotyping of reassortants and intermediate clones during the preparation of LAIV candidates, and can expedite the selection of vaccine virus candidates.

Epidemiology of canine distemper virus in wild raccoon dogs (Nyctereutes procyonoides) from South Korea.

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Cha, Se-Yeoun; Kim, Eun-Ju; Kang, Min; Jang, Sang-Ho; Lee, Hae-Beom; Jang, Hyung-Kwan

2012-09-01

Raccoon dogs (Nyctereutes procyonoides) are widespread and common in South Korea. In 2011, we obtained serum samples from 102 wild raccoon dogs to survey their exposure to canine distemper virus (CDV). Forty-five of the 102 animals (44.1%) were seropositive. Field cases of canine distemper in wild raccoon dogs from 2010 to 2011 were investigated. Fourteen cases of CDV infection were identified by a commercially available CDV antigen detection kit. These cases were used for virus isolation and molecular analysis. Sequence analysis of hemagglutinin genes indicated that all viruses isolated belonged to the Asia-2 genotype. H protein residues which are related to the receptor and host specificity (residues 530 and 549) were analyzed. A glutamic acid (E) residue is present at 530 in all isolates. At 549, a histidine (H) residue was found in five isolates and tyrosine (Y) residue was found in 6 isolates. Our study demonstrated that CDV infection was widespread in wild raccoon dogs in South Korea. Copyright © 2012 Elsevier Ltd. All rights reserved.

Synthetically derived bat influenza A-like viruses reveal a cell type- but not species-specific tropism.

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Moreira, Étori Aguiar; Locher, Samira; Kolesnikova, Larissa; Bolte, Hardin; Aydillo, Teresa; García-Sastre, Adolfo; Schwemmle, Martin; Zimmer, Gert

2016-10-24

Two novel influenza A-like viral genome sequences have recently been identified in Central and South American fruit bats and provisionally designated "HL17NL10" and "HL18NL11." All efforts to isolate infectious virus from bats or to generate these viruses by reverse genetics have failed to date. Recombinant vesicular stomatitis virus (VSV) encoding the hemagglutinin-like envelope glycoproteins HL17 or HL18 in place of the VSV glycoprotein were generated to identify cell lines that are susceptible to bat influenza A-like virus entry. More than 30 cell lines derived from various species were screened but only a few cell lines were found to be susceptible, including Madin-Darby canine kidney type II (MDCK II) cells. The identification of cell lines susceptible to VSV chimeras allowed us to recover recombinant HL17NL10 and HL18NL11 viruses from synthetic DNA. Both influenza A-like viruses established a productive infection in MDCK II cells; however, HL18NL11 replicated more efficiently than HL17NL10 in this cell line. Unlike conventional influenza A viruses, bat influenza A-like viruses started the infection preferentially at the basolateral membrane of polarized MDCK II cells; however, similar to conventional influenza A viruses, bat influenza A-like viruses were released primarily from the apical site. The ability of HL18NL11 or HL17NL10 viruses to infect canine and human cells might reflect a zoonotic potential of these recently identified bat viruses.

The antigenic property of the H5N1 avian influenza viruses isolated in central China

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Zou Wei

2012-08-01

Full Text Available Abstract Background Three influenza pandemics outbroke in the last century accompanied the viral antigen shift and drift, resulting in the change of antigenic property and the low cross protective ability of the existed antibody to the newly emerged pandemic virus, and eventually the death of millions of people. The antigenic characterizations of the viruses isolated in central China in 2004 and 2006–2007 were investigated in the present study. Results Hemagglutinin inhibition assay and neutralization assay displayed differential antigenic characteristics of the viruses isolated in central China in two periods (2004 and 2006–2007. HA genes of the viruses mainly located in two branches in phylogeny analysis. 53 mutations of the deduced amino acids of the HA genes were divided into 4 patterns. Mutations in pattern 2 and 3 showed the main difference between viruses isolated in 2004 and 2006–2007. Meanwhile, most amino acids in pattern 2 and 3 located in the globular head of the HA protein, and some of the mutations evenly distributed at the epitope sites. Conclusions The study demonstrated that a major antigenic drift had occurred in the viruses isolated in central China. And monitoring the antigenic property should be the priority in preventing the potential pandemic of H5N1 avian influenza virus.

Membrane fusion-competent virus-like proteoliposomes and proteinaceous supported bilayers made directly from cell plasma membranes.

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Costello, Deirdre A; Hsia, Chih-Yun; Millet, Jean K; Porri, Teresa; Daniel, Susan

2013-05-28

Virus-like particles are useful materials for studying virus-host interactions in a safe manner. However, the standard production of pseudovirus based on the vesicular stomatitis virus (VSV) backbone is an intricate procedure that requires trained laboratory personnel. In this work, a new strategy for creating virus-like proteoliposomes (VLPLs) and virus-like supported bilayers (VLSBs) is presented. This strategy uses a cell blebbing technique to induce the formation of nanoscale vesicles from the plasma membrane of BHK cells expressing the hemagglutinin (HA) fusion protein of influenza X-31. These vesicles and supported bilayers contain HA and are used to carry out single particle membrane fusion events, monitored using total internal reflection fluorescence microscopy. The results of these studies show that the VLPLs and VLSBs contain HA proteins that are fully competent to carry out membrane fusion, including the formation of a fusion pore and the release of fluorophores loaded into vesicles. This new strategy for creating spherical and planar geometry virus-like membranes has many potential applications. VLPLs could be used to study fusion proteins of virulent viruses in a safe manner, or they could be used as therapeutic delivery particles to transport beneficial proteins coexpressed in the cells to a target cell. VLSBs could facilitate high throughput screening of antiviral drugs or pathogen-host cell interactions.

Real-Time PCR Assay To Detect Smallpox Virus

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Sofi Ibrahim, M.; Kulesh, David A.; Saleh, Sharron S.; Damon, Inger K.; Esposito, Joseph J.; Schmaljohn, Alan L.; Jahrling, Peter B.

2003-01-01

We developed a highly sensitive and specific assay for the rapid detection of smallpox virus DNA on both the Smart Cycler and LightCycler platforms. The assay is based on TaqMan chemistry with the orthopoxvirus hemagglutinin gene used as the target sequence. With genomic DNA purified from variola virus Bangladesh 1975, the limit of detection was estimated to be approximately 25 copies on both machines. The assay was evaluated in a blinded study with 322 coded samples that included genomic DNA from 48 different isolates of variola virus; 25 different strains and isolates of camelpox, cowpox, ectromelia, gerbilpox, herpes, monkeypox, myxoma, rabbitpox, raccoonpox, skunkpox, vaccinia, and varicella-zoster viruses; and two rickettsial species at concentrations mostly ranging from 100 fg/μl to 1 ng/μl. Contained within those 322 samples were variola virus DNA, obtained from purified viral preparations, at concentrations of 1 fg/μl to 1 ng/μl. On the Smart Cycler platform, 2 samples with false-positive results were detected among the 116 samples not containing variola virus tested; i.e., the overall specificity of the assay was 98.3%. On the LightCycler platform, five samples with false-positive results were detected (overall specificity, 95.7%). Of the 206 samples that contained variola virus DNA ranging in concentrations from 100 fg/μl to 1 ng/μl, 8 samples were considered negative on the Smart Cycler platform and 1 sample was considered negative on the LightCycler platform. Thus, the clinical sensitivities were 96.1% for the Smart Cycler instrument and 99.5% for the LightCycler instrument. The vast majority of these samples were derived from virus-infected cell cultures and variola virus-infected tissues; thus, the DNA material contained both viral DNA and cellular DNA. Of the 43 samples that contained purified variola virus DNA ranging in concentration from 1 fg/μl to 1 ng/μl, the assay correctly detected the virus in all 43 samples on both the Smart Cycler

Detection of antibodies against H5 and H7 strains in birds: evaluation of influenza pseudovirus particle neutralization tests

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Sofie Wallerström

2014-01-01

Full Text Available Introduction: Avian influenza viruses circulate in bird populations, and it is important to maintain and uphold our knowledge of the viral strains that are currently of interest in this context. Here, we describe the use of hemagglutinin-pseudotype retroviruses based on highly pathogenic influenza viruses for the screening of avian sera for influenza A antibodies. Our aim was also to determine whether the pseudovirus neutralization tests that we assessed were sensitive and simple to use compared to the traditional methods, including hemagglutination inhibition assays and microneutralization tests. Material and methods: H5 and H7 pseudovirus neutralization tests were evaluated by using serum from infected rabbits. Subsequently, the assays were further investigated using a panel of serum samples from avian species. The panel contained samples that were seropositive for five different hemagglutinin subtypes as well as influenza A seronegative samples. Results and discussion: The results suggest that the pseudovirus neutralization test is an alternative to hemagglutination inhibition assays, as we observed comparable titers to those of both standard microneutralizations assays as well as hemagglutinin inhibition assays. When evaluated by a panel of avian sera, the method also showed its capability to recognize antibodies directed toward low-pathogenic H5 and H7. Hence, we conclude that it is possible to use pseudoviruses based on highly pathogenic avian influenza viruses to screen avian sera for antibodies directed against influenza A subtypes H5 and H7.

Cross-Neutralising Nanobodies Bind to a Conserved Pocket in the Hemagglutinin Stem Region Identified Using Yeast Display and Deep Mutational Scanning.

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Tiziano Gaiotto

Full Text Available Cross-neutralising monoclonal antibodies against influenza hemagglutinin (HA are of considerable interest as both therapeutics and diagnostic tools. We have recently described five different single domain antibodies (nanobodies which share this cross-neutralising activity and suggest their small size, high stability, and cleft binding properties may present distinct advantages over equivalent conventional antibodies. We have used yeast display in combination with deep mutational scanning to give residue level resolution of positions in the antibody-HA interface which are crucial for binding. In addition, we have mapped positions within HA predicted to have minimal effect on antibody binding when mutated. Our cross-neutralising nanobodies were shown to bind to a highly conserved pocket in the HA2 domain of A(H1N1pdm09 influenza virus overlapping with the fusion peptide suggesting their mechanism of action is through the inhibition of viral membrane fusion. We also note that the epitope overlaps with that of CR6261 and F10 which are human monoclonal antibodies in clinical development as immunotherapeutics. Although all five nanobodies mapped to the same highly conserved binding pocket we observed differences in the size of the epitope footprint which has implications in comparing the relative genetic barrier each nanobody presents to a rapidly evolving influenza virus. To further refine our epitope map, we have re-created naturally occurring mutations within this HA stem epitope and tested their effect on binding using yeast display. We have shown that a D46N mutation in the HA2 stem domain uniquely interferes with binding of R2b-E8. Further testing of this substitution in the context of full length purified HA from 1918 H1N1 pandemic (Spanish flu, 2009 H1N1 pandemic (swine flu and highly pathogenic avian influenza H5N1 demonstrated binding which correlated with D46 whereas binding to seasonal H1N1 strains carrying N46 was absent. In addition, our

Structure of Epstein-Barr Virus Glycoprotein 42 Suggests a Mechanism for Triggering Receptor-Activated Virus Entry

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Kirschner, Austin N.; Sorem, Jessica; Longnecker, Richard; Jardetzky, Theodore S.; (NWU); (Stanford-MED)

2009-05-26

Epstein-Barr virus requires glycoproteins gH/gL, gB, and gp42 to fuse its lipid envelope with B cells. Gp42 is a type II membrane protein consisting of a flexible N-terminal region, which binds gH/gL, and a C-terminal lectin-like domain that binds to the B-cell entry receptor human leukocyte antigen (HLA) class II. Gp42 triggers membrane fusion after HLA binding, a process that requires simultaneous binding to gH/gL and a functional hydrophobic pocket in the lectin domain adjacent to the HLA binding site. Here we present the structure of gp42 in its unbound form. Comparisons to the previously determined structure of a gp42:HLA complex reveals additional N-terminal residues forming part of the gH/gL binding site and structural changes in the receptor binding domain. Although the core of the lectin domain remains similar, significant shifts in two loops and an {alpha} helix bordering the essential hydrophobic pocket suggest a structural mechanism for triggering fusion.

Using multiple linear regression and physicochemical changes of amino acid mutations to predict antigenic variants of influenza A/H3N2 viruses.

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Cui, Haibo; Wei, Xiaomei; Huang, Yu; Hu, Bin; Fang, Yaping; Wang, Jia

2014-01-01

Among human influenza viruses, strain A/H3N2 accounts for over a quarter of a million deaths annually. Antigenic variants of these viruses often render current vaccinations ineffective and lead to repeated infections. In this study, a computational model was developed to predict antigenic variants of the A/H3N2 strain. First, 18 critical antigenic amino acids in the hemagglutinin (HA) protein were recognized using a scoring method combining phi (ϕ) coefficient and information entropy. Next, a prediction model was developed by integrating multiple linear regression method with eight types of physicochemical changes in critical amino acid positions. When compared to other three known models, our prediction model achieved the best performance not only on the training dataset but also on the commonly-used testing dataset composed of 31878 antigenic relationships of the H3N2 influenza virus.

Morphological and biochemical characterization of the membranous hepatitis C virus replication compartment.

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Paul, David; Hoppe, Simone; Saher, Gesine; Krijnse-Locker, Jacomine; Bartenschlager, Ralf

2013-10-01

Like all other positive-strand RNA viruses, hepatitis C virus (HCV) induces rearrangements of intracellular membranes that are thought to serve as a scaffold for the assembly of the viral replicase machinery. The most prominent membranous structures present in HCV-infected cells are double-membrane vesicles (DMVs). However, their composition and role in the HCV replication cycle are poorly understood. To gain further insights into the biochemcial properties of HCV-induced membrane alterations, we generated a functional replicon containing a hemagglutinin (HA) affinity tag in nonstructural protein 4B (NS4B), the supposed scaffold protein of the viral replication complex. By using HA-specific affinity purification we isolated NS4B-containing membranes from stable replicon cells. Complementing biochemical and electron microscopy analyses of purified membranes revealed predominantly DMVs, which contained viral proteins NS3 and NS5A as well as enzymatically active viral replicase capable of de novo synthesis of HCV RNA. In addition to viral factors, co-opted cellular proteins, such as vesicle-associated membrane protein-associated protein A (VAP-A) and VAP-B, that are crucial for viral RNA replication, as well as cholesterol, a major structural lipid of detergent-resistant membranes, are highly enriched in DMVs. Here we describe the first isolation and biochemical characterization of HCV-induced DMVs. The results obtained underline their central role in the HCV replication cycle and suggest that DMVs are sites of viral RNA replication. The experimental approach described here is a powerful tool to more precisely define the molecular composition of membranous replication factories induced by other positive-strand RNA viruses, such as picorna-, arteri- and coronaviruses.

Role of Poultry in the Spread of Novel H7N9 Influenza Virus in China

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Pantin-Jackwood, Mary J.; Miller, Patti J.; Spackman, Erica; Swayne, David E.; Susta, Leonardo; Costa-Hurtado, Mar

2014-01-01

ABSTRACT The recent outbreak of H7N9 influenza in China has resulted in many human cases with a high fatality rate. Poultry are the likely source of infection for humans on the basis of sequence analysis and virus isolations from live bird markets, but it is not clear which species of birds are most likely to be infected and shedding levels of virus sufficient to infect humans. Intranasal inoculation of chickens, Japanese quail, pigeons, Pekin ducks, Mallard ducks, Muscovy ducks, and Embden geese with 106 50% egg infective doses of the A/Anhui/1/2013 virus resulted in infection but no clinical disease signs. Virus shedding was much higher and prolonged in quail and chickens than in the other species. Quail effectively transmitted the virus to direct contacts, but pigeons and Pekin ducks did not. In all species, virus was detected at much higher titers from oropharyngeal swabs than cloacal swabs. The hemagglutinin gene from samples collected from selected experimentally infected birds was sequenced, and three amino acid differences were commonly observed when the sequence was compared to the sequence of A/Anhui/1/2013: N123D, N149D, and L217Q. Leucine at position 217 is highly conserved for human isolates and is associated with α2,6-sialic acid binding. Different amino acid combinations were observed, suggesting that the inoculum had viral subpopulations that were selected after passage in birds. These experimental studies corroborate the finding that certain poultry species are reservoirs of the H7N9 influenza virus and that the virus is highly tropic for the upper respiratory tract, so testing of bird species should preferentially be conducted with oropharyngeal swabs for the best sensitivity. IMPORTANCE The recent outbreak of H7N9 influenza in China has resulted in a number of human infections with a high case fatality rate. The source of the viral outbreak is suspected to be poultry, but definitive data on the source of the infection are not available. This

Genetically distant American Canine distemper virus lineages have recently caused epizootics with somewhat different characteristics in raccoons living around a large suburban zoo in the USA

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Lednicky, John A; Dubach, Jean; Kinsel, Michael J; Meehan, Thomas P; Bocchetta, Maurizio; Hungerford, Laura L; Sarich, Nicolene A; Witecki, Kelley E; Braid, Michael D; Pedrak, Casandra; Houde, Christiane M

2004-01-01

Background Mortality rates have differed during distemper outbreaks among free-ranging raccoons (Procyon lotor) living around a large Chicago-area zoo, and appeared higher in year 2001 than in 1998 and 2000. We hypothesized that a more lethal variant of the local Canine distemper virus (CDV) lineage had emerged in 2001, and sought the genetic basis that led to increased virulence. However, a more complex model surfaced during preliminary analyses of CDV genomic sequences in infected tissues and of virus isolated in vitro from the raccoons. Results Phylogenetic analyses of subgenomic CDV fusion (F) -, phosphoprotein (P) -, and complete hemagglutinin (H) – gene sequences indicated that distinct American CDV lineages caused the distemper epizootics. The 1998 outbreak was caused by viruses that are likely from an old CDV lineage that includes CDV Snyder Hill and Lederle, which are CDV strains from the early 1950's. The 2000 and 2001 viruses appear to stem from the lineage of CDV A75/17, which was isolated in the mid 1970's. Only the 2001 viruses formed large syncytia in brain and/or lung tissue, and during primary isolation in-vitro in Vero cells, demonstrating at least one phenotypic property by which they differed from the other viruses. Conclusions Two different American CDV lineages caused the raccoon distemper outbreaks. The 1998 viruses are genetically distant to the 2000/2001 viruses. Since CDV does not cause persistent infections, the cycling of different CDV lineages within the same locale suggests multiple reintroductions of the virus to area raccoons. Our findings establish a precedent for determining whether the perceived differences in mortality rates are actual and attributable in part to inherent differences between CDV strains arising from different CDV lineages. PMID:15507154

Genetically distant American Canine distemper virus lineages have recently caused epizootics with somewhat different characteristics in raccoons living around a large suburban zoo in the USA

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Lednicky John A

2004-09-01

Full Text Available Abstract Background Mortality rates have differed during distemper outbreaks among free-ranging raccoons (Procyon lotor living around a large Chicago-area zoo, and appeared higher in year 2001 than in 1998 and 2000. We hypothesized that a more lethal variant of the local Canine distemper virus (CDV lineage had emerged in 2001, and sought the genetic basis that led to increased virulence. However, a more complex model surfaced during preliminary analyses of CDV genomic sequences in infected tissues and of virus isolated in vitro from the raccoons. Results Phylogenetic analyses of subgenomic CDV fusion (F -, phosphoprotein (P -, and complete hemagglutinin (H – gene sequences indicated that distinct American CDV lineages caused the distemper epizootics. The 1998 outbreak was caused by viruses that are likely from an old CDV lineage that includes CDV Snyder Hill and Lederle, which are CDV strains from the early 1950's. The 2000 and 2001 viruses appear to stem from the lineage of CDV A75/17, which was isolated in the mid 1970's. Only the 2001 viruses formed large syncytia in brain and/or lung tissue, and during primary isolation in-vitro in Vero cells, demonstrating at least one phenotypic property by which they differed from the other viruses. Conclusions Two different American CDV lineages caused the raccoon distemper outbreaks. The 1998 viruses are genetically distant to the 2000/2001 viruses. Since CDV does not cause persistent infections, the cycling of different CDV lineages within the same locale suggests multiple reintroductions of the virus to area raccoons. Our findings establish a precedent for determining whether the perceived differences in mortality rates are actual and attributable in part to inherent differences between CDV strains arising from different CDV lineages.

Induction of influenza-specific mucosal immunity by an attenuated recombinant Sendai virus.

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Thuc-vy L Le

2011-04-01

Full Text Available Many pathogens initiate infection at the mucosal surfaces; therefore, induction of mucosal immune responses is a first level of defense against infection and is the most powerful means of protection. Although intramuscular injection is widely used for vaccination and is effective at inducing circulating antibodies, it is less effective at inducing mucosal antibodies.Here we report a novel recombinant, attenuated Sendai virus vector (GP42-H1 in which the hemagglutinin (HA gene of influenza A virus was introduced into the Sendai virus genome as an additional gene. Infection of CV-1 cells by GP42-H1 resulted in cell surface expression of the HA protein. Intranasal immunization of mice with 1,000 plaque forming units (pfu of GP42-H1 induced HA-specific IgG and IgA antibodies in the blood, bronchoalveolar lavage fluid, fecal pellet extracts and saliva. The HA-specific antibody titer induced by GP42-H1 closely resembles the titer induced by sublethal infection by live influenza virus; however, in contrast to infection by influenza virus, immunization with GP42-H1 did not result in disease symptoms or the loss of body weight. In mice that were immunized with GP42-H1 and then challenged with 5LD(50 (1250 pfu of influenza virus, no significant weight loss was observed and other visual signs of morbidity were not detected.These results demonstrate that the GP42-H1 Sendai virus recombinant is able to confer full protection from lethal infection by influenza virus, supporting the conclusion that it is a safe and effective mucosal vaccine vector.

Intercontinental circulation of human influenza A(H1N2) reassortant viruses during the 2001-2002 influenza season.

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Xu, Xiyan; Smith, Catherine B; Mungall, Bruce A; Lindstrom, Stephen E; Hall, Henrietta E; Subbarao, Kanta; Cox, Nancy J; Klimov, Alexander

2002-11-15

Reassortant influenza A viruses bearing the H1 subtype of hemagglutinin (HA) and the N2 subtype of neuraminidase (NA) were isolated from humans in the United States, Canada, Singapore, Malaysia, India, Oman, Egypt, and several countries in Europe during the 2001-2002 influenza season. The HAs of these H1N2 viruses were similar to that of the A/New Caledonia/20/99(H1N1) vaccine strain both antigenically and genetically, and the NAs were antigenically and genetically related to those of recent human H3N2 reference strains, such as A/Moscow/10/99(H3N2). All 6 internal genes of the H1N2 reassortants examined originated from an H3N2 virus. This article documents the first widespread circulation of H1N2 reassortants on 4 continents. The current influenza vaccine is expected to provide good protection against H1N2 viruses, because it contains the A/New Caledonia/20/99(H1N1) and A/Moscow/10/99(H3N2)-like viruses, which have H1 and N2 antigens that are similar to those of recent H1N2 viruses.

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

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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

Broad-spectrum detection of H5 subtype influenza A viruses with a new fluorescent immunochromatography system.

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Akira Sakurai

Full Text Available Immunochromatography (IC is an antigen-detection assay that plays an important role in the rapid diagnosis of influenza virus because the protocol is short time and easy to use. Despite the usability of IC, the sensitivity is approximately 10(3 pfu per reaction. In addition, antigen-antibody interaction-based method cannot be used for the detection of influenza viruses with major antigenic change. In this study, we established the use of fluorescent immunochromatography (FLIC to detect a broad spectrum of H5 subtype influenza A viruses. This method has improved sensitivity 10-100 fold higher than traditional IC because of the use of fluorescent conjugated beads. Our Type-E FLIC kit detected all of the H5 subtype influenza viruses that were examined, as well as recombinant hemagglutinin (HA proteins (rHAs belonging to the Eurasian H5 subtype viruses and the Type-N diagnosed North American H5 subtype influenza A viruses. Thus, this kit has the improved potential to detect H5 subtype influenza viruses of different clades with both Type-E and Type-N FLIC kits. Compared with PCR-based diagnosis, FLIC has a strong advantage in usability, because the sample preparation required for FLIC is only mix-and-drop without any additional steps such as RNA extraction. Our results can provide new strategies against the spread and transmission of HPAI H5N1 viruses in birds and mammals including humans.

Evidence from lateral mobility studies for dynamic interactions of a mutant influenza hemagglutinin with coated pits.

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Fire, E; Zwart, D E; Roth, M G; Henis, Y I

1991-12-01

Replacement of cysteine at position 543 by tyrosine in the influenza virus hemagglutinin (HA) protein enables the endocytosis of the mutant protein (Tyr 543) through coated pits (Lazarovits, J., and M. G. Roth. 1988. Cell. 53:743-752). To investigate the interactions between Tyr 543 and the clathrin coats in the plasma membrane of live cells, we performed fluorescence photobleaching recovery measurements comparing the lateral mobilities of Tyr 543 (which enters coated pits) and wild-type HA (HA wt, which is excluded from coated pits), following their expression in CV-1 cells by SV-40 vectors. While both proteins exhibited the same high mobile fractions, the lateral diffusion rate of Tyr 543 was significantly slower than that of HA wt. Incubation of the cells in a sucrose-containing hypertonic medium, a treatment that disperses the membrane-associated coated pits, resulted in similar lateral mobilities for Tyr 543 and HA wt. These findings indicate that the lateral motion of Tyr 543 (but not of HA wt) is inhibited by transient interactions with coated pits (which are essentially immobile on the time scale of the lateral mobility measurements). Acidification of the cytoplasm by prepulsing the cells with NH4Cl (a treatment that arrests the pinching-off of coated vesicles from the plasma membrane and alters the clathrin lattice morphology) led to immobilization of a significant part of the Tyr 543 molecules, presumably due to their entrapment in coated pits for the entire duration of the lateral mobility measurement. Furthermore, in both untreated and cytosol-acidified cells, the restrictions on Tyr 543 mobility were less pronounced in the cold, suggesting that the mobility-restricting interactions are temperature dependent and become weaker at low temperatures. From these studies we conclude the following. (a) Lateral mobility measurements are capable of detecting interactions of transmembrane proteins with coated pits in intact cells. (b) The interactions of Tyr 543

Mouse Saliva Inhibits Transit of Influenza Virus to the Lower Respiratory Tract by Efficiently Blocking Influenza Virus Neuraminidase Activity.

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Gilbertson, Brad; Ng, Wy Ching; Crawford, Simon; McKimm-Breschkin, Jenny L; Brown, Lorena E

2017-07-15

We previously identified a novel inhibitor of influenza virus in mouse saliva that halts the progression of susceptible viruses from the upper to the lower respiratory tract of mice in vivo and neutralizes viral infectivity in MDCK cells. Here, we investigated the viral target of the salivary inhibitor by using reverse genetics to create hybrid viruses with some surface proteins derived from an inhibitor-sensitive strain and others from an inhibitor-resistant strain. These viruses demonstrated that the origin of the viral neuraminidase (NA), but not the hemagglutinin or matrix protein, was the determinant of susceptibility to the inhibitor. Comparison of the NA sequences of a panel of H3N2 viruses with differing sensitivities to the salivary inhibitor revealed that surface residues 368 to 370 (N2 numbering) outside the active site played a key role in resistance. Resistant viruses contained an EDS motif at this location, and mutation to either EES or KDS, found in highly susceptible strains, significantly increased in vitro susceptibility to the inhibitor and reduced the ability of the virus to progress to the lungs when the viral inoculum was initially confined to the upper respiratory tract. In the presence of saliva, viral strains with a susceptible NA could not be efficiently released from the surfaces of infected MDCK cells and had reduced enzymatic activity based on their ability to cleave substrate in vitro This work indicates that the mouse has evolved an innate inhibitor similar in function, though not in mechanism, to what humans have created synthetically as an antiviral drug for influenza virus. IMPORTANCE Despite widespread use of experimental pulmonary infection of the laboratory mouse to study influenza virus infection and pathogenesis, to our knowledge, mice do not naturally succumb to influenza. Here, we show that mice produce their own natural form of neuraminidase inhibitor in saliva that stops the virus from reaching the lungs, providing a

Genetic and antigenic characterization of influenza A virus circulating in Danish swine during the past decade

DEFF Research Database (Denmark)

Fobian, Kristina; Kirk, Isa Kristina; Breum, Solvej Østergaard

Influenza A virus has been endemic in Danish swine for the last 30 years, with H1N1 and H1N2 being the dominating subtypes. The purpose of this study was to investigate the genetic and antigenic evolution of the influenza viruses found in Danish swine during the last 10 years. A total of 78 samples...... to the complex epidemiology of circulating swine influenza virus in Denmark and indicates that vaccine development targeted against Danish H1N1 and H1N2 need only to include few components for the induction of cross protection against the predominant strains. The study was supported by grants from “European......-synonymous substitutions for H1, N1 and N2 were found to be in agreement with previously observed values for Eurasian swine lineages. Calculation of possible glycosylation sites in the hemagglutinin gene revealed that the H1N2 and H1N1 subtypes had three well conserved glycosylation sites in common. The results of the HI...

Humoral and cell-mediated immune responses in DNA immunized mink challenged with wild-type canine distemper virus.

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Nielsen, Line; Søgaard, Mette; Karlskov-Mortensen, Peter; Jensen, Trine Hammer; Jensen, Tove Dannemann; Aasted, Bent; Blixenkrone-Møller, Merete

2009-07-30

The aim of the study was to investigate the different phases of the immune response after DNA immunization with the hemagglutinin and nucleoprotein genes from canine distemper virus (CDV). Although attenuated live CDV vaccines have effectively reduced the incidence of disease, canine distemper is still a problem worldwide. The broad host range of CDV creates a constant viral reservoir among wildlife animals. Our results demonstrated early humoral and cell-mediated immune responses (IFN-gamma) in DNA vaccinated mink compared to mock-vaccinated mink after challenge with a Danish wild-type CDV. The DNA vaccine-induced immunity protected the natural host against disease development.

H5N2 Highly Pathogenic Avian Influenza Viruses from the US 2014-2015 outbreak have an unusually long pre-clinical period in turkeys

OpenAIRE

Spackman, Erica; Pantin-Jackwood, Mary J.; Kapczynski, Darrell R.; Swayne, David E.; Suarez, David L.

2016-01-01

Background From December 2014 through June 2015, the US experienced the most costly highly pathogenic avian influenza (HPAI) outbreak to date. Most cases in commercial poultry were caused by an H5N2 strain which was a reassortant with 5 Eurasian lineage genes, including a clade 2.3.4.4 goose/Guangdong/1996 lineage hemagglutinin, and 3 genes from North American wild waterfowl low pathogenicity avian influenza viruses. The outbreak primarily affected turkeys and table-egg layer type chickens. T...

Detection by hemi-nested reverse transcription polymerase chain reaction and genetic characterization of wild type strains of Canine distemper virus in suspected infected dogs.

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Di Francesco, Cristina E; Di Francesco, Daniela; Di Martino, Barbara; Speranza, Roberto; Santori, Domenico; Boari, Andrea; Marsilio, Fulvio

2012-01-01

A new highly sensitive and specific hemi-nested reverse transcription polymerase chain reaction (RT-PCR) assay was applied to detect nucleoprotein (NP) gene of Canine distemper virus (CDV) in samples collected from dogs showing respiratory, gastrointestinal, and neurological signs. Thirty-eight out of 86 samples were positive suggesting that despite the vaccination, canine distemper may still represent a high risk to the canine population. The 968 base pair (bp) fragments from the hemagglutinin (H) gene of 10 viral strains detected in positive samples were amplified and analyzed by restriction fragment length polymorphism (RFLP) using AluI and PsiI enzymes in order to differentiate among vaccine and wild-type CDV strains and to characterize the field viral strains. The products of the both enzymatic digestions allowed identification all viruses as wild strains of CDV. In addition, the RFLP analysis with AluI provided additional information about the identity level among the strains analyzed on the basis of the positions of the cleavage site in the nucleotide sequences of the H gene. The method could be a more useful and simpler method for molecular studies of CDV strains.

Punctuated Evolution of Influenza Virus Neuraminidase (A/H1N1 under Opposing Migration and Vaccination Pressures

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J. C. Phillips

2014-01-01

Full Text Available Influenza virus contains two highly variable envelope glycoproteins, hemagglutinin (HA and neuraminidase (NA. The structure and properties of HA, which is responsible for binding the virus to the cell that is being infected, change significantly when the virus is transmitted from avian or swine species to humans. Here we focus first on the simpler problem of the much smaller human individual evolutionary amino acid mutational changes in NA, which cleaves sialic acid groups and is required for influenza virus replication. Our thermodynamic panorama shows that very small amino acid changes can be monitored very accurately across many historic (1945–2011 Uniprot and NCBI strains using hydropathicity scales to quantify the roughness of water film packages. Quantitative sequential analysis is most effective with the fractal differential hydropathicity scale based on protein self-organized criticality (SOC. Our analysis shows that large-scale vaccination programs have been responsible for a very large convergent reduction in common influenza severity in the last century. Hydropathic analysis is capable of interpreting and even predicting trends of functional changes in mutation prolific viruses directly from amino acid sequences alone. An engineered strain of NA1 is described which could well be significantly less virulent than current circulating strains.

Wildlife Reservoirs of Canine Distemper Virus Resulted in a Major Outbreak in Danish Farmed Mink (Neovison vison)

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Trebbien, Ramona; Chriel, Mariann; Struve, Tina; Hjulsager, Charlotte Kristiane; Larsen, Gitte; Larsen, Lars Erik

2014-01-01

A major outbreak of canine distemper virus (CDV) in Danish farmed mink (Neovison vison) started in the late summer period of 2012. At the same time, a high number of diseased and dead wildlife species such as foxes, raccoon dogs, and ferrets were observed. To track the origin of the outbreak virus full-length sequencing of the receptor binding surface protein hemagglutinin (H) was performed on 26 CDV's collected from mink and 10 CDV's collected from wildlife species. Subsequent phylogenetic analyses showed that the virus circulating in the mink farms and wildlife were highly identical with an identity at the nucleotide level of 99.45% to 100%. The sequences could be grouped by single nucleotide polymorphisms according to geographical distribution of mink farms and wildlife. The signaling lymphocytic activation molecule (SLAM) receptor binding region in most viruses from both mink and wildlife contained G at position 530 and Y at position 549; however, three mink viruses had an Y549H substitution. The outbreak viruses clustered phylogenetically in the European lineage and were highly identical to wildlife viruses from Germany and Hungary (99.29% – 99.62%). The study furthermore revealed that fleas (Ceratophyllus sciurorum) contained CDV and that vertical transmission of CDV occurred in a wild ferret. The study provides evidence that wildlife species, such as foxes, play an important role in the transmission of CDV to farmed mink and that the virus may be maintained in the wild animal reservoir between outbreaks. PMID:24454897

Wildlife reservoirs of canine distemper virus resulted in a major outbreak in Danish farmed mink (Neovison vison.

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Ramona Trebbien

Full Text Available A major outbreak of canine distemper virus (CDV in Danish farmed mink (Neovison vison started in the late summer period of 2012. At the same time, a high number of diseased and dead wildlife species such as foxes, raccoon dogs, and ferrets were observed. To track the origin of the outbreak virus full-length sequencing of the receptor binding surface protein hemagglutinin (H was performed on 26 CDV's collected from mink and 10 CDV's collected from wildlife species. Subsequent phylogenetic analyses showed that the virus circulating in the mink farms and wildlife were highly identical with an identity at the nucleotide level of 99.45% to 100%. The sequences could be grouped by single nucleotide polymorphisms according to geographical distribution of mink farms and wildlife. The signaling lymphocytic activation molecule (SLAM receptor binding region in most viruses from both mink and wildlife contained G at position 530 and Y at position 549; however, three mink viruses had an Y549H substitution. The outbreak viruses clustered phylogenetically in the European lineage and were highly identical to wildlife viruses from Germany and Hungary (99.29% - 99.62%. The study furthermore revealed that fleas (Ceratophyllus sciurorum contained CDV and that vertical transmission of CDV occurred in a wild ferret. The study provides evidence that wildlife species, such as foxes, play an important role in the transmission of CDV to farmed mink and that the virus may be maintained in the wild animal reservoir between outbreaks.

Vaccination has minimal impact on the intrahost diversity of H3N2 influenza viruses.

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Kari Debbink

2017-01-01

Full Text Available While influenza virus diversity and antigenic drift have been well characterized on a global scale, the factors that influence the virus' rapid evolution within and between human hosts are less clear. Given the modest effectiveness of seasonal vaccination, vaccine-induced antibody responses could serve as a potent selective pressure for novel influenza variants at the individual or community level. We used next generation sequencing of patient-derived viruses from a randomized, placebo-controlled trial of vaccine efficacy to characterize the diversity of influenza A virus and to define the impact of vaccine-induced immunity on within-host populations. Importantly, this study design allowed us to isolate the impact of vaccination while still studying natural infection. We used pre-season hemagglutination inhibition and neuraminidase inhibition titers to quantify vaccine-induced immunity directly and to assess its impact on intrahost populations. We identified 166 cases of H3N2 influenza over 3 seasons and 5119 person-years. We obtained whole genome sequence data for 119 samples and used a stringent and empirically validated analysis pipeline to identify intrahost single nucleotide variants at ≥1% frequency. Phylogenetic analysis of consensus hemagglutinin and neuraminidase sequences showed no stratification by pre-season HAI and NAI titer, respectively. In our study population, we found that the vast majority of intrahost single nucleotide variants were rare and that very few were found in more than one individual. Most samples had fewer than 15 single nucleotide variants across the entire genome, and the level of diversity did not significantly vary with day of sampling, vaccination status, or pre-season antibody titer. Contrary to what has been suggested in experimental systems, our data indicate that seasonal influenza vaccination has little impact on intrahost diversity in natural infection and that vaccine-induced immunity may be only a

Insights into genetic diversity and biological propensities of potentially zoonotic avian influenza H9N2 viruses circulating in Egypt.

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Naguib, Mahmoud M; Arafa, Abdel-Satar; Parvin, Rokshana; Beer, Martin; Vahlenkamp, Thomas; Harder, Timm C

2017-11-01

Low pathogenic avian influenza (LPAI) H9N2 viruses have established endemic status in Egyptian poultry populations since 2012. Recently, four cases of human H9N2 virus infections in Egypt demonstrated the zoonotic potential of these viruses. Egyptian H9N2 viruses obtained from 2011 to 2014 phylogenetically grouped into three clusters (1-3) within subclade B of the G1 lineage. Antigenically, a close clustering of the Egyptian H9N2 viruses with other recent G1-B like H9N2 strains and a significant antigenic distance from viruses outside the G1-B lineage was evident. Recent Egyptian LPAIV H9N2 showed a tendency to increased binding with erythrocytes expressing α 2,6-linked sialic acid which correlated with the Q226L amino acid substitution at the receptor binding unit of the hemagglutinin (Q234L, H9 numbering). Sequence analyses of the N2 neuraminidase (NA) revealed substitutions in the NA hemadsorption site similar to the N2 of prepandemic H3N2/1968, but no distinct antigenic or functional characteristics of the H9N2 NA associated with increased zoonotic potential could be identified. Copyright © 2017 Elsevier Inc. All rights reserved.

Evaluation of the zoonotic potential of multiple subgroups of clade 2.3.4.4 influenza A (H5N8) virus.

Science.gov (United States)

Lee, Yu-Na; Lee, Eun-Kyoung; Song, Byung-Min; Heo, Gyeong-Beom; Woo, Sang-Hee; Cheon, Sun-Ha; Lee, Youn-Jeong

2018-03-01

Clade 2.3.4.4 H5N8 highly pathogenic avian influenza viruses (HPAIVs) have spread worldwide. Phylogenetic analysis identified two genetic groups of the H5N8 HPAIVs in South Korea; group A evolved further into four subgroups. Here, we examined the zoonotic potential, both in vivo and in vitro, of genetically distinct subgroups of H5N8 HPAIVs isolated in South Korea. When compared with other subgroups, A/mallard/Korea/H2102/2015 (H2102) virus caused relatively severe disease in mice at high doses. In ferrets, all H5N8 viruses replicated restrictively in the respiratory tract and did not induce significant clinical signs of influenza infection. In vitro studies, all viruses displayed a hemagglutinin phenotype that was poorly adapted for infection of mammals, although the H2102 virus exhibited higher replication kinetics at 33°C than the others. Although H5N8 HPAIVs have not yet acquired all the characteristics required for adaptation to mammals, their ability to evolve continuously underscores the need for timely risk assessment. Copyright © 2018 Elsevier Inc. All rights reserved.

PLAQUE ASSAY OF NEWCASTLE DISEASE VIRUS

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B. Sardjono

2012-09-01

Full Text Available The Newcastle disease virus (NDV was isolated from a 3 months-old indigenous chicken (buras or kampung chicken which showed clinical signs of Newcastle disease (ND. For viral isolation a small part of the spleen and lung were inoculated into 10 days-old embryonated chicken eggs. The physical characteristics of the isolate (A/120 were studied. The hemagglutination of chicken red blood cell showed slow elution, thermostability of hemagglutinin at 56°C was 120 minutes. The vims was able to agglutinate horse erythrocytes but not those of sheep. The biological characteristics on mean death time (MDT of embryonated chicken egg and plaque morphology on chicken embryo fibroblast (CEF primary cell cultures were studied. The MDT was 56 hours, the isolate was velogenic NDV. There were three different plaque morphologies on CEF : 2 mm clear plaques, 1 mm clear plaques, and minute clear plaques which were visible only with microscopic examination.

Re-emergence of H3N2 strains carrying potential neutralizing mutations at the N-linked glycosylation site at the hemagglutinin head, post the 2009 H1N1 pandemic.

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Ushirogawa, Hiroshi; Naito, Tadasuke; Tokunaga, Hirotoshi; Tanaka, Toshihiro; Nakano, Takashi; Terada, Kihei; Ohuchi, Masanobu; Saito, Mineki

2016-08-08

Seasonally prevalent H1N1 and H3N2 influenza A viruses have evolved by antigenic drift; this evolution has resulted in the acquisition of asparagine (N)-linked glycosylation sites (NGSs) in the globular head of hemagglutinin (HA), thereby affecting the antigenic and receptor-binding properties, as well as virulence. An epidemiological survey indicated that although the traditional seasonal H1N1 strain had disappeared, H3N2 became predominant again in the seasons (2010-11 and 2011-12) immediately following the H1N1 pandemic of 2009. Interestingly, although the 2009 pandemic H1N1 strain (H1N1pdm09) lacks additional NGSs, clinically isolated H3N2 strains obtained during these seasons gained N (Asn) residues at positions 45 and 144 of HA that forms additional NGSs. To investigate whether these NGSs are associated with re-emergence of H3N2 within the subtype, we tested the effect of amino acid substitutions on neutralizing activity by using the antisera raised against H3N2 strains with or without additional NGSs. Furthermore, because the N residue at position 144 of HA was identified as the site of mismatch between the vaccine and epidemic strains of 2011-2012, we generated mutant viruses by reverse genetics and tested the functional importance of this particular NGS for antibody-mediated neutralization by intranasal inoculation of mice. The results indicated that amino acid substitution at residue 144 significantly affected neutralization activity, acting as an escape mutation. Our data suggest that the newly acquired NGSs in the HA globular head may play an important role in the re-emergence of endemic seasonal H3N2 strain by aiding the escape from humoral immunity.

Recombinant infectious bronchitis virus (IBV) H120 vaccine strain expressing the hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) protects chickens against IBV and NDV challenge.

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Yang, Xin; Zhou, Yingshun; Li, Jianan; Fu, Li; Ji, Gaosheng; Zeng, Fanya; Zhou, Long; Gao, Wenqian; Wang, Hongning

2016-05-01

Infectious bronchitis (IB) and Newcastle disease (ND) are common viral diseases of chickens, which are caused by infectious bronchitis virus (IBV) and Newcastle disease virus (NDV), respectively. Vaccination with live attenuated strains of IBV-H120 and NDV-LaSota are important for the control of IB and ND. However, conventional live attenuated vaccines are expensive and result in the inability to differentiate between infected and vaccinated chickens. Therefore, there is an urgent need to develop new efficacious vaccines. In this study, using a previously established reverse genetics system, we generated a recombinant IBV virus based on the IBV H120 vaccine strain expressing the haemagglutinin-neuraminidase (HN) protein of NDV. The recombinant virus, R-H120-HN/5a, exhibited growth dynamics, pathogenicity and viral titers that were similar to those of the parental IBV H120, but it had acquired hemagglutination activity from NDV. Vaccination of SPF chickens with the R-H120-HN/5a virus induced a humoral response at a level comparable to that of the LaSota/H120 commercial bivalent vaccine and provided significant protection against challenge with virulent IBV and NDV. In summary, the results of this study indicate that the IBV H120 strain could serve as an effective tool for designing vaccines against IB and other infectious diseases, and the generation of IBV R-H120-HN/5a provides a solid foundation for the development of an effective bivalent vaccine against IBV and NDV.

Application of virus-like particles (VLP) to NMR characterization of viral membrane protein interactions

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Antanasijevic, Aleksandar; Kingsley, Carolyn [University of Illinois at Chicago, Department of Biochemistry and Molecular Genetics (United States); Basu, Arnab; Bowlin, Terry L. [Microbiotix Inc. (United States); Rong, Lijun [University of Illinois at Chicago, Department of Microbiology and Immunology (United States); Caffrey, Michael, E-mail: caffrey@uic.edu [University of Illinois at Chicago, Department of Biochemistry and Molecular Genetics (United States)

2016-03-15

The membrane proteins of viruses play critical roles in the virus life cycle and are attractive targets for therapeutic intervention. Virus-like particles (VLP) present the possibility to study the biochemical and biophysical properties of viral membrane proteins in their native environment. Specifically, the VLP constructs contain the entire protein sequence and are comprised of native membrane components including lipids, cholesterol, carbohydrates and cellular proteins. In this study we prepare VLP containing full-length hemagglutinin (HA) or neuraminidase (NA) from influenza and characterize their interactions with small molecule inhibitors. Using HA-VLP, we first show that VLP samples prepared using the standard sucrose gradient purification scheme contain significant amounts of serum proteins, which exhibit high potential for non-specific interactions, thereby complicating NMR studies of ligand-target interactions. We then show that the serum contaminants may be largely removed with the addition of a gel filtration chromatography step. Next, using HA-VLP we demonstrate that WaterLOGSY NMR is significantly more sensitive than Saturation Transfer Difference (STD) NMR for the study of ligand interactions with membrane bound targets. In addition, we compare the ligand orientation to HA embedded in VLP with that of recombinant HA by STD NMR. In a subsequent step, using NA-VLP we characterize the kinetic and binding properties of substrate analogs and inhibitors of NA, including study of the H274Y-NA mutant, which leads to wide spread resistance to current influenza antivirals. In summary, our work suggests that VLP have high potential to become standard tools in biochemical and biophysical studies of viral membrane proteins, particularly when VLP are highly purified and combined with control VLP containing native membrane proteins.

Large-scale analysis of B-cell epitopes on influenza virus hemagglutinin - implications for cross-reactivity of neutralizing antibodies

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Sun, Jing; Kudahl, Ulrich J.; Simon, Christian

2014-01-01

Influenza viruses continue to cause substantial morbidity and mortality worldwide. Fast gene mutation on surface proteins of influenza virus result in increasing resistance to current vaccines and available antiviral drugs. Broadly neutralizing antibodies (bnAbs) represent targets for prophylacti......, and differences between historical influenza strains, we enhance our preparedness and the ability to respond to the emerging pandemic threats....... a method to assess the likely cross-reactivity potential of bnAbs for influenza strains, either newly emerged or existing. Our method catalogs influenza strains by a new concept named discontinuous peptide, and then provide assessment of cross-reactivity. Potentially cross-reactive strains are those...

Experimental adaptation of wild-type canine distemper virus (CDV to the human entry receptor CD150.

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Maria Bieringer

Full Text Available Canine distemper virus (CDV, a close relative of measles virus (MV, is widespread and well known for its broad host range. When the goal of measles eradication may be achieved, and when measles vaccination will be stopped, CDV might eventually cross the species barrier to humans and emerge as a new human pathogen. In order to get an impression how fast such alterations may occur, we characterized required adaptive mutations to the human entry receptors CD150 (SLAM and nectin-4 as first step to infect human target cells. Recombinant wild-type CDV-A75/17(red adapted quickly to growth in human H358 epithelial cells expressing human nectin-4. Sequencing of the viral attachment proteins (hemagglutinin, H, and fusion protein, F genes revealed that no adaptive alteration was required to utilize human nectin-4. In contrast, the virus replicated only to low titres (10(2 pfu/ml in Vero cells expressing human CD150 (Vero-hSLAM. After three passages using these cells virus was adapted to human CD150 and replicated to high titres (10(5 pfu/ml. Sequence analyses revealed that only one amino acid exchange in the H-protein at position 540 Asp→Gly (D540G was required for functional adaptation to human CD150. Structural modelling suggests that the adaptive mutation D540G in H reflects the sequence alteration from canine to human CD150 at position 70 and 71 from Pro to Leu (P70L and Gly to Glu (G71E, and compensates for the gain of a negative charge in the human CD150 molecule. Using this model system our data indicate that only a minimal alteration, in this case one adaptive mutation, is required for adaptation of CDV to the human entry receptors, and help to understand the molecular basis why this adaptive mutation occurs.

Exploring the early stages of the pH-induced conformational change of influenza hemagglutinin.

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Zhou, Yu; Wu, Chao; Zhao, Lifeng; Huang, Niu

2014-10-01

Hemagglutinin (HA) mediates the membrane fusion process of influenza virus through its pH-induced conformational change. However, it remains challenging to study its structure reorganization pathways in atomic details. Here, we first applied continuous constant pH molecular dynamics approach to predict the pK(a) values of titratable residues in H2 subtype HA. The calculated net-charges in HA1 globular heads increase from 0e (pH 7.5) to +14e (pH 4.5), indicating that the charge repulsion drives the detrimerization of HA globular domains. In HA2 stem regions, critical pH sensors, such as Glu103(2), His18(1), and Glu89(1), are identified to facilitate the essential structural reorganizations in the fusing pathways, including fusion peptide release and interhelical loop transition. To probe the contribution of identified pH sensors and unveil the early steps of pH-induced conformational change, we carried out conventional molecular dynamics simulations in explicit water with determined protonation state for each titratable residue in different environmental pH conditions. Particularly, energy barriers involving previously uncharacterized hydrogen bonds and hydrophobic interactions are identified in the fusion peptide release pathway. Nevertheless, comprehensive comparisons across HA family members indicate that different HA subtypes might employ diverse pH sensor groups along with different fusion pathways. Finally, we explored the fusion inhibition mechanism of antibody CR6261 and small molecular inhibitor TBHQ, and discovered a novel druggable pocket in H2 and H5 subtypes. Our results provide the underlying mechanism for the pH-driven conformational changes and also novel insight for anti-flu drug development. © 2014 Wiley Periodicals, Inc.

Resting lymphocyte transduction with measles virus glycoprotein pseudotyped lentiviral vectors relies on CD46 and SLAM

International Nuclear Information System (INIS)

Zhou Qi; Schneider, Irene C.; Gallet, Manuela; Kneissl, Sabrina; Buchholz, Christian J.

2011-01-01

The measles virus (MV) glycoproteins hemagglutinin (H) and fusion (F) were recently shown to mediate transduction of resting lymphocytes by lentiviral vectors. MV vaccine strains use CD46 or signaling lymphocyte activation molecule (SLAM) as receptor for cell entry. A panel of H protein mutants derived from vaccine strain or wild-type MVs that lost or gained CD46 or SLAM receptor usage were investigated for their ability to mediate gene transfer into unstimulated T lymphocytes. The results demonstrate that CD46 is sufficient for efficient vector particle association with unstimulated lymphocytes. For stable gene transfer into these cells, however, both MV receptors were found to be essential.

Virological surveillance and phylogenetic analysis of the PB2 genes of influenza viruses isolated from wild water birds flying from their nesting lakes in Siberia to Hokkaido, Japan in autumn.

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Samad, Rozanah Asmah Abdul; Sakoda, Yoshihiro; Tsuda, Yoshimi; Simulundu, Edgar; Manzoor, Rashid; Okamatsu, Masatoshi; Ito, Kimihito; Kida, Hiroshi

2011-02-01

Recent introduction of H5N1 highly pathogenic avian influenza virus (HPAIV) in wild birds from poultry in Eurasia signaled the possibility that this virus may perpetuate in nature. Surveillance of avian influenza especially in migratory birds, therefore, has been conducted to provide information on the viruses brought by them to Hokkaido, Japan, from their nesting lakes in Siberia in autumn. During 2008-2009, 62 influenza viruses of 21 different combinations of hemagglutinin (HA) and neuraminidase (NA) subtypes were isolated. Up to September 2010, no HPAIV has been found, indicating that H5N1 HPAIV has not perpetuated at least dominantly in the lakes where ducks nest in summer in Siberia. The PB2 genes of 54 influenza viruses out of 283 influenza viruses isolated in Hokkaido in 2000-2009 were phylogenetically analysed. None of the genes showed close relation to those of H5N1 HPAIVs that were detected in wild birds found dead in Eurasia on the way back to their northern territory in spring.

Genetic diversity of the 2009 pandemic influenza A(H1N1 viruses in Finland.

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Niina Ikonen

Full Text Available BACKGROUND: In Finland, the first infections caused by the 2009 pandemic influenza A(H1N1 virus were identified on May 10. During the next three months almost all infections were found from patients who had recently traveled abroad. In September 2009 the pandemic virus started to spread in the general population, leading to localized outbreaks and peak epidemic activity was reached during weeks 43-48. METHODS/RESULTS: The nucleotide sequences of the hemagglutinin (HA and neuraminidase (NA genes from viruses collected from 138 patients were determined. The analyzed viruses represented mild and severe infections and different geographic regions and time periods. Based on HA and NA gene sequences, the Finnish pandemic viruses clustered in four groups. Finnish epidemic viruses and A/California/07/2009 vaccine virus strain varied from 2-8 and 0-5 amino acids in HA and NA molecules, respectively, giving a respective maximal evolution speed of 1.4% and 1.1%. Most amino acid changes in HA and NA molecules accumulated on the surface of the molecule and were partly located in antigenic sites. Three severe infections were detected with a mutation at HA residue 222, in two viruses with a change D222G, and in one virus D222Y. Also viruses with change D222E were identified. All Finnish pandemic viruses were sensitive to oseltamivir having the amino acid histidine at residue 275 of the neuraminidase molecule. CONCLUSIONS: The Finnish pandemic viruses were quite closely related to A/California/07/2009 vaccine virus. Neither in the HA nor in the NA were changes identified that may lead to the selection of a virus with increased epidemic potential or exceptionally high virulence. Continued laboratory-based surveillance of the 2009 pandemic influenza A(H1N1 is important in order to rapidly identify drug resistant viruses and/or virus variants with potential ability to cause severe forms of infection and an ability to circumvent vaccine-induced immunity.

Antigenic variation of H1N1, H1N2 and H3N2 swine influenza viruses in Japan and Vietnam.

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Takemae, Nobuhiro; Nguyen, Tung; Ngo, Long Thanh; Hiromoto, Yasuaki; Uchida, Yuko; Pham, Vu Phong; Kageyama, Tsutomu; Kasuo, Shizuko; Shimada, Shinichi; Yamashita, Yasutaka; Goto, Kaoru; Kubo, Hideyuki; Le, Vu Tri; Van Vo, Hung; Do, Hoa Thi; Nguyen, Dang Hoang; Hayashi, Tsuyoshi; Matsuu, Aya; Saito, Takehiko

2013-04-01

The antigenicity of the influenza A virus hemagglutinin is responsible for vaccine efficacy in protecting pigs against swine influenza virus (SIV) infection. However, the antigenicity of SIV strains currently circulating in Japan and Vietnam has not been well characterized. We examined the antigenicity of classical H1 SIVs, pandemic A(H1N1)2009 (A(H1N1)pdm09) viruses, and seasonal human-lineage SIVs isolated in Japan and Vietnam. A hemagglutination inhibition (HI) assay was used to determine antigenic differences that differentiate the recent Japanese H1N2 and H3N2 SIVs from the H1N1 and H3N2 domestic vaccine strains. Minor antigenic variation between pig A(H1N1)pdm09 viruses was evident by HI assay using 13 mAbs raised against homologous virus. A Vietnamese H1N2 SIV, whose H1 gene originated from a human strain in the mid-2000s, reacted poorly with post-infection ferret serum against human vaccine strains from 2000-2010. These results provide useful information for selection of optimal strains for SIV vaccine production.

[Phylogenetic analysis of human/swine/avian gene reassortant H1N2 influenza A virus isolated from a pig in China].

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Chen, Yixiang; Meng, Xueqiong; Liu, Qi; Huang, Xia; Huang, Shengbin; Liu, Cuiquan; Shi, Kaichuang; Guo, Jiangang; Chen, Fangfang; Hu, Liping

2008-04-01

Our aim in this study was to determine the genetic characterization and probable origin of the H1N2 swine influenza virus (A/Swine/Guangxi/13/2006) (Sw/GX/13/06) from lung tissue of a pig in Guangxi province, China. Eight genes of Sw/GX/13/06 were cloned and genetically analyzed. The hemagglutinin (HA), nucleoprotein (NP), matrix (M) and non-structural (NS) genes of Sw/GX/13/06 were most closely related to genes from the classical swine H1N1 influenza virus lineage. The neuraminidase (NA) and PB1 genes were most closely related to the corresponding genes from the human influenza H3N2 virus lineage. The remaining two genes PA and PB2 polymerase genes were most closely related to the genes from avian influenza virus lineage. Phylogenetic analyses revealed that Sw/GX/13/06 was a human/swine/avian H1N2 virus, and closely related to H1N2 viruses isolated from pigs in United States (1999-2001) and Korea (2002). To our knowledge, Sw/GX/13/06 was the first triple-reassortant H1N2 influenza A virus isolated from a pig in China. Whether the Sw/GX/13/06 has a potential threat to breeding farm and human health remains to be further investigated.

Zoonotic Risk, Pathogenesis, and Transmission of Avian-Origin H3N2 Canine Influenza Virus.

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Sun, Hailiang; Blackmon, Sherry; Yang, Guohua; Waters, Kaitlyn; Li, Tao; Tangwangvivat, Ratanaporn; Xu, Yifei; Shyu, Daniel; Wen, Feng; Cooley, Jim; Senter, Lucy; Lin, Xiaoxu; Jarman, Richard; Hanson, Larry; Webby, Richard; Wan, Xiu-Feng

2017-11-01

Two subtypes of influenza A virus (IAV), avian-origin canine influenza virus (CIV) H3N2 (CIV-H3N2) and equine-origin CIV H3N8 (CIV-H3N8), are enzootic in the canine population. Dogs have been demonstrated to seroconvert in response to diverse IAVs, and naturally occurring reassortants of CIV-H3N2 and the 2009 H1N1 pandemic virus (pdmH1N1) have been isolated. We conducted a thorough phenotypic evaluation of CIV-H3N2 in order to assess its threat to human health. Using ferret-generated antiserum, we determined that CIV-H3N2 is antigenically distinct from contemporary human H3N2 IAVs, suggesting that there may be minimal herd immunity in humans. We assessed the public health risk of CIV-H3N2 × pandemic H1N1 (pdmH1N1) reassortants by characterizing their in vitro genetic compatibility and in vivo pathogenicity and transmissibility. Using a luciferase minigenome assay, we quantified the polymerase activity of all possible 16 ribonucleoprotein (RNP) complexes (PB2, PB1, PA, NP) between CIV-H3N2 and pdmH1N1, identifying some combinations that were more active than either parental virus complex. Using reverse genetics and fixing the CIV-H3N2 hemagglutinin (HA), we found that 51 of the 127 possible reassortant viruses were viable and able to be rescued. Nineteen of these reassortant viruses had high-growth phenotypes in vitro , and 13 of these replicated in mouse lungs. A single reassortant with the NP and HA gene segments from CIV-H3N2 was selected for characterization in ferrets. The reassortant was efficiently transmitted by contact but not by the airborne route and was pathogenic in ferrets. Our results suggest that CIV-H3N2 reassortants may pose a moderate risk to public health and that the canine host should be monitored for emerging IAVs. IMPORTANCE IAV pandemics are caused by the introduction of novel viruses that are capable of efficient and sustained transmission into a human population with limited herd immunity. Dogs are a a potential mixing vessel for avian

Broadly-reactive human monoclonal antibodies elicited following pandemic H1N1 influenza virus exposure protect mice from highly pathogenic H5N1 challenge.

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Nachbagauer, Raffael; Shore, David; Yang, Hua; Johnson, Scott K; Gabbard, Jon D; Tompkins, S Mark; Wrammert, Jens; Wilson, Patrick C; Stevens, James; Ahmed, Rafi; Krammer, Florian; Ellebedy, Ali H

2018-06-13

Broadly cross-reactive antibodies that recognize conserved epitopes within the influenza virus hemagglutinin (HA) stalk domain are of particular interest for their potential use as therapeutic and prophylactic agents against multiple influenza virus subtypes including zoonotic virus strains. Here, we characterized four human HA stalk-reactive monoclonal antibodies (mAbs) for their binding breadth and affinity, in vitro neutralization capacity, and in vivo protective potential against an highly pathogenic avian influenza virus. The monoclonal antibodies were isolated from individuals shortly following infection with (70-1F02 and 1009-3B05) or vaccination against (05-2G02 and 09-3A01) A(H1N1)pdm09. Three of the mAbs bound HAs from multiple strains of group 1 viruses, and one mAb, 05-2G02, bound to both group 1 and group 2 influenza A HAs. All four antibodies prophylactically protected mice against a lethal challenge with the highly pathogenic A/Vietnam/1203/04 (H5N1) strain. Two mAbs, 70-1F02 and 09-3A01, were further tested for their therapeutic efficacy against the same strain and showed good efficacy in this setting as well. One mAb, 70-1F02, was co-crystallized with H5 HA and showed similar heavy chain only interactions as a the previously described anti-stalk antibody CR6261. Finally, we showed that antibodies that compete with these mAbs are prevalent in serum from an individual recently infected with A(H1N1)pdm09 virus. The antibodies described here can be developed into broad-spectrum antiviral therapeutics that could be used to combat infections with zoonotic or emerging pandemic influenza viruses. IMPORTANCE The rise in zoonotic infections of humans with emerging influenza viruses is a worldwide public health concern. The majority of recent zoonotic human influenza cases were caused by H7N9 and H5Nx viruses and were associated with high morbidity and mortality. In addition, seasonal influenza viruses are estimated to cause up to 650,000 deaths annually

Novel genotypes of H9N2 influenza A viruses isolated from poultry in Pakistan containing NS genes similar to highly pathogenic H7N3 and H5N1 viruses.

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Munir Iqbal

2009-06-01

Full Text Available The impact of avian influenza caused by H9N2 viruses in Pakistan is now significantly more severe than in previous years. Since all gene segments contribute towards the virulence of avian influenza virus, it was imperative to investigate the molecular features and genetic relationships of H9N2 viruses prevalent in this region. Analysis of the gene sequences of all eight RNA segments from 12 viruses isolated between 2005 and 2008 was undertaken. The hemagglutinin (HA sequences of all isolates were closely related to H9N2 viruses isolated from Iran between 2004 and 2007 and contained leucine instead of glutamine at position 226 in the receptor binding pocket, a recognised marker for the recognition of sialic acids linked alpha2-6 to galactose. The neuraminidase (NA of two isolates contained a unique five residue deletion in the stalk (from residues 80 to 84, a possible indication of greater adaptation of these viruses to the chicken host. The HA, NA, nucleoprotein (NP, and matrix (M genes showed close identity with H9N2 viruses isolated during 1999 in Pakistan and clustered in the A/Quail/Hong Kong/G1/97 virus lineage. In contrast, the polymerase genes clustered with H9N2 viruses from India, Iran and Dubai. The NS gene segment showed greater genetic diversity and shared a high level of similarity with NS genes from either H5 or H7 subtypes rather than with established H9N2 Eurasian lineages. These results indicate that during recent years the H9N2 viruses have undergone extensive genetic reassortment which has led to the generation of H9N2 viruses of novel genotypes in the Indian sub-continent. The novel genotypes of H9N2 viruses may play a role in the increased problems observed by H9N2 to poultry and reinforce the continued need to monitor H9N2 infections for their zoonotic potential.

Subtype-Specific Influenza A Virus Antibodies in Canada Geese (Branta canadensis)

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Kistler, Whitney M.; Stallknecht, David E.; DeLiberto, Thomas J.; Van Why, Kyle; Yabsley, Michael J.

2015-01-01

Historically, surveillance for influenza A viruses (IAVs) in wild birds has relied on viral detection assays. This was largely due to poor performance of serological assays in wild birds; however, recently developed commercial serological assays have improved the ability to detect IAV antibodies in wild birds. Serological surveillance for IAV antibodies in Canada geese (Branta canadensis) has shown that, despite a low prevalence of virus isolations, Canada geese are frequently exposed to IAVs and that exposure increases with latitude, which follows virus isolation prevalence patterns observed in dabbling ducks. The objectives of this study were to further evaluate IAV antibodies in Canada geese using a subtype-specific serological assay to determine if Canada geese are exposed to subtypes that commonly circulate in dabbling ducks. We collected serum samples from Canada geese in Minnesota, New Jersey, Pennsylvania, and Wisconsin and tested for antibodies to IAVs using a blocking ELISA. Positive samples were further tested by hemagglutination inhibition for 10 hemagglutinin IAV subtypes (H1–H10). Overall, we detected antibodies to NP in 24% (714/2,919) of geese. Antibodies to H3, H4, H5, and H6 subtypes predominated, with H5 being detected most frequently. A decrease in H5 HI antibody prevalence and titers was observed from 2009 to 2012. We also detected similar exposure pattern in Canada geese from New Jersey, Minnesota, Washington and Wisconsin. Based on the published literature, H3, H4, and H6 viruses are the most commonly reported IAVs from dabbling ducks. These results indicate that Canada geese also are frequently exposed to viruses of the same HA subtypes; however, the high prevalence of antibodies to H5 viruses was not expected as H5 IAVs are generally not well represented in reported isolates from ducks. PMID:25845755

Seasonal influenza split vaccines confer partial cross-protection against heterologous influenza virus in ferrets when combined with the CAF01 adjuvant

DEFF Research Database (Denmark)

Christensen, Dennis; Christensen, Jan P.; Korsholm, Karen S.

2018-01-01

Influenza epidemics occur annually, and estimated 5-10% of the adult population and 20-30% of children will become ill from influenza infection. Seasonal vaccines primarily work through the induction of neutralizing antibodies against the principal surface antigen hemagglutinin (HA). This important...... role of HA-specific antibodies explains why previous pandemics have emerged when new HAs have appeared in circulating human viruses. It has long been recognized that influenza virus-specific CD4(+) T cells are important in protection from infection through direct effector mechanisms or by providing...... help to B cells and CD8(+) T cells. However, the seasonal influenza vaccine is poor at inducing CD4(+) T-cell responses and needs to be combined with an adjuvant facilitating this response. In this study, we applied the ferret model to investigate the cross-protective efficacy of a heterologous...