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Sample records for h5 hemagglutinin protein

  1. 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

    ABSTRACT

    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.

  2. Acid Stability of the Hemagglutinin Protein Regulates H5N1 Influenza Virus Pathogenicity

    Energy Technology Data Exchange (ETDEWEB)

    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.

  3. Sensitization with vaccinia virus encoding H5N1 hemagglutinin restores immune potential against H5N1 influenza virus.

    Science.gov (United States)

    Yasui, Fumihiko; Itoh, Yasushi; Ikejiri, Ai; Kitabatake, Masahiro; Sakaguchi, Nobuo; Munekata, Keisuke; Shichinohe, Shintaro; Hayashi, Yukiko; Ishigaki, Hirohito; Nakayama, Misako; Sakoda, Yoshihiro; Kida, Hiroshi; Ogasawara, Kazumasa; Kohara, Michinori

    2016-11-28

    H5N1 highly pathogenic avian influenza (H5N1 HPAI) virus causes elevated mortality compared with seasonal influenza viruses like H1N1 pandemic influenza (H1N1 pdm) virus. We identified a mechanism associated with the severe symptoms seen with H5N1 HPAI virus infection. H5N1 HPAI virus infection induced a decrease of dendritic cell number in the splenic extrafollicular T-cell zone and impaired formation of the outer layers of B-cell follicles, resulting in insufficient levels of antibody production after infection. However, in animals vaccinated with a live recombinant vaccinia virus expressing the H5 hemagglutinin, infection with H5N1 HPAI virus induced parafollicular dendritic cell accumulation and efficient antibody production. These results indicate that a recombinant vaccinia encoding H5 hemagglutinin gene does not impair dendritic cell recruitment and can be a useful vaccine candidate.

  4. Purification and production of monospecific antibody to the hemagglutinin from Subtype H5N1 influenza virus

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    Simson Tarigan

    2010-12-01

    Full Text Available The purpose of this study was to purify the hemagglutinin from H5N1 virus and to generate monospecific antibody appropriate for production of sensitive and specific immunoassay for H5N1 avian influenza. For this purpose, a local isolate H5N1 virus (A/Ck/West Java/Hamd/2006 was propagated in chicken embryos. The viral pellet was dissolved in a Triton-X-100 solution, undissolved viral particles were pelleted by ultracentrifuge, and the supernatant containing viral surface glycoproteins (Hemagglutinin and neuraminidase was collected. The neuraminidase in the supernatant was absorbed by passing the supernatant through an Oxamic-acid-superose column. After dialyzing extensively, the filtrate was further fractionated with an anion exchange chromatography (Q-sepharose column. Proteins adsorbed by the column were eluted stepwisely with 0.10, 0.25, 0.25 and 0.75 M NaCl in 20 mM Tris, ph 8. Hemagglutinin (H5 was found to be eluted from the column with the 0.5 M NaCl elution buffer. The purified H5 was free from other viral proteins based on immunoassays using commercial antibodies to H5N1 nucleoprotein and neuraminidase. When used as ELISA’s coating antigen, the purified H5 proved to be sensitive and specific for hemagglutinin H5. Cross reactions with other type-A-influenza virus, H6, H7 dan H9, were negligibly low. For the production of monospecific antiserum, the purified H5 was separated with SDS-PAGE, the band containing the H5 monomer was cut out , homogenised and injected into rabbits. The antiserum was capable of detecting the presence of inactivated H5N1 virus in a very dilute suspension, with a detection limit of 0.04 heagglutination (HA unit. The purified hemagglutinin and the serum raised against it should be useful for developing specific, sensitive and affordable immunoassay for H5N1 avian influenza.

  5. 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 ...

  6. 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. ... 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 ...

  7. 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

  8. 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.

  9. 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

  10. 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...

  11. 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...

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

    Science.gov (United States)

    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.

  13. 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

  14. 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.

  15. 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.

  16. 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.

  17. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  19. Expression, identification, and immunological study of H5N1 influenza virus hemagglutinin (HA) protein%H5N1亚型流感病毒血凝素(HA)蛋白的表达鉴定及免疫原性研究

    Institute of Scientific and Technical Information of China (English)

    孟令楠; 任志广; 冀显亮; 于竟杰; 李元果; 李胜楠; 于志君; 孙伟阳; 国娇

    2016-01-01

    目的 为了获得H5N1HA蛋白,利用昆虫杆状病毒表达系统表达了A/meerkat/Shanghai/SH-1/2012(clade 2.3.2.1)的HA基因并获得重组H5N1亚型流感病毒HA蛋白. 方法 将H5N1亚型2.3.2.1谱系的HA蛋白基因扩增并克隆到pFastBacⅠ载体中,构建重组穿梭质粒,转染昆虫sf9细胞后获得重组杆状病毒,对大量培养后表达的H5N1 HA蛋白进行SDS-PAGE、Western blot和间接免疫荧光检测.分别在0d和14d用纯化的表达产物HA蛋白免疫小鼠,免疫两周后进行H5N1攻毒试验,记录小鼠体重变化率及生存率. 结果 测序鉴定重组表达载体构建正确;SDS-PAGE显示纯化的表达蛋白分子质量为65 ku;Western blot分析该蛋白能够被兔抗流感病毒IgG识别;鸡抗流感病毒抗体间接免疫荧光法测定HA蛋白的表达,在感染HA的昆虫细胞中出现特异性荧光;攻毒试验表明HA蛋白能够在小鼠体内诱导产生免疫应答并且保护H5N1流感病毒的致死性攻击. 结论 成功的表达了 H5N1的HA蛋白,并且此HA蛋白能成功的诱导小鼠体内的免疫反应,并起到良好的保护作用.

  20. Pleiotropic effects of hemagglutinin amino acid substitutions of H5 influenza escape mutants

    International Nuclear Information System (INIS)

    Rudneva, Irina A.; Timofeeva, Tatiana A.; Ignatieva, Anna V.; Shilov, Aleksandr A.; Krylov, Petr S.; Ilyushina, Natalia A.; Kaverin, Nikolai V.

    2013-01-01

    In the present study we assessed pleiotropic characteristics of the antibody-selected mutations. We examined pH optimum of fusion, temperatures of HA heat inactivation, and in vitro and in vivo replication kinetics of the previously obtained influenza H5 escape mutants. Our results showed that HA1 N142K mutation significantly lowered the pH of fusion optimum. Mutations of the escape mutants located in the HA lateral loop significantly affected H5 HA thermostability (P<0.05). HA changes at positions 131, 144, 145, and 156 and substitutions at positions 131, 142, 145, and 156 affected the replicative ability of H5 escape mutants in vitro and in vivo, respectively. Overall, a co-variation between antigenic specificity and different HA phenotypic properties has been demonstrated. We believe that the monitoring of pleiotropic effects of the HA mutations found in H5 escape mutants is essential for accurate prediction of mutants with pandemic potential. - Highlights: • HA1 N142K mutation significantly lowered the pH of fusion optimum. • Mutations located in the HA lateral loop significantly affected H5 HA thermostability. • HA changes at positions 131, 142, 144, 145, and 156 affected the replicative ability of H5 mutants. • Acquisition of glycosylation site could lead to the emergence of multiple pleiotropic effects

  1. Pleiotropic effects of hemagglutinin amino acid substitutions of H5 influenza escape mutants

    Energy Technology Data Exchange (ETDEWEB)

    Rudneva, Irina A.; Timofeeva, Tatiana A.; Ignatieva, Anna V.; Shilov, Aleksandr A.; Krylov, Petr S. [D.I. Ivanovsky Institute of Virology, 123098 Moscow (Russian Federation); Ilyushina, Natalia A., E-mail: Natalia.Ilyushina@fda.hhs.gov [FDA CDER, 29 Lincoln Drive, Bethesda, MD 20892 (United States); Kaverin, Nikolai V., E-mail: nik.kaverin@gmail.com [D.I. Ivanovsky Institute of Virology, 123098 Moscow (Russian Federation)

    2013-12-15

    In the present study we assessed pleiotropic characteristics of the antibody-selected mutations. We examined pH optimum of fusion, temperatures of HA heat inactivation, and in vitro and in vivo replication kinetics of the previously obtained influenza H5 escape mutants. Our results showed that HA1 N142K mutation significantly lowered the pH of fusion optimum. Mutations of the escape mutants located in the HA lateral loop significantly affected H5 HA thermostability (P<0.05). HA changes at positions 131, 144, 145, and 156 and substitutions at positions 131, 142, 145, and 156 affected the replicative ability of H5 escape mutants in vitro and in vivo, respectively. Overall, a co-variation between antigenic specificity and different HA phenotypic properties has been demonstrated. We believe that the monitoring of pleiotropic effects of the HA mutations found in H5 escape mutants is essential for accurate prediction of mutants with pandemic potential. - Highlights: • HA1 N142K mutation significantly lowered the pH of fusion optimum. • Mutations located in the HA lateral loop significantly affected H5 HA thermostability. • HA changes at positions 131, 142, 144, 145, and 156 affected the replicative ability of H5 mutants. • Acquisition of glycosylation site could lead to the emergence of multiple pleiotropic effects.

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

    Science.gov (United States)

    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.

  3. Radioimmunoassay of measles virus hemagglutinin protein G

    International Nuclear Information System (INIS)

    Lund, G.A.; Salmi, A.A.

    1982-01-01

    Guinea pig and rabbit antisera from animals immunized with purified measles virus hemagglutinin (G) protein were used to establish a solid-phase four-layer radioimmunoassay for quantitative measurement of the G protein. The sensitivity of the assay was 2 ng of purified G protein, and 200 μg of protein from uninfected Vero cells neither decreased the sensitivity nor reacted non-specifically in the assay. Radioimmunoassay standard dose-response curves were established and unknown values interpolated from these using the logit program of a desktop computer. Using this procedure, a measles virus growth curve in infected Vero cells was determined by measurement of G protein production. Under these same conditions, hemagglutination was not sensitive enough to detect early hemagglutinin production. Viral antigens in canine distemper virus, Newcastle disease virus, parainfluenza viruses 1-4, simian virus 5, and respiratory syncytial virus-infected cell lysates did not cross-react in the radioimmunoassay. A small degree of cross-reactivity was detected with mumps viral antigens, both with Vero cell-derived (wild-type strain) and egg-derived (Enders strain) purified virus preparations and with a cell lysate antigen prepared from wild-type mumps virus-infected Vero cells. (Auth.)

  4. Radioimmunoassay of measles virus hemagglutinin protein G

    Energy Technology Data Exchange (ETDEWEB)

    Lund, G A; Salmi, A A [Turku Univ. (Finland)

    1982-08-01

    Guinea pig and rabbit antisera from animals immunized with purified measles virus hemagglutinin (G) protein were used to establish a solid-phase four-layer radioimmunoassay for quantitative measurement of the G protein. The sensitivity of the assay was 2 ng of purified G protein, and 200 ..mu..g of protein from uninfected Vero cells neither decreased the sensitivity nor reacted non-specifically in the assay. Radioimmunoassay standard dose-response curves were established and unknown values interpolated from these using the logit program of a desktop computer. Using this procedure, a measles virus growth curve in infected Vero cells was determined by measurement of G protein production. Under these same conditions, hemagglutination was not sensitive enough to detect early hemagglutinin production. Viral antigens in canine distemper virus, Newcastle disease virus, parainfluenza viruses 1-4, simian virus 5, and respiratory syncytial virus-infected cell lysates did not cross-react in the radioimmunoassay. A small degree of cross-reactivity was detected with mumps viral antigens, both with Vero cell-derived (wild-type strain) and egg-derived (Enders strain) purified virus preparations and with a cell lysate antigen prepared from wild-type mumps virus-infected Vero cells.

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

    Directory of Open Access Journals (Sweden)

    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.

  6. Dual function of the hemagglutinin H5 fused to chicken CD154 in a potential strategy of DIVA against avian influenza disease: preliminary study

    Directory of Open Access Journals (Sweden)

    A.G. Pose

    2015-09-01

    Full Text Available In this study we demonstrated that the vaccine candidate against avian influenza virus H5N1 based on the hemagglutinin H5 (HA fused to the chicken CD154 (HACD can also be used for differentiating infected from vaccinated animals (DIVA. As the strategy of DIVA requires at least two proteins, we obtained a variant of the nucleoprotein (NP49-375 in E. coli. After its purification by IMAC, the competence of the proteins NP49-375 and HACD as coating antigens in indirect ELISA assays were tested by using the sera of chickens immunized with the proteins HA and HACD and the reference sera from several avian influenza subtypes. Together with these sera, the sera from different species of birds and the sera of chickens infected with other avian viral diseases were analyzed by competition ELISA assays coated with the proteins NP49-375 and HACD. The results showed that the segment CD154 in the chimeric protein HACD did not interfere with the recognition of the molecule HA by its specific antibodies. Also, we observed variable detection levels when the reference sera were analyzed in the ELISA plates coated with the protein NP49-375. Moreover, only the antibodies of the reference serum subtype H5 were detected in the ELISA plates coated with the protein HACD. The competition ELISA assays showed percentages of inhibition of 88-91% for the positives sera and less than 20% for the negative sera. We fixed the cut-off value of these assays at 25%. No antibody detection was observed in the sera from different species of birds or the sera of chickens infected with other avian viral diseases. This study supported the fact that the ELISA assays using the proteins NP49-375 and HACD could be valuable tools for avian influenza surveillance and as a strategy of DIVA for counteracting the highly pathogenic avian influenza virus H5N1 outbreaks.

  7. Dual function of the hemagglutinin H5 fused to chicken CD154 in a potential strategy of DIVA against avian influenza disease: preliminary study.

    Science.gov (United States)

    Pose, A G; Rodríguez, E S; Méndez, A C; Gómez, J N; Redondo, A V; Rodríguez, E R; Ramos, E M G; Gutiérrez, A Á; Moltó, M P R; Roche, D G; Ugalde, Y S; López, A M

    2015-01-01

    In this study we demonstrated that the vaccine candidate against avian influenza virus H5N1 based on the hemagglutinin H5 (HA) fused to the chicken CD154 (HACD) can also be used for differentiating infected from vaccinated animals (DIVA). As the strategy of DIVA requires at least two proteins, we obtained a variant of the nucleoprotein (NP49-375) in E. coli. After its purification by IMAC, the competence of the proteins NP49-375 and HACD as coating antigens in indirect ELISA assays were tested by using the sera of chickens immunized with the proteins HA and HACD and the reference sera from several avian influenza subtypes. Together with these sera, the sera from different species of birds and the sera of chickens infected with other avian viral diseases were analyzed by competition ELISA assays coated with the proteins NP49-375 and HACD. The results showed that the segment CD154 in the chimeric protein HACD did not interfere with the recognition of the molecule HA by its specific antibodies. Also, we observed variable detection levels when the reference sera were analyzed in the ELISA plates coated with the protein NP49-375. Moreover, only the antibodies of the reference serum subtype H5 were detected in the ELISA plates coated with the protein HACD. The competition ELISA assays showed percentages of inhibition of 88-91% for the positives sera and less than 20% for the negative sera. We fixed the cut-off value of these assays at 25%. No antibody detection was observed in the sera from different species of birds or the sera of chickens infected with other avian viral diseases. This study supported the fact that the ELISA assays using the proteins NP49-375 and HACD could be valuable tools for avian influenza surveillance and as a strategy of DIVA for counteracting the highly pathogenic avian influenza virus H5N1 outbreaks.

  8. 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

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    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

  12. 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.

  13. 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

  14. The Length of N-Glycans of Recombinant H5N1 Hemagglutinin Influences the Oligomerization and Immunogenicity of Vaccine Antigen

    Directory of Open Access Journals (Sweden)

    Edyta Kopera

    2017-04-01

    Full Text Available Hemagglutinin glycoprotein (HA is a principle influenza vaccine antigen. Recombinant HA-based vaccines become a potential alternative for traditional approach. Complexity and variation of HA N-glycosylation are considered as the important factors for the vaccine design. The number and location of glycan moieties in the HA molecule are also crucial. Therefore, we decided to study the effect of N-glycosylation pattern on the H5 antigen structure and its ability to induce immunological response. We also decided to change neither the number nor the position of the HA glycosylation sites but only the glycan length. Two variants of the H5 antigen with high mannose glycosylation (H5hm and with low-mannose glycosylation (H5Man5 were prepared utilizing different Pichia strains. Our structural studies demonstrated that only the highly glycosylated H5 antigen formed high molecular weight oligomers similar to viral particles. Further, the H5hm was much more immunogenic for mice than H5Man5. In summary, our results suggest that high mannose glycosylation of vaccine antigen is superior to the low glycosylation pattern. Our findings have strong implications for the recombinant HA-based influenza vaccine design.

  15. Hemagglutinin pseudotyped lentiviral particles: characterization of a new method for avian H5N1 influenza sero-diagnosis.

    OpenAIRE

    Nefkens , Isabelle; Garcia , Jean-Michel; Ling , Chu Shui; Lagarde , Nadège; Nicholls , John; Tang , Dong Jiang; Peiris , Malik; Buchy , Philippe; Altmeyer , Ralf

    2007-01-01

    BACKGROUND: Highly pathogenic avian influenza (HPAI) H5N1 has spread globally in birds and infected over 270 humans with an apparently high mortality rate. Serologic studies to determine the extent of asymptomatic H5N1 infection in humans and other mammals and to investigate the immunogenicity of current H5N1 vaccine candidates have been hampered by the biosafety requirements needed for H5N1 micro-neutralization tests. OBJECTIVE: Development of a serodiagnostic tool for highly pathogenic infl...

  16. Progress toward a universal H5N1 vaccine: a recombinant modified vaccinia virus Ankara-expressing trivalent hemagglutinin vaccine.

    Directory of Open Access Journals (Sweden)

    Mookkan Prabakaran

    Full Text Available The rapid evolution of new sublineages of H5N1 influenza poses the greatest challenge in control of H5N1 infection by currently existing vaccines. To overcome this, an MVAtor vector expressing three H5HA antigens A/Vietnam/1203/04, A/Indonesia/669/06 and A/Anhui/01/05 (MVAtor-tri-HA vector was developed to elicit broad cross-protection against diverse clades by covering amino acid variations in the major neutralizing epitopes of HA among H5N1 subtypes.BALB/c mice and guinea pigs were immunized i.m. with 8×107 TCID50/animal of MVAtor-tri-HA vector. The immunogenicity and cross-protective immunity of the MVAtor-tri-HA vector was evaluated against diverse clades of H5N1 strains.The results showed that mice immunized with MVAtor-tri-HA vector induced robust cross-neutralizing immunity to diverse H5N1 clades. In addition, the MVAtor-tri-HA vector completely protected against 10 MLD50 of a divergent clade of H5N1 infection (clade 7. Importantly, the serological surveillance of post-vaccinated guinea pig sera demonstrated that MVAtor-tri-HA vector was able to elicit strong cross-clade neutralizing immunity against twenty different H5N1 strains from six clades that emerged between 1997 and 2012.The present findings revealed that incorporation of carefully selected HA genes from divergent H5N1 strains within a single vector could be an effective approach in developing a vaccine with broad coverage to prevent infection during a pandemic situation.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    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

  19. 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.

  20. 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.

  1. Application of recombinant hemagglutinin proteins as alternative antigen standards for pandemic influenza vaccines.

    Science.gov (United States)

    Choi, Yejin; Kwon, Seong Yi; Oh, Ho Jung; Shim, Sunbo; Chang, Seokkee; Chung, Hye Joo; Kim, Do Keun; Park, Younsang; Lee, Younghee

    2017-09-01

    The single radial immunodiffusion (SRID) assay, used to quantify hemagglutinin (HA) in influenza vaccines, requires reference reagents; however, because centralized production of reference reagents may slow the emergency deployment of vaccines, alternatives are needed. We investigated the production of HA proteins using recombinant DNA technology, rather than a traditional egg-based production process. The HA proteins were then used in an SRID assay as a reference antigen. We found that HA can be quantified in both egg-based and cell-based influenza vaccines when recombinant HAs (rHAs) are used as the reference antigen. Furthermore, we confirmed that rHAs obtained from strains with pandemic potential, such as H5N1, H7N3, H7N9, and H9N2 strains, can be utilized in the SRID assay. The rHA production process takes just one month, in contrast to the traditional process that takes three to four months. The use of rHAs may reduce the time required to produce reference reagents and facilitate timely introduction of vaccines during emergencies.

  2. In silico approach towards H5N1 virus protein and transcriptomics ...

    African Journals Online (AJOL)

    H5N1 influenza A virus is a serious threat to human population. With a considerable mortality rate, strategies for coping with the infection are being developed. Our research group and some others investigated the potential therapeutic and preventive measures for tackling H5N1 infections. Protein based and transcriptomics ...

  3. Rapid Diagnostic Assay for Intact Influenza Virus Using a High Affinity Hemagglutinin Binding Protein.

    Science.gov (United States)

    Anderson, Caitlin E; Holstein, Carly A; Strauch, Eva-Maria; Bennett, Steven; Chevalier, Aaron; Nelson, Jorgen; Fu, Elain; Baker, David; Yager, Paul

    2017-06-20

    Influenza is a ubiquitous and recurring infection that results in approximately 500 000 deaths globally each year. Commercially available rapid diagnostic tests are based upon detection of the influenza nucleoprotein, which are limited in that they are unable to differentiate by species and require an additional viral lysis step. Sample preprocessing can be minimized or eliminated by targeting the intact influenza virus, thereby reducing assay complexity and leveraging the large number of hemagglutinin proteins on the surface of each virus. Here, we report the development of a paper-based influenza assay that targets the hemagglutinin protein; the assay employs a combination of antibodies and novel computationally designed, recombinant affinity proteins as the capture and detection agents. This system leverages the customizability of recombinant protein design to target the conserved receptor-binding pocket of the hemagglutinin protein and to match the trimeric nature of hemagglutinin for improved avidity. Using this assay, we demonstrate the first instance of intact influenza virus detection using a combination of antibody and affinity proteins within a porous network. The recombinant head region binder based assays yield superior analytical sensitivity as compared to the antibody based assay, with lower limits of detection of 3.54 × 10 7 and 1.34 × 10 7 CEID 50 /mL for the mixed and all binder stacks, respectively. Not only does this work describe the development of a novel influenza assay, it also demonstrates the power of recombinant affinity proteins for use in rapid diagnostic assays.

  4. Homosubtypic and heterosubtypic antibodies against highly pathogenic avian influenza H5N1 recombinant proteins in H5N1 survivors and non-H5N1 subjects.

    Science.gov (United States)

    Noisumdaeng, Pirom; Pooruk, Phisanu; Prasertsopon, Jarunee; Assanasen, Susan; Kitphati, Rungrueng; Auewarakul, Prasert; Puthavathana, Pilaipan

    2014-04-01

    Six recombinant vaccinia viruses containing HA, NA, NP, M or NS gene insert derived from a highly pathogenic avian influenza H5N1 virus, and the recombinant vaccinia virus harboring plasmid backbone as the virus control were constructed. The recombinant proteins were characterized for their expression and subcellular locations in TK(-) cells. Antibodies to the five recombinant proteins were detected in all 13 sequential serum samples collected from four H5N1 survivors during four years of follow-up; and those directed to rVac-H5 HA and rVac-NA proteins were found in higher titers than those directed to the internal proteins as revealed by indirect immunofluorescence assay. Although all 28 non-H5N1 subjects had no neutralizing antibodies against H5N1 virus, they did have cross-reactive antibodies to those five recombinant proteins. A significant increase in cross-reactive antibody titer to rVac-H5 HA and rVac-NA was found in paired blood samples from patients infected with the 2009 pandemic virus. Copyright © 2014 Elsevier Inc. All rights reserved.

  5. 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.

    Science.gov (United States)

    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.

  6. Protective Efficacy of Newcastle Disease Virus Expressing Soluble Trimeric Hemagglutinin against Highly Pathogenic H5N1 Influenza in Chickens and Mice

    NARCIS (Netherlands)

    Cornelissen, A.H.M.; Leeuw, de O.S.; Tacken, M.G.J.; Klos, H.C.; Vries, de R.P.; Boer-Luijtze, de E.A.; Zoelen-Bos, van D.J.; Rigter, A.; Rottier, P.J.M.; Moormann, R.J.M.; Haan, de C.A.M.

    2012-01-01

    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

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

    Science.gov (United States)

    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.

  8. 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...

  9. Effect of pH 5 enzyme from liver on the protein synthesis by mammary gland subcellular fractions in vitro

    International Nuclear Information System (INIS)

    Singh, Jaspal; Singh, Ajit; Ganguli, N.C.

    1976-01-01

    The effect of pH 5 enzyme fraction of liver on the protein synthesizing activity of the subcellular fractions of the mammary gland has been investigated. Results indicate that (1) lactating liver pH 5 enzyme stimulates protein synthesis which is enhanced by the addition of ATP-generating system and (2) the enzyme fractions from the non-lactating liver inhibits the protein synthesis by mammary fractions, but in some cases like mitochondrial and supernatant fractions of mammary it elevates the synthesis when supplemented with ATP-generating system. Chlorella protein hydrolysate- 14 C was used as a tracer and rabits were used as experimental animals. (M.G.B.)

  10. 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.

  11. Computational design of trimeric influenza-neutralizing proteins targeting the hemagglutinin receptor binding site

    Energy Technology Data Exchange (ETDEWEB)

    Strauch, Eva-Maria; Bernard, Steffen M.; La, David; Bohn, Alan J.; Lee, Peter S.; Anderson, Caitlin E.; Nieusma, Travis; Holstein, Carly A.; Garcia, Natalie K.; Hooper, Kathryn A.; Ravichandran, Rashmi; Nelson, Jorgen W.; Sheffler, William; Bloom, Jesse D.; Lee, Kelly K.; Ward, Andrew B.; Yager, Paul; Fuller, Deborah H.; Wilson, Ian A.; Baker , David (UWASH); (Scripps); (FHCRC)

    2017-06-12

    Many viral surface glycoproteins and cell surface receptors are homo-oligomers1, 2, 3, 4, and thus can potentially be targeted by geometrically matched homo-oligomers that engage all subunits simultaneously to attain high avidity and/or lock subunits together. The adaptive immune system cannot generally employ this strategy since the individual antibody binding sites are not arranged with appropriate geometry to simultaneously engage multiple sites in a single target homo-oligomer. We describe a general strategy for the computational design of homo-oligomeric protein assemblies with binding functionality precisely matched to homo-oligomeric target sites5, 6, 7, 8. In the first step, a small protein is designed that binds a single site on the target. In the second step, the designed protein is assembled into a homo-oligomer such that the designed binding sites are aligned with the target sites. We use this approach to design high-avidity trimeric proteins that bind influenza A hemagglutinin (HA) at its conserved receptor binding site. The designed trimers can both capture and detect HA in a paper-based diagnostic format, neutralizes influenza in cell culture, and completely protects mice when given as a single dose 24 h before or after challenge with influenza.

  12. 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.

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

    Science.gov (United States)

    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.

  14. Potential of the virion-associated peptidoglycan hydrolase HydH5 and its derivative fusion proteins in milk biopreservation.

    Directory of Open Access Journals (Sweden)

    Lorena Rodríguez-Rubio

    Full Text Available Bacteriophage lytic enzymes have recently attracted considerable interest as novel antimicrobials against Gram-positive bacteria. In this work, antimicrobial activity in milk of HydH5 [a virion-associated peptidoglycan hydrolase (VAPGH encoded by the Staphylococcus aureus bacteriophage vB_SauS-phiIPLA88], and three different fusion proteins created between HydH5 and lysostaphin has been assessed. The lytic activity of the five proteins (HydH5, HydH5Lyso, HydH5SH3b, CHAPSH3b and lysostaphin was confirmed using commercial whole extended shelf-life milk (ESL in challenge assays with 10(4 CFU/mL of the strain S. aureus Sa9. HydH5, HydH5Lyso and HydH5SH3b (3.5 µM kept the staphylococcal viable counts below the control cultures for 6 h at 37°C. The effect is apparent just 15 minutes after the addition of the lytic enzyme. Of note, lysostaphin and CHAPSH3b showed the highest staphylolytic protection as they were able to eradicate the initial staphylococcal challenge immediately or 15 min after addition, respectively, at lower concentration (1 µM at 37°C. CHAPSH3b showed the same antistaphyloccal effect at room temperature (1.65 µM. No re-growth was observed for the remainder of the experiment (up to 6 h. CHAPSH3b activity (1.65 µM was also assayed in raw (whole and skim and pasteurized (whole and skim milk. Pasteurization of milk clearly enhanced CHAPSH3b staphylolytic activity in both whole and skim milk at both temperatures. This effect was most dramatic at room temperature as this protein was able to reduce S. aureus viable counts to undetectable levels immediately after addition with no re-growth detected for the duration of the experiment (360 min. Furthermore, CHAPSH3b protein is known to be heat tolerant and retained some lytic activity after pasteurization treatment and after storage at 4°C for 3 days. These results might facilitate the use of the peptidoglycan hydrolase HydH5 and its derivative fusions, particularly CHAPSH3b, as

  15. Potential of the Virion-Associated Peptidoglycan Hydrolase HydH5 and Its Derivative Fusion Proteins in Milk Biopreservation

    Science.gov (United States)

    Rodríguez-Rubio, Lorena; Martínez, Beatriz; Donovan, David M.; García, Pilar; Rodríguez, Ana

    2013-01-01

    Bacteriophage lytic enzymes have recently attracted considerable interest as novel antimicrobials against Gram-positive bacteria. In this work, antimicrobial activity in milk of HydH5 [a virion-associated peptidoglycan hydrolase (VAPGH) encoded by the Staphylococcus aureus bacteriophage vB_SauS-phiIPLA88], and three different fusion proteins created between HydH5 and lysostaphin has been assessed. The lytic activity of the five proteins (HydH5, HydH5Lyso, HydH5SH3b, CHAPSH3b and lysostaphin) was confirmed using commercial whole extended shelf-life milk (ESL) in challenge assays with 104 CFU/mL of the strain S. aureus Sa9. HydH5, HydH5Lyso and HydH5SH3b (3.5 µM) kept the staphylococcal viable counts below the control cultures for 6 h at 37°C. The effect is apparent just 15 minutes after the addition of the lytic enzyme. Of note, lysostaphin and CHAPSH3b showed the highest staphylolytic protection as they were able to eradicate the initial staphylococcal challenge immediately or 15 min after addition, respectively, at lower concentration (1 µM) at 37°C. CHAPSH3b showed the same antistaphyloccal effect at room temperature (1.65 µM). No re-growth was observed for the remainder of the experiment (up to 6 h). CHAPSH3b activity (1.65 µM) was also assayed in raw (whole and skim) and pasteurized (whole and skim) milk. Pasteurization of milk clearly enhanced CHAPSH3b staphylolytic activity in both whole and skim milk at both temperatures. This effect was most dramatic at room temperature as this protein was able to reduce S. aureus viable counts to undetectable levels immediately after addition with no re-growth detected for the duration of the experiment (360 min). Furthermore, CHAPSH3b protein is known to be heat tolerant and retained some lytic activity after pasteurization treatment and after storage at 4°C for 3 days. These results might facilitate the use of the peptidoglycan hydrolase HydH5 and its derivative fusions, particularly CHAPSH3b, as biocontrol agents

  16. Role of position 627 of PB2 and the multibasic cleavage site of the hemagglutinin in the virulence of H5N1 avian influenza virus in chickens and ducks.

    Directory of Open Access Journals (Sweden)

    Karel A Schat

    Full Text Available Highly pathogenic H5N1 avian influenza viruses have caused major disease outbreaks in domestic and free-living birds with transmission to humans resulting in 59% mortality amongst 564 cases. The mutation of the amino acid at position 627 of the viral polymerase basic-2 protein (PB2 from glutamic acid (E in avian isolates to lysine (K in human isolates is frequently found, but it is not known if this change affects the fitness and pathogenicity of the virus in birds. We show here that horizontal transmission of A/Vietnam/1203/2004 H5N1 (VN/1203 virus in chickens and ducks was not affected by the change of K to E at PB2-627. All chickens died between 21 to 48 hours post infection (pi, while 70% of the ducks survived infection. Virus replication was detected in chickens within 12 hours pi and reached peak titers in spleen, lung and brain between 18 to 24 hours for both viruses. Viral antigen in chickens was predominantly in the endothelium, while in ducks it was present in multiple cell types, including neurons, myocardium, skeletal muscle and connective tissues. Virus replicated to a high titer in chicken thrombocytes and caused upregulation of TLR3 and several cell adhesion molecules, which may explain the rapid virus dissemination and location of viral antigen in endothelium. Virus replication in ducks reached peak values between 2 and 4 days pi in spleen, lung and brain tissues and in contrast to infection in chickens, thrombocytes were not involved. In addition, infection of chickens with low pathogenic VN/1203 caused neuropathology, with E at position PB2-627 causing significantly higher infection rates than K, indicating that it enhances virulence in chickens.

  17. In silico approach towards H5N1 virus protein and transcriptomics ...

    African Journals Online (AJOL)

    Arli Parikesit

    2013-05-22

    May 22, 2013 ... possible by three lead compound derivatives of Wang et al. (2010). Henceforth ... as drug or vaccines. FUTURE PROSPECTS: INTEGRATION OF PROTEIN- ... option as well (Parikesit et al., 2011). It would be interes-.

  18. A replicating modified vaccinia tiantan strain expressing an avian-derived influenza H5N1 hemagglutinin induce broadly neutralizing antibodies and cross-clade protective immunity in mice.

    Directory of Open Access Journals (Sweden)

    Haixia Xiao

    Full Text Available To combat the possibility of a zoonotic H5N1 pandemic in a timely fashion, it is necessary to develop a vaccine that would confer protection against homologous and heterologous human H5N1 influenza viruses. Using a replicating modified vaccinia virus Tian Tan strain (MVTT as a vaccine vector, we constructed MVTTHA-QH and MVTTHA-AH, which expresses the H5 gene of a goose-derived Qinghai strain A/Bar-headed Goose/Qinghai/1/2005 or human-derived Anhui Strain A/Anhui/1/2005. The immunogenicity profiles of both vaccine candidates were evaluated. Vaccination with MVTTHA-QH induced a significant level of neutralizing antibodies (Nabs against a homologous strain and a wide range of H5N1 pseudoviruses (clades 1, 2.1, 2.2, 2.3.2, and 2.3.4. Neutralization tests (NT and Haemagglutination inhibition (HI antibodies inhibit the live autologous virus as well as a homologous A/Xingjiang/1/2006 and a heterologous A/Vietnam/1194/2004, representing two human isolates from clade 2.2 and clade 1, respectively. Importantly, mice vaccinated with intranasal MVTTHA-QH were completely protected from challenge with lethal dosages of A/Bar-headed Goose/Qinghai/1/2005 and the A/Viet Nam/1194/2004, respectively, but not control mice that received a mock MVTTS vaccine. However, MVTTHA-AH induced much lower levels of NT against its autologous strain. Our results suggest that it is feasible to use the H5 gene from A/Bar-headed Goose/Qinghai/1/2005 to construct an effective vaccine, when using MVTT as a vector, to prevent infections against homologous and genetically divergent human H5N1 influenza viruses.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    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.

  2. 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

  3. Cell-to-Cell Measles Virus Spread between Human Neurons Is Dependent on Hemagglutinin and Hyperfusogenic Fusion Protein.

    Science.gov (United States)

    Sato, Yuma; Watanabe, Shumpei; Fukuda, Yoshinari; Hashiguchi, Takao; Yanagi, Yusuke; Ohno, Shinji

    2018-03-15

    Measles virus (MV) usually causes acute infection but in rare cases persists in the brain, resulting in subacute sclerosing panencephalitis (SSPE). Since human neurons, an important target affected in the disease, do not express the known MV receptors (signaling lymphocyte activation molecule [SLAM] and nectin 4), how MV infects neurons and spreads between them is unknown. Recent studies have shown that many virus strains isolated from SSPE patients possess substitutions in the extracellular domain of the fusion (F) protein which confer enhanced fusion activity. Hyperfusogenic viruses with such mutations, unlike the wild-type MV, can induce cell-cell fusion even in SLAM- and nectin 4-negative cells and spread efficiently in human primary neurons and the brains of animal models. We show here that a hyperfusogenic mutant MV, IC323-F(T461I)-EGFP (IC323 with a fusion-enhancing T461I substitution in the F protein and expressing enhanced green fluorescent protein), but not the wild-type MV, spreads in differentiated NT2 cells, a widely used human neuron model. Confocal time-lapse imaging revealed the cell-to-cell spread of IC323-F(T461I)-EGFP between NT2 neurons without syncytium formation. The production of virus particles was strongly suppressed in NT2 neurons, also supporting cell-to-cell viral transmission. The spread of IC323-F(T461I)-EGFP was inhibited by a fusion inhibitor peptide as well as by some but not all of the anti-hemagglutinin antibodies which neutralize SLAM- or nectin-4-dependent MV infection, suggesting the presence of a distinct neuronal receptor. Our results indicate that MV spreads in a cell-to-cell manner between human neurons without causing syncytium formation and that the spread is dependent on the hyperfusogenic F protein, the hemagglutinin, and the putative neuronal receptor for MV. IMPORTANCE Measles virus (MV), in rare cases, persists in the human central nervous system (CNS) and causes subacute sclerosing panencephalitis (SSPE) several

  4. Characterization of Hemagglutinin Negative Botulinum Progenitor Toxins

    Directory of Open Access Journals (Sweden)

    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.

  5. 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.

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

    International Nuclear Information System (INIS)

    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

  7. 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.

  8. Pseudomonas fluorescens filamentous hemagglutinin, an iron-regulated protein, is an important virulence factor that modulates bacterial pathogenicity

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    Yuan-yuan Sun

    2016-08-01

    Full Text Available Pseudomonas fluorescens is a common bacterial pathogen to a wide range of aquaculture animals including various species of fish. In this study, we employed proteomic analysis and identified filamentous hemagglutinin (FHA as an iron-responsive protein secreted by TSS, a pathogenic P. fluorescens isolate. In vitro study showed that compared to the wild type, the fha mutant TSSfha (i exhibited a largely similar vegetative growth profile but significantly retarded in the ability of biofilm growth and producing extracellular matrix, (ii displayed no apparent flagella and motility, (iii was defective in the attachment to host cells and unable to form self-aggregation, (iv displayed markedly reduced capacity of hemagglutination and surviving in host serum. In vivo infection analysis revealed that TSSfha was significantly attenuated in the ability of dissemination in fish tissues and inducing host mortality, and that antibody blocking of the natural FHA produced by the wild type TSS impaired the infectivity of the pathogen. Furthermore, when introduced into turbot as a subunit vaccine, recombinant FHA elicited a significant protection against lethal TSS challenge. Taken together, these results indicate for the first time that P. fluorescens FHA is a key virulence factor essential to multiple biological processes associated with pathogenicity.

  9. Construction and heterologous expression of a truncated Haemagglutinin (HA) protein from the avian influenza virus H5N1 in Escherichia coli.

    Science.gov (United States)

    Chee Wei, T; Nurul Wahida, A G; Shaharum, S

    2014-12-01

    Malaysia first reported H5N1 poultry case in 2004 and subsequently outbreak in poultry population in 2007. Here, a recombinant gene encoding of peptide epitopes, consisting fragments of HA1, HA2 and a polybasic cleavage site of H5N1 strain Malaysia, was amplified and cloned into pET-47b(+) bacterial expression vector. DNA sequencing and alignment analysis confirmed that the gene had no alteration and in-frame to the vector. Then, His-tagged truncated HA protein was expressed in Escherichia coli BL21 (DE3) under 1 mM IPTG induction. The protein expression was optimized under a time-course induction study and further purified using Ni-NTA agarose under reducing condition. Migration size of protein was detected at 15 kDa by Western blot using anti-His tag monoclonal antibody and demonstrated no discrepancy compared to its calculated molecular weight.

  10. An H5N1-based matrix protein 2 ectodomain tetrameric peptide vaccine provides cross-protection against lethal infection with H7N9 influenza virus.

    Science.gov (United States)

    Leung, Ho-Chuen; Chan, Chris Chung-Sing; Poon, Vincent Kwok-Man; Zhao, Han-Jun; Cheung, Chung-Yan; Ng, Fai; Huang, Jian-Dong; Zheng, Bo-Jian

    2015-04-01

    In March 2013, a patient infected with a novel avian influenza A H7N9 virus was reported in China. Since then, there have been 458 confirmed infection cases and 177 deaths. The virus contains several human-adapted markers, indicating that H7N9 has pandemic potential. The outbreak of this new influenza virus highlighted the need for the development of universal influenza vaccines. Previously, we demonstrated that a tetrameric peptide vaccine based on the matrix protein 2 ectodomain (M2e) of the H5N1 virus (H5N1-M2e) could protect mice from lethal infection with different clades of H5N1 and 2009 pandemic H1N1 influenza viruses. In this study, we investigated the cross-protection of H5N1-M2e against lethal infection with the new H7N9 virus. Although five amino acid differences existed at positions 13, 14, 18, 20, and 21 between M2e of H5N1 and H7N9, H5N1-M2e vaccination with either Freund's adjuvant or the Sigma adjuvant system (SAS) induced a high level of anti-M2e antibody, which cross-reacted with H7N9-M2e peptide. A mouse-adapted H7N9 strain, A/Anhui/01/2013m, was used for lethal challenge in animal experiments. H5N1-M2e vaccination provided potent cross-protection against lethal challenge of the H7N9 virus. Reduced viral replication and histopathological damage of mouse lungs were also observed in the vaccinated mice. Our results suggest that the tetrameric H5N1-M2e peptide vaccine could protect against different subtypes of influenza virus infections. Therefore, this vaccine may be an ideal candidate for developing a universal vaccine to prevent the reemergence of avian influenza A H7N9 virus and the emergence of potential novel reassortants of influenza virus.

  11. D701N mutation in the PB2 protein contributes to the pathogenicity of H5N1 avian influenza viruses but not transmissibility in guinea pigs

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    Peirong eJiao

    2014-11-01

    Full Text Available H5N1 highly pathogenic avian influenza virus (HPAIV of clade 2.3.2 has been circulating in waterfowl in Southern China since 2003. Our previous studies showed that certain H5N1 HPAIV isolates within clade 2.3.2 from Southern China had high pathogenicity in different birds. Guinea pigs have been successfully used as models to evaluate the transmissibility of AIVs and other species of influenza viruses in mammalian hosts. However, few studies have reported pathogenicity and transmissibility of H5N1 HPAIVs of this clade in guinea pigs. In this study, we selected an H5N1 HPAIV isolate, A/duck/Guangdong/357/2008, to investigate the pathogenicity and transmissibility of the virus in guinea pigs. The virus had high pathogenicity in mice; additionally, it only replicated in some tissues of the guinea pigs without production of clinical signs, but was transmissible among guinea pigs. Interestingly, virus isolates from co-caged guinea pigs had the D701N mutation in the PB2 protein. These mutant viruses showed higher pathogenicity in mice and higher replication capability in guinea pigs but did not demonstrate enhanced the transmissibility among guinea pigs. These findings indicate the transmission of the H5N1 virus between mammals could induce virus mutations, and the mutant viruses might have higher pathogenicity in mammals without higher transmissibility. Therefore, the continued evaluation of the pathogenicity and transmissibility of avian influenza virus (AIVs in mammals is critical to the understanding of the evolutionary characteristics of AIVs and the emergence of potential pandemic strains.

  12. Preparation of a standardized, efficacious agricultural H5N3 vaccine by reverse genetics

    International Nuclear Information System (INIS)

    Liu Ming; Wood, John M.; Ellis, Trevor; Krauss, Scott; Seiler, Patrick; Johnson, Christie; Hoffmann, Erich; Humberd, Jennifer; Hulse, Diane; Zhang Yun; Webster, Robert G.; Perez, Daniel R.

    2003-01-01

    Options for the control of emerging and reemerging H5N1 influenza viruses include improvements in biosecurity and the use of inactivated vaccines. Commercially available H5N2 influenza vaccine prevents disease signs and reduces virus load but does not completely prevent virus shedding after challenge with H5N1 virus. By using reverse genetics, we prepared an H5N3 vaccine whose hemagglutinin is 99.6% homologous to that of A/CK/HK/86.3/02 (H5N1). We used the internal genes of A/PR/8/34 and the H5 of A/Goose/HK/437.4/99 (H5N1) after deletion of basic amino acids from its connecting peptide region. The resulting virus was not lethal to chicken embryos and grew to high HA titers in eggs, allowing preparation of HA protein-standardized vaccine in unconcentrated allantoic fluid. The N3 neuraminidase, derived from A/Duck/Germany/1215/73 (H2N3), permitted discrimination between vaccinated and naturally infected birds. The virus construct failed to replicate in quail and chickens. Similar to parental A/PR/8/34 (H1N1), it replicated in mice and ferrets and spread to the brains of mice; therefore, it should not be used as a live-attenuated vaccine. The H5N3 vaccine, at doses of 1.2 μg HA, induced HI antibodies in chickens and prevented death, signs of disease, and markedly reduced virus shedding after challenge with A/CK/HK/86.3/02 (H5N1) but did not provide sterilizing immunity. Thus, reverse genetics allows the inexpensive preparation of standardized, efficacious H5N3 poultry vaccines that may also reduce the reemergence of H5N1 genotypes

  13. Characterization of avian influenza H5N1 virosome

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    Chatchai Sarachai

    2014-04-01

    Full Text Available The purpose of this study was to prepare and characterize virosome containing envelope proteins of the avian influenza (H5N1 virus. The virosome was prepared by the solubilization of virus with octaethyleneglycol mono (n-dodecyl ether (C12E8 followed by detergent removal with SM2 Bio-Beads. Biochemical analysis by SDS-PAGE and western blotting, indicated that avian influenza H5N1 virosome had similar characteristics to the parent virus and contained both the hemagglutinin (HA, 60-75 kDa and neuraminidase (NA, 220 kDa protein, with preserved biological activity, such as hemagglutination activity. The virosome structure was analyzed by negative stained transmission electron microscope (TEM demonstrated that the spherical shapes of vesicles with surface glycoprotein spikes were harbored. In conclusion, the biophysical properties of the virosome were similar to the parent virus, and the use of octaethyleneglycol mono (n-dodecyl ether to solubilize viral membrane, followed by removal of detergent using polymer beads adsorption (Bio-Beads SM2 was the preferable method for obtaining avian influenza virosome. The outcome of this study might be useful for further development veterinary virus vaccines.

  14. 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

  15. 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.

  16. Immune escape mutants of Highly Pathogenic Avian Influenza H5N1 selected using polyclonal sera: identification of key amino acids in the HA protein.

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    Ioannis Sitaras

    Full Text Available Evolution of Avian Influenza (AI viruses--especially of the Highly Pathogenic Avian Influenza (HPAI H5N1 subtype--is a major issue for the poultry industry. HPAI H5N1 epidemics are associated with huge economic losses and are sometimes connected to human morbidity and mortality. Vaccination (either as a preventive measure or as a means to control outbreaks is an approach that splits the scientific community, due to the risk of it being a potential driving force in HPAI evolution through the selection of mutants able to escape vaccination-induced immunity. It is therefore essential to study how mutations are selected due to immune pressure. To this effect, we performed an in vitro selection of mutants from HPAI A/turkey/Turkey/1/05 (H5N1, using immune pressure from homologous polyclonal sera. After 42 rounds of selection, we identified 5 amino acid substitutions in the Haemagglutinin (HA protein, most of which were located in areas of antigenic importance and suspected to be prone to selection pressure. We report that most of the mutations took place early in the selection process. Finally, our antigenic cartography studies showed that the antigenic distance between the selected isolates and their parent strain increased with passage number.

  17. 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.

  18. 78 FR 9355 - Influenza Viruses Containing the Hemagglutinin From the Goose/Guangdong/1/96 Lineage

    Science.gov (United States)

    2013-02-08

    ... DEPARTMENT OF HEALTH AND HUMAN SERVICES [Docket: CDC-2012-0010] 42 CFR Part 73 Influenza Viruses... influenza (HPAI) H5N1 viruses that contain a hemagglutinin (HA) from the Goose/Guangdong/1/96 lineage, and... concerning highly pathogenic avian influenza (HPAI) H5N1 viruses that contain a hemagglutinin (HA) from the...

  19. Efficacy of gamma irradiation on H5N1 for the preparation of hemagglutination Inhibition test antigen

    International Nuclear Information System (INIS)

    Chaisingh, Arunee; Thammasart, Suree; Kamolsiripichaiporn, Somjai; Piadang Nattayana

    2006-09-01

    The result of the efficiencies of gamma irradiation at the dose of 10-60 kGy on highly pathogenic avian influenza virus, H5N1 (Thai isolate) revealed that gamma irradiation at the dose of 10 and 20 kGy could reduce the infectivity of Hanna but gamma irradiation at 30-60 kGy could inactivate H5N1 virus completely. All doses of gamma irradiation used in this experiment had no effect on antigenicity of hemagglutinin protein. Thus, gamma irradiation at the dose of 30- 60 kGy could be use safely for the antigen preparation to detect the antibody against H5N1 virus.

  20. Induction of long-term protective immune responses by influenza H5N1 virus-like particles.

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    Sang-Moo Kang

    Full Text Available Recurrent outbreaks of highly pathogenic H5N1 avian influenza virus pose a threat of eventually causing a pandemic. Early vaccination of the population would be the single most effective measure for the control of an emerging influenza pandemic.Influenza virus-like particles (VLPs produced in insect cell-culture substrates do not depend on the availability of fertile eggs for vaccine manufacturing. We produced VLPs containing influenza A/Viet Nam1203/04 (H5N1 hemagglutinin, neuraminidase, and matrix proteins, and investigated their preclinical immunogenicity and protective efficacy. Mice immunized intranasally with H5N1 VLPs developed high levels of H5N1 specific antibodies and were 100% protected against a high dose of homologous H5N1 virus infection at 30 weeks after immunization. Protection is likely to be correlated with humoral and cellular immunologic memory at systemic and mucosal sites as evidenced by rapid anamnestic responses to re-stimulation with viral antigen in vivo and in vitro.These results provide support for clinical evaluation of H5N1 VLP vaccination as a public health intervention to mitigate a possible pandemic of H5N1 influenza.

  1. Mucosal and systemic immune responses elicited by recombinant Lactococcus lactis expressing a fusion protein composed of pertussis toxin and filamentous hemagglutinin from Bordetella pertussis.

    Science.gov (United States)

    Torkashvand, Ali; Bahrami, Fariborz; Adib, Minoo; Ajdary, Soheila

    2018-05-05

    We constructed a food-grade expression system harboring a F1S1 fusion protein of Bordetella pertussis to be produced in Lactococcus lactis NZ3900 as a new oral vaccine model against whooping cough, caused by B. pertussis. F1S1 was composed of N-terminally truncated S1 subunit of pertussis toxin and type I immunodominant domain of filamentous hemagglutinin which are both known as protective immunogens against pertussis. The recombinant L. lactis was administered via oral or intranasal routes to BALB/c mice and the related specific systemic and mucosal immune responses were then evaluated. The results indicated significantly higher levels of specific IgA in the lung extracts and IgG in sera of mucosally-immunized mice, compared to their controls. It was revealed that higher levels of IgG2a, compared to IgG1, were produced in all mucosally-immunized mice. Moreover, immunized mice developed Th1 responses with high levels of IFN-γ production by the spleen cells. These findings provide evidence for L. lactis to be used as a suitable vehicle for expression and delivery of F1S1 fusion protein to mucosa and induction of appropriate systemic and mucosal immune responses against pertussis. Copyright © 2018 Elsevier Ltd. All rights reserved.

  2. 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.

  3. Contemporary avian influenza A virus subtype H1, H6, H7, H10, and H15 hemagglutinin genes encode a mammalian virulence factor similar to the 1918 pandemic virus H1 hemagglutinin.

    Science.gov (United States)

    Qi, Li; Pujanauski, Lindsey M; Davis, A Sally; Schwartzman, Louis M; Chertow, Daniel S; Baxter, David; Scherler, Kelsey; Hartshorn, Kevan L; Slemons, Richard D; Walters, Kathie-Anne; Kash, John C; Taubenberger, Jeffery K

    2014-11-18

    Zoonotic avian influenza virus infections may lead to epidemics or pandemics. The 1918 pandemic influenza virus has an avian influenza virus-like genome, and its H1 hemagglutinin was identified as a key mammalian virulence factor. A chimeric 1918 virus expressing a contemporary avian H1 hemagglutinin, however, displayed murine pathogenicity indistinguishable from that of the 1918 virus. Here, isogenic chimeric avian influenza viruses were constructed on an avian influenza virus backbone, differing only by hemagglutinin subtype expressed. Viruses expressing the avian H1, H6, H7, H10, and H15 subtypes were pathogenic in mice and cytopathic in normal human bronchial epithelial cells, in contrast to H2-, H3-, H5-, H9-, H11-, H13-, H14-, and H16-expressing viruses. Mouse pathogenicity was associated with pulmonary macrophage and neutrophil recruitment. These data suggest that avian influenza virus hemagglutinins H1, H6, H7, H10, and H15 contain inherent mammalian virulence factors and likely share a key virulence property of the 1918 virus. Consequently, zoonotic infections with avian influenza viruses bearing one of these hemagglutinins may cause enhanced disease in mammals. Influenza viruses from birds can cause outbreaks in humans and may contribute to the development of pandemics. The 1918 pandemic influenza virus has an avian influenza virus-like genome, and its main surface protein, an H1 subtype hemagglutinin, was identified as a key mammalian virulence factor. In a previous study, a 1918 virus expressing an avian H1 gene was as virulent in mice as the reconstructed 1918 virus. Here, a set of avian influenza viruses was constructed, differing only by hemagglutinin subtype. Viruses with the avian H1, H6, H7, H10, and H15 subtypes caused severe disease in mice and damaged human lung cells. Consequently, infections with avian influenza viruses bearing one of these hemagglutinins may cause enhanced disease in mammals, and therefore surveillance for human infections

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  6. The nuclear export protein of H5N1 influenza A viruses recruits Matrix 1 (M1) protein to the viral ribonucleoprotein to mediate nuclear export.

    Science.gov (United States)

    Brunotte, Linda; Flies, Joe; Bolte, Hardin; Reuther, Peter; Vreede, Frank; Schwemmle, Martin

    2014-07-18

    In influenza A virus-infected cells, replication and transcription of the viral genome occurs in the nucleus. To be packaged into viral particles at the plasma membrane, encapsidated viral genomes must be exported from the nucleus. Intriguingly, the nuclear export protein (NEP) is involved in both processes. Although NEP stimulates viral RNA synthesis by binding to the viral polymerase, its function during nuclear export implicates interaction with viral ribonucleoprotein (vRNP)-associated M1. The observation that both interactions are mediated by the C-terminal moiety of NEP raised the question whether these two features of NEP are linked functionally. Here we provide evidence that the interaction between M1 and the vRNP depends on the NEP C terminus and its polymerase activity-enhancing property for the nuclear export of vRNPs. This suggests that these features of NEP are linked functionally. Furthermore, our data suggest that the N-terminal domain of NEP interferes with the stability of the vRNP-M1-NEP nuclear export complex, probably mediated by its highly flexible intramolecular interaction with the NEP C terminus. On the basis of our data, we propose a new model for the assembly of the nuclear export complex of Influenza A vRNPs. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Comprehensive global amino acid sequence analysis of PB1F2 protein of influenza A H5N1 viruses and the influenza A virus subtypes responsible for the 20th‐century pandemics

    Science.gov (United States)

    Pasricha, Gunisha; Mishra, Akhilesh C.; Chakrabarti, Alok K.

    2012-01-01

    Please cite this paper as: Pasricha et al. (2012) Comprehensive global amino acid sequence analysis of PB1F2 protein of influenza A H5N1 viruses and the Influenza A virus subtypes responsible for the 20th‐century pandemics. Influenza and Other Respiratory Viruses 7(4), 497–505. Background  PB1F2 is the 11th protein of influenza A virus translated from +1 alternate reading frame of PB1 gene. Since the discovery, varying sizes and functions of the PB1F2 protein of influenza A viruses have been reported. Selection of PB1 gene segment in the pandemics, variable size and pleiotropic effect of PB1F2 intrigued us to analyze amino acid sequences of this protein in various influenza A viruses. Methods  Amino acid sequences for PB1F2 protein of influenza A H5N1, H1N1, H2N2, and H3N2 subtypes were obtained from Influenza Research Database. Multiple sequence alignments of the PB1F2 protein sequences of the aforementioned subtypes were used to determine the size, variable and conserved domains and to perform mutational analysis. Results  Analysis showed that 96·4% of the H5N1 influenza viruses harbored full‐length PB1F2 protein. Except for the 2009 pandemic H1N1 virus, all the subtypes of the 20th‐century pandemic influenza viruses contained full‐length PB1F2 protein. Through the years, PB1F2 protein of the H1N1 and H3N2 viruses has undergone much variation. PB1F2 protein sequences of H5N1 viruses showed both human‐ and avian host‐specific conserved domains. Global database of PB1F2 protein revealed that N66S mutation was present only in 3·8% of the H5N1 strains. We found a novel mutation, N84S in the PB1F2 protein of 9·35% of the highly pathogenic avian influenza H5N1 influenza viruses. Conclusions  Varying sizes and mutations of the PB1F2 protein in different influenza A virus subtypes with pandemic potential were obtained. There was genetic divergence of the protein in various hosts which highlighted the host‐specific evolution of the virus

  8. An Adjuvanted A(H5N1) Subvirion Vaccine Elicits Virus-Specific Antibody Response and Improves Protection Against Lethal Influenza Viral Challenge in Mouse Model of Protein Energy Malnutrition.

    Science.gov (United States)

    Jones, Enitra N; Amoah, Samuel; Cao, Weiping; Sambhara, Suryaprakash; Gangappa, Shivaprakash

    2017-09-15

    Protein energy malnutrition (PEM) increases susceptibility to infectious diseases, including influenza infection, but no studies have addressed the potential influences of PEM on the immunogenicity and protective efficacy of avian influenza A(H5N1) vaccine. We investigated the role of PEM on vaccine-mediated protection after a lethal challenge with recombinant A(H5N1) virus using isocaloric diets providing either adequate protein (AP; 18% protein) or very low protein (VLP; 2% protein) in an established murine model of influenza vaccination. We demonstrated that mice maintained on a VLP diet succumb to lethal challenge at greater rates than mice maintained on an AP diet, despite comparable immunization regimens. Importantly, there was no virus-induced mortality in both VLP and AP groups of mice when either group was immunized with adjuvanted low-dose A(H5N1) subvirion vaccine. Our results suggest that adjuvanted vaccination in populations where PEM is endemic may be one strategy to boost vaccination-promoted immunity and improve outcomes associated with highly pathogenic A(H5N1). 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.

  9. Live, Attenuated Influenza A H5N1 Candidate Vaccines Provide Broad Cross-Protection in Mice and Ferrets

    Science.gov (United States)

    Mills, Kimberly L; Jin, Hong; Duke, Greg; Lu, Bin; Luke, Catherine J; Murphy, Brian; Swayne, David E; Kemble, George; Subbarao, Kanta

    2006-01-01

    Background Recent outbreaks of highly pathogenic influenza A H5N1 viruses in humans and avian species that began in Asia and have spread to other continents underscore an urgent need to develop vaccines that would protect the human population in the event of a pandemic. Methods and Findings Live, attenuated candidate vaccines possessing genes encoding a modified H5 hemagglutinin (HA) and a wild-type (wt) N1 neuraminidase from influenza A H5N1 viruses isolated in Hong Kong and Vietnam in 1997, 2003, and 2004, and remaining gene segments derived from the cold-adapted (ca) influenza A vaccine donor strain, influenza A/Ann Arbor/6/60 ca (H2N2), were generated by reverse genetics. The H5N1 ca vaccine viruses required trypsin for efficient growth in vitro, as predicted by the modification engineered in the gene encoding the HA, and possessed the temperature-sensitive and attenuation phenotypes specified by the internal protein genes of the ca vaccine donor strain. More importantly, the candidate vaccines were immunogenic in mice. Four weeks after receiving a single dose of 106 50% tissue culture infectious doses of intranasally administered vaccines, mice were fully protected from lethality following challenge with homologous and antigenically distinct heterologous wt H5N1 viruses from different genetic sublineages (clades 1, 2, and 3) that were isolated in Asia between 1997 and 2005. Four weeks after receiving two doses of the vaccines, mice and ferrets were fully protected against pulmonary replication of homologous and heterologous wt H5N1 viruses. Conclusions The promising findings in these preclinical studies of safety, immunogenicity, and efficacy of the H5N1 ca vaccines against antigenically diverse H5N1 vaccines provide support for their careful evaluation in Phase 1 clinical trials in humans. PMID:16968127

  10. Live, attenuated influenza A H5N1 candidate vaccines provide broad cross-protection in mice and ferrets.

    Directory of Open Access Journals (Sweden)

    Amorsolo L Suguitan

    2006-09-01

    Full Text Available Recent outbreaks of highly pathogenic influenza A H5N1 viruses in humans and avian species that began in Asia and have spread to other continents underscore an urgent need to develop vaccines that would protect the human population in the event of a pandemic.Live, attenuated candidate vaccines possessing genes encoding a modified H5 hemagglutinin (HA and a wild-type (wt N1 neuraminidase from influenza A H5N1 viruses isolated in Hong Kong and Vietnam in 1997, 2003, and 2004, and remaining gene segments derived from the cold-adapted (ca influenza A vaccine donor strain, influenza A/Ann Arbor/6/60 ca (H2N2, were generated by reverse genetics. The H5N1 ca vaccine viruses required trypsin for efficient growth in vitro, as predicted by the modification engineered in the gene encoding the HA, and possessed the temperature-sensitive and attenuation phenotypes specified by the internal protein genes of the ca vaccine donor strain. More importantly, the candidate vaccines were immunogenic in mice. Four weeks after receiving a single dose of 10(6 50% tissue culture infectious doses of intranasally administered vaccines, mice were fully protected from lethality following challenge with homologous and antigenically distinct heterologous wt H5N1 viruses from different genetic sublineages (clades 1, 2, and 3 that were isolated in Asia between 1997 and 2005. Four weeks after receiving two doses of the vaccines, mice and ferrets were fully protected against pulmonary replication of homologous and heterologous wt H5N1 viruses.The promising findings in these preclinical studies of safety, immunogenicity, and efficacy of the H5N1 ca vaccines against antigenically diverse H5N1 vaccines provide support for their careful evaluation in Phase 1 clinical trials in humans.

  11. Comprehensive global amino acid sequence analysis of PB1F2 protein of influenza A H5N1 viruses and the influenza A virus subtypes responsible for the 20th-century pandemics.

    Science.gov (United States)

    Pasricha, Gunisha; Mishra, Akhilesh C; Chakrabarti, Alok K

    2013-07-01

    PB1F2 is the 11th protein of influenza A virus translated from +1 alternate reading frame of PB1 gene. Since the discovery, varying sizes and functions of the PB1F2 protein of influenza A viruses have been reported. Selection of PB1 gene segment in the pandemics, variable size and pleiotropic effect of PB1F2 intrigued us to analyze amino acid sequences of this protein in various influenza A viruses. Amino acid sequences for PB1F2 protein of influenza A H5N1, H1N1, H2N2, and H3N2 subtypes were obtained from Influenza Research Database. Multiple sequence alignments of the PB1F2 protein sequences of the aforementioned subtypes were used to determine the size, variable and conserved domains and to perform mutational analysis. Analysis showed that 96·4% of the H5N1 influenza viruses harbored full-length PB1F2 protein. Except for the 2009 pandemic H1N1 virus, all the subtypes of the 20th-century pandemic influenza viruses contained full-length PB1F2 protein. Through the years, PB1F2 protein of the H1N1 and H3N2 viruses has undergone much variation. PB1F2 protein sequences of H5N1 viruses showed both human- and avian host-specific conserved domains. Global database of PB1F2 protein revealed that N66S mutation was present only in 3·8% of the H5N1 strains. We found a novel mutation, N84S in the PB1F2 protein of 9·35% of the highly pathogenic avian influenza H5N1 influenza viruses. Varying sizes and mutations of the PB1F2 protein in different influenza A virus subtypes with pandemic potential were obtained. There was genetic divergence of the protein in various hosts which highlighted the host-specific evolution of the virus. However, studies are required to correlate this sequence variability with the virulence and pathogenicity. © 2012 John Wiley & Sons Ltd.

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

    Science.gov (United States)

    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.

  13. 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.

  14. 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...

  15. A study of analysis PB1-F2 protein of Influenza Viruses A/H1N1pdm09, A/ H3N2, and A/H5N1

    Directory of Open Access Journals (Sweden)

    Hana Apsari Pawestri

    2016-07-01

    Full Text Available Abstrak Tujuan. Protein PB1-F2 (polymerase basic 1-frame 2 adalah protein terbaru yang ditemukan pada virus Influenza dan telah terbukti berperan dalam induksi kematian sel dan patogenitas. Tujuan dari tulisan ini adalah untuk menganalisis protein PB1-F2 pada virus Influenza A/H5N1 dan A/H1N1pdm09. Metode. Kami melakukan pencarian data yang relevan yaitu sekuens gen virus Influenza A/H5N1 dan A/H1N1pdm09 dari Gen Bank National Center for Biotechnology Information (NCBI selama tahun 1997-2015. Data yang digunakan adalah data sekuens nukleotida gen PB1 (polymerase basic1 virus influenza A/H5N1 dan A/H1N1pdm09. Kemudian dilakukan analisis alignment untuk mengetahui variasi protein dan mutasi yang berhubungan dengan patogenitas dan virulensi. Hasil. Kami melakukan penelitian terhadap sekuens PB1-F2 sebanyak 3262 influenza A/H5N1 dan 2472 Influenza A/H1N1pdm09. Hasil analisis menunjukkan bahwa semua sekuens A/H5N1 memiliki panjang yang penuh sebanyak 90 asam amino, kecuali influenza pandemi 2009 hanya memiliki panjang 87 asam amino. Kemudian, ditemukan mutasi yang berhubungan dengan virulensi yang ditunjukan dengan perubahan asam amino Asparagin (N menjadi Serin (S. Mutasi tersebut terjadi pada Influenza A/H5N1 sebanyak 8.5% dan Influenza A/H1N1pdm09 sebanyak 0.5%. Kesimpulan. Ditemukan beberapa variasi panjang asam amino dan mutasi penting pada sekuens PB1-F2 dari subtipe yang berbeda yaitu influenza A/H5N1 dan A/H1N1pdm09  yang mengindikasikan seleksi spesifik karena introduksi dan adaptasi terhadap inang yang berbeda. Diperlukan penelitian lanjutan untuk lebih memahami variasi dan kontribusi protein PB1-F2 tersebut terhadap virulensi dan patogenitas virus Influenza. Kata kunci : Patogenesis, Virus Influenza, Protein  PB1-F2 Abstract Aim. Influenza virus PB1-F2 (polymerase basic 1-frame 2 protein is a novel protein previously shown to be involved in cell death induction and pathogenesis. Here we analysis the PB1-F2 protein of Influenza virus A/H

  16. A study of analysis PB1-F2 protein of Influenza Viruses A/H1N1pdm09, A/ H3N2, and A/H5N1

    Directory of Open Access Journals (Sweden)

    Hana Apsari Pawestri

    2016-07-01

    Full Text Available Abstrak Tujuan. Protein PB1-F2 (polymerase basic 1-frame 2 adalah protein terbaru yang ditemukan pada virus Influenza dan telah terbukti berperan dalam induksi kematian sel dan patogenitas. Tujuan dari tulisan ini adalah untuk menganalisis protein PB1-F2 pada virus Influenza A/H5N1 dan A/H1N1pdm09. Metode. Kami melakukan pencarian data yang relevan yaitu sekuens gen virus Influenza A/H5N1 dan A/H1N1pdm09 dari Gen Bank National Center for Biotechnology Information (NCBI selama tahun 1997-2015. Data yang digunakan adalah data sekuens nukleotida gen PB1 (polymerase basic1 virus influenza A/H5N1 dan A/H1N1pdm09. Kemudian dilakukan analisis alignment untuk mengetahui variasi protein dan mutasi yang berhubungan dengan patogenitas dan virulensi. Hasil. Kami melakukan penelitian terhadap sekuens PB1-F2 sebanyak 3262 influenza A/H5N1 dan 2472 Influenza A/H1N1pdm09. Hasil analisis menunjukkan bahwa semua sekuens A/H5N1 memiliki panjang yang penuh sebanyak 90 asam amino, kecuali influenza pandemi 2009 hanya memiliki panjang 87 asam amino. Kemudian, ditemukan mutasi yang berhubungan dengan virulensi yang ditunjukan dengan perubahan asam amino Asparagin (N menjadi Serin (S. Mutasi tersebut terjadi pada Influenza A/H5N1 sebanyak 8.5% dan Influenza A/H1N1pdm09 sebanyak 0.5%. Kesimpulan. Ditemukan beberapa variasi panjang asam amino dan mutasi penting pada sekuens PB1-F2 dari subtipe yang berbeda yaitu influenza A/H5N1 dan A/H1N1pdm09  yang mengindikasikan seleksi spesifik karena introduksi dan adaptasi terhadap inang yang berbeda. Diperlukan penelitian lanjutan untuk lebih memahami variasi dan kontribusi protein PB1-F2 tersebut terhadap virulensi dan patogenitas virus Influenza. Kata kunci : Patogenesis, Virus Influenza, Protein  PB1-F2 Abstract Aim. Influenza virus PB1-F2 (polymerase basic 1-frame 2 protein is a novel protein previously shown to be involved in cell death induction and pathogenesis. Here we analysis the PB1-F2 protein of Influenza virus A/H

  17. Multivalent HA DNA vaccination protects against highly pathogenic H5N1 avian influenza infection in chickens and mice.

    Directory of Open Access Journals (Sweden)

    Srinivas Rao

    Full Text Available Sustained outbreaks of highly pathogenic avian influenza (HPAI H5N1 in avian species increase the risk of reassortment and adaptation to humans. The ability to contain its spread in chickens would reduce this threat and help maintain the capacity for egg-based vaccine production. While vaccines offer the potential to control avian disease, a major concern of current vaccines is their potency and inability to protect against evolving avian influenza viruses.The ability of DNA vaccines encoding hemagglutinin (HA proteins from different HPAI H5N1 serotypes was evaluated for its ability to elicit neutralizing antibodies and to protect against homologous and heterologous HPAI H5N1 strain challenge in mice and chickens after DNA immunization by needle and syringe or with a pressure injection device. These vaccines elicited antibodies that neutralized multiple strains of HPAI H5N1 when given in combinations containing up to 10 HAs. The response was dose-dependent, and breadth was determined by the choice of the influenza virus HA in the vaccine. Monovalent and trivalent HA vaccines were tested first in mice and conferred protection against lethal H5N1 A/Vietnam/1203/2004 challenge 68 weeks after vaccination. In chickens, protection was observed against heterologous strains of HPAI H5N1 after vaccination with a trivalent H5 serotype DNA vaccine with doses as low as 5 microg DNA given twice either by intramuscular needle injection or with a needle-free device.DNA vaccines offer a generic approach to influenza virus immunization applicable to multiple animal species. In addition, the ability to substitute plasmids encoding different strains enables rapid adaptation of the vaccine to newly evolving field isolates.

  18. 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.

  19. Gene expression profiles in primary duodenal chick cells following transfection with avian influenza virus H5 DNA plasmid encapsulated in silver nanoparticles

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    Jazayeri SD

    2013-02-01

    Full Text Available Seyed Davoud Jazayeri,1 Aini Ideris,1,2 Kamyar Shameli,3 Hassan Moeini,1 Abdul Rahman Omar1,21Institute of Bioscience, 2Faculty of Veterinary Medicine, 3Faculty of Science, Universiti Putra Malaysia, Serdang, Selangor, MalaysiaAbstract: In order to develop a systemically administered safe and effective nonviral gene delivery system against avian influenza virus (AIV that induced cytokine expression, the hemagglutinin (H5 gene of AIV, A/Ck/Malaysia/5858/04 (H5N1 and green fluorescent protein were cloned into a coexpression vector pIRES (pIREGFP-H5 and formulated using green synthesis of silver nanoparticles (AgNPs with poly(ethylene glycol and transfected into primary duodenal cells taken from 18-day-old specific-pathogen-free chick embryos. The AgNPs were prepared using moderated temperature and characterized for particle size, surface charge, ultraviolet-visible spectra, DNA loading, and stability. AgNPs and AgNP-pIREGFP-H5 were prepared in the size range of 13.9 nm and 25 nm with a positive charge of +78 ± 0.6 mV and +40 ± 6.2 mV, respectively. AgNPs with a positive surface charge could encapsulate pIREGFP-H5 efficiently. The ultraviolet-visible spectra for AgNP-pIREGFP-H5 treated with DNase I showed that the AgNPs were able to encapsulate pIREGFP-H5 efficiently. Polymerase chain reaction showed that AgNP-pIREGFP-H5 entered into primary duodenal cells rapidly, as early as one hour after transfection. Green fluorescent protein expression was observed after 36 hours, peaked at 48 hours, and remained stable for up to 60 hours. In addition, green fluorescent protein expression generally increased with increasing DNA concentration and time. Cells were transfected using Lipocurax in vitro transfection reagent as a positive control. A multiplex quantitative mRNA gene expression assay in the transfected primary duodenal cells via the transfection reagent and AgNPs with pIREGFP-H5 revealed expression of interleukin (IL-18, IL-15, and IL-12

  20. Structure-activity relationships of 3-O-β-chacotriosyl oleanic acid derivatives as entry inhibitors for highly pathogenic H5N1 influenza virus.

    Science.gov (United States)

    Li, Sumei; Jia, Xiuhua; Shen, Xintian; Wei, Zhuwen; Jiang, Zhiyan; Liao, Yixian; Guo, Yiming; Zheng, Xiaojun; Zhong, Guohua; Song, Gaopeng

    2017-08-15

    Highly pathogenic H5N1 virus (H5N1) entry is a key target for the development of novel anti-influenza agents with new mechanisms of action. In our continuing efforts to identify novel potential anti-H5N1 entry inhibitors, a series of 3-O-β-chacotriosyl oleanolic acid analogs have been designed, synthesized and evaluated as H5N1 entry inhibitors based on two small molecule inhibitors 1 and 2 previously discovered by us. The anti-H5N1 entry activities were determined based on HA/HIV and VSVG/HIV entry assays. Compound 15 displayed the most promising anti-H5N1 entry activities with average IC 50 values of 4.05μM and good selective index (22.9). Detailed structure-activity relationships (SARs) studies suggested that either the introduction of an additional oxo group to position 11 at OA or alteration of the C-3 configuration of OA from 3β- to 3α-forms can significantly enhance the selective index while maintaining their antiviral activities in vitro. Molecular simulation analysis confirmed that the compounds exert their inhibitory activity through binding tightly to hemagglutinin (HA2) protein near the fusion peptide and prevent virus entry. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Oligomeric recombinant H5 HA1 vaccine produced in bacteria protects ferrets from homologous and heterologous wild-type H5N1 influenza challenge and controls viral loads better than subunit H5N1 vaccine by eliciting high-affinity antibodies.

    Science.gov (United States)

    Verma, Swati; Dimitrova, Milena; Munjal, Ashok; Fontana, Juan; Crevar, Corey J; Carter, Donald M; Ross, Ted M; Khurana, Surender; Golding, Hana

    2012-11-01

    Recombinant hemagglutinin from influenza viruses with pandemic potential can be produced rapidly in various cell substrates. In this study, we compared the functionality and immunogenicity of bacterially produced oligomeric or monomeric HA1 proteins from H5N1 (A/Vietnam/1203/04) with those of the egg-based licensed subunit H5N1 (SU-H5N1) vaccine in ferrets challenged with homologous or heterologous H5N1 highly pathogenic influenza strains. Ferrets were vaccinated twice with the oligomeric or monomeric rHA1 or with SU-H5N1 (Sanofi Pasteur) emulsified with Titermax adjuvant and were challenged with wild-type homologous (A/Vietnam/1203/04; clade 1) or heterologous (A/Whooperswan/Mongolia/244/2005; clade 2.2) virus. Only the oligomeric rHA1 (not the monomeric rHA1) immunogen and the SU-H5N1 vaccine provided protection against the lethality and morbidity of homologous and heterologous highly pathogenic H5N1. Oligomeric rHA1 generated more cross-neutralizing antibodies and higher levels of serum antibody binding to HA1, with stronger avidity and a better IgG/IgM ratio, than monomeric HA1 and SU-H5N1 vaccines, as determined by surface plasmon resonance (SPR). Importantly, viral loads after heterologous H5N1 challenge were more efficiently controlled in ferrets vaccinated with the oligomeric rHA1 immunogen than in SU-H5N1-vaccinated ferrets. The reduction of viral loads in the nasal washes correlated strongly with higher-avidity antibodies to oligomeric rHA1 derived from H5N1 clade 1 and clade 2.2 viruses, as measured by SPR. This is the first study to show the role of antibody avidity for the HA1 globular head domain in reduction of viral loads in the upper respiratory tract, which could significantly reduce viral transmission.

  2. 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.

  3. Highly immunogenic prime–boost DNA vaccination protects chickens against challenge with homologous and heterologous H5N1 virus

    Directory of Open Access Journals (Sweden)

    Anna Stachyra

    2014-01-01

    Full Text Available Highly pathogenic avian influenza viruses (HPAIVs cause huge economic losses in the poultry industry because of high mortality rate in infected flocks and trade restrictions. Protective antibodies, directed mainly against hemagglutinin (HA, are the primary means of protection against influenza outbreaks. A recombinant DNA vaccine based on the sequence of H5 HA from the H5N1/A/swan/Poland/305-135V08/2006 strain of HPAIV was prepared. Sequence manipulation included deletion of the proteolytic cleavage site to improve protein stability, codon usage optimization to improve translation and stability of RNA in host cells, and cloning into a commercially available vector to enable expression in animal cells. Naked plasmid DNA was complexed with a liposomal carrier and the immunization followed the prime–boost strategy. The immunogenic potential of the DNA vaccine was first proved in broilers in near-to-field conditions resembling a commercial farm. Next, the protective activity of the vaccine was confirmed in SPF layer-type chickens. Experimental infections (challenge experiments indicated that 100% of vaccinated chickens were protected against H5N1 of the same clade and that 70% of them were protected against H5N1 influenza virus of a different clade. Moreover, the DNA vaccine significantly limited (or even eliminated transmission of the virus to contact control chickens. Two intramuscular doses of DNA vaccine encoding H5 HA induced a strong protective response in immunized chicken. The effective protection lasted for a minimum 8 weeks after the second dose of the vaccine and was not limited to the homologous H5N1 virus. In addition, the vaccine reduced shedding of the virus.

  4. Research of H5N6 Treatment by Comparing with H6N1 and H10N8 by Using Decision Tree and Apriori Algorithm

    Directory of Open Access Journals (Sweden)

    Kim Sunghyun

    2016-01-01

    Full Text Available Since 2003, 608 people in 15 countries have infected with human-infectious AI viruses and 359 of them died. Especially, in China, H6N1 and H10N8 viruses were wide-spread and a lot of people were infected and died. Recently, H5N6 virus emerged in China and the number of patients has been increasing gradually. Therefore, this research compared amino acid strain of Matrix Protein, Hemagglutinin, Neuraminidase and Nucleoprotein of H5N6, H6N1 and H10N8, by using Decision tree and Apriori Algorithm, to figure out their similarity and devise the treatment. In result, Matrix protein and Nucleoprotein sequences of H5N6 were similar with those of H6N1 and H10N8. Therefore, this research concluded that the treatment targeting those proteins of H6N1 and H10N8 will be also effective to H5N6.

  5. Purification of neuraminidase from Influenza virus subtype H5N1

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    Simson Tariga

    2009-03-01

    Full Text Available Influenza-virus neuraminidase plays vital role in the survival of the organisms. Vaccination of animals with this glycoprotein confers immune responses so that enable it to protect the animals from incoming infection. Supplementation of conventional vaccines with this glycoprotein increases the protection and longevity of the vaccine. Purified neuraminidase can also be used to develop serological tests for differentiation of serologically positive animals due to infection or to vaccination. In this study purification of neuraminidase from influenza virus subtype H5N1 was described. Triton x-100 and Octyl β-D-glucopyranoside were used to extract and diluted the glycoprotein membrane. The enzymatic activity of the neuraminidase was assayed using a fluorochrome substrate, 4-methylumbelliferyl-a-D-N-acetyl neuraminic acid, which was found to be simple, sensitive and suitable for the purification purpose. The neuraminidase was absorbed selectively on an oxamic-acid agarose column. The purity of neuraminidase eluted from this affinity column was high. A higher purity of the neuraminidase was obtained by further separation with gel filtration on Superdex-200. The purified neuraminidase was enzymatically active and did not contain any detectable haemagglutinin, either by haemagglutination assay or by monospecific antibodies raised against H5N1 hemagglutinin. The purified neuraminidase was recognized strongly by antibodies raised against an internal but only weakly by that against C-terminal regions of the neuraminidase protein of H5N1-influenza virus. The purified neuraminidase was in tetrameric forms but dissociated into monomeric form on reducing condition, or mostly dimeric form on non-reducing SDS-PAGE.

  6. Human Coronavirus HKU1 Spike Protein Uses O-Acetylated Sialic Acid as an Attachment Receptor Determinant and Employs Hemagglutinin-Esterase Protein as a Receptor-Destroying Enzyme.

    Science.gov (United States)

    Huang, Xingchuan; Dong, Wenjuan; Milewska, Aleksandra; Golda, Anna; Qi, Yonghe; Zhu, Quan K; Marasco, Wayne A; Baric, Ralph S; Sims, Amy C; Pyrc, Krzysztof; Li, Wenhui; Sui, Jianhua

    2015-07-01

    Human coronavirus (hCoV) HKU1 is one of six hCoVs identified to date and the only one with an unidentified cellular receptor. hCoV-HKU1 encodes a hemagglutinin-esterase (HE) protein that is unique to the group a betacoronaviruses (group 2a). The function of HKU1-HE remains largely undetermined. In this study, we examined binding of the S1 domain of hCoV-HKU1 spike to a panel of cells and found that the S1 could specifically bind on the cell surface of a human rhabdomyosarcoma cell line, RD. Pretreatment of RD cells with neuraminidase (NA) and trypsin greatly reduced the binding, suggesting that the binding was mediated by sialic acids on glycoproteins. However, unlike other group 2a CoVs, e.g., hCoV-OC43, for which 9-O-acetylated sialic acid (9-O-Ac-Sia) serves as a receptor determinant, HKU1-S1 bound with neither 9-O-Ac-Sia-containing glycoprotein(s) nor rat and mouse erythrocytes. Nonetheless, the HKU1-HE was similar to OC43-HE, also possessed sialate-O-acetylesterase activity, and acted as a receptor-destroying enzyme (RDE) capable of eliminating the binding of HKU1-S1 to RD cells, whereas the O-acetylesterase-inactive HKU1-HE mutant lost this capacity. Using primary human ciliated airway epithelial (HAE) cell cultures, the only in vitro replication model for hCoV-HKU1 infection, we confirmed that pretreatment of HAE cells with HE but not the enzymatically inactive mutant blocked hCoV-HKU1 infection. These results demonstrate that hCoV-HKU1 exploits O-Ac-Sia as a cellular attachment receptor determinant to initiate the infection of host cells and that its HE protein possesses the corresponding sialate-O-acetylesterase RDE activity. Human coronaviruses (hCoV) are important human respiratory pathogens. Among the six hCoVs identified to date, only hCoV-HKU1 has no defined cellular receptor. It is also unclear whether hemagglutinin-esterase (HE) protein plays a role in viral entry. In this study, we found that, similarly to other members of the group 2a CoVs, sialic

  7. 77 FR 63783 - Influenza Viruses Containing the Hemagglutinin from the Goose/Guangdong/1/96 Lineage

    Science.gov (United States)

    2012-10-17

    ... DEPARTMENT OF HEALTH AND HUMAN SERVICES 42 CFR Part 73 [Docket: CDC-2012-0010] Influenza Viruses... questions concerning highly pathogenic avian influenza (HPAI) H5N1 viruses that contain a hemagglutinin (HA... avian influenza (HPAI) H5N1 viruses with a mortality rate that exceeds 50 percent in hospitalized...

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

    Science.gov (United States)

    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...

  9. Rapid and highly informative diagnostic assay for H5N1 influenza viruses.

    Directory of Open Access Journals (Sweden)

    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.

  10. Detection and Characterization of Clade 1 Reassortant H5N1 Viruses Isolated from Human Cases in Vietnam during 2013.

    Directory of Open Access Journals (Sweden)

    Sharmi W Thor

    Full Text Available Highly pathogenic avian influenza (HPAI H5N1 is endemic in Vietnamese poultry and has caused sporadic human infection in Vietnam since 2003. Human infections with HPAI H5N1 are of concern due to a high mortality rate and the potential for the emergence of pandemic viruses with sustained human-to-human transmission. Viruses isolated from humans in southern Vietnam have been classified as clade 1 with a single genome constellation (VN3 since their earliest detection in 2003. This is consistent with detection of this clade/genotype in poultry viruses endemic to the Mekong River Delta and surrounding regions. Comparison of H5N1 viruses detected in humans from southern Vietnamese provinces during 2012 and 2013 revealed the emergence of a 2013 reassortant virus with clade 1.1.2 hemagglutinin (HA and neuraminidase (NA surface protein genes but internal genes derived from clade 2.3.2.1a viruses (A/Hubei/1/2010-like; VN12. Closer analysis revealed mutations in multiple genes of this novel genotype (referred to as VN49 previously associated with increased virulence in animal models and other markers of adaptation to mammalian hosts. Despite the changes identified between the 2012 and 2013 genotypes analyzed, their virulence in a ferret model was similar. Antigenically, the 2013 viruses were less cross-reactive with ferret antiserum produced to the clade 1 progenitor virus, A/Vietnam/1203/2004, but reacted with antiserum produced against a new clade 1.1.2 WHO candidate vaccine virus (A/Cambodia/W0526301/2012 with comparable hemagglutination inhibition titers as the homologous antigen. Together, these results indicate changes to both surface and internal protein genes of H5N1 viruses circulating in southern Vietnam compared to 2012 and earlier viruses.

  11. 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.

  12. Protection of White Leghorn chickens by U.S. emergency H5 vaccination against clade 2.3.4.4 H5N2 high pathogenicity avian influenza virus.

    Science.gov (United States)

    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.

  13. The antigenic property of the H5N1 avian influenza viruses isolated in central China

    Directory of Open Access Journals (Sweden)

    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.

  14. h5-HT1B receptor-mediated constitutive Gαi3-protein activation in stably transfected Chinese hamster ovary cells: an antibody capture assay reveals protean efficacy of 5-HT

    Science.gov (United States)

    Newman-Tancredi, Adrian; Cussac, Didier; Marini, Laetitia; Touzard, Manuelle; Millan, Mark J

    2003-01-01

    Serotonin 5-HT1B receptors couple to G-proteins of the Gi/o family. However, their activation of specific G-protein subtypes is poorly characterised. Using an innovative antibody capture/guanosine-5′-0-(3-[35S]thio)-triphosphate ([35S]GTPγS) binding strategy, we characterised Gαi3 subunit activation by h5-HT1B receptors stably expressed in Chinese hamster ovary (CHO) cells. The agonists, 5-HT, alniditan and BMS181,101, stimulated Gαi3, whereas methiothepin and SB224,289 behaved as inverse agonists. The selective 5-HT1B receptor ligand, S18127, modestly stimulated Gαi3 and reversed the actions of both 5-HT and methiothepin. S18127 (1 μM) also produced parallel, dextral shifts of the 5-HT and methiothepin isotherms. Isotopic dilution experiments ([35S]GTPγS versus GTPγS) revealed high-affinity [35S]GTPγS binding to Gαi3 subunits in the absence of receptor ligands indicating constitutive activity. High-affinity [35S]GTPγS binding was increased 2.8-fold by 5-HT with an increase in the affinity of GTPγS for Gαi3 subunits. In contrast, methiothepin halved the number of high-affinity binding sites and decreased their affinity. h5-HT1B receptor-mediated Gαi3 subunit activation was dependent on the concentration of NaCl. At 300 mM, 5-HT stimulated [35S]GTPγS binding, basal Gαi3 activation was low and methiothepin was inactive. In contrast, at 10 mM NaCl, basal activity was enhanced and the inverse agonist activity of methiothepin was accentuated. Under these conditions, 5-HT decreased Gαi3 activation. In conclusion, at h5-HT1B receptors expressed in CHO cells: (i) inverse agonist induced inhibition of Gαi3, and its reversal by S18127, reveals constitutive activation of this Gα subunit; (ii) constitutive Gαi3 activation can be quantified by isotopic dilution [35S]GTPγS binding and (iii) decreasing NaCl concentrations enhances Gαi3 activation and leads to protean agonist properties of 5-HT: that is a switch to inhibition of Gαi3. PMID:12684263

  15. h5-HT(1B) receptor-mediated constitutive Galphai3-protein activation in stably transfected Chinese hamster ovary cells: an antibody capture assay reveals protean efficacy of 5-HT.

    Science.gov (United States)

    Newman-Tancredi, Adrian; Cussac, Didier; Marini, Laetitia; Touzard, Manuelle; Millan, Mark J

    2003-03-01

    1. Serotonin 5-HT(1B) receptors couple to G-proteins of the Gi/o family. However, their activation of specific G-protein subtypes is poorly characterised. Using an innovative antibody capture/guanosine-5'-0-(3-[(35)S]thio)-triphosphate ([(35)S]GTPgammaS) binding strategy, we characterised Galpha(i3) subunit activation by h5-HT(1B) receptors stably expressed in Chinese hamster ovary (CHO) cells. 2. The agonists, 5-HT, alniditan and BMS181,101, stimulated Galpha(i3), whereas methiothepin and SB224,289 behaved as inverse agonists. The selective 5-HT(1B) receptor ligand, S18127, modestly stimulated Galpha(i3) and reversed the actions of both 5-HT and methiothepin. S18127 (1 micro M) also produced parallel, dextral shifts of the 5-HT and methiothepin isotherms. 3. Isotopic dilution experiments ([(35)S]GTPgammaS versus GTPgammaS) revealed high-affinity [(35)S]GTPgammaS binding to Galpha(i3) subunits in the absence of receptor ligands indicating constitutive activity. High-affinity [(35)S]GTPgammaS binding was increased 2.8-fold by 5-HT with an increase in the affinity of GTPgammaS for Galpha(i3) subunits. In contrast, methiothepin halved the number of high-affinity binding sites and decreased their affinity. 4. h5-HT(1B) receptor-mediated Galpha(i3) subunit activation was dependent on the concentration of NaCl. At 300 mM, 5-HT stimulated [(35)S]GTPgammaS binding, basal Galpha(i3) activation was low and methiothepin was inactive. In contrast, at 10 mM NaCl, basal activity was enhanced and the inverse agonist activity of methiothepin was accentuated. Under these conditions, 5-HT decreased Galpha(i3) activation. 5. In conclusion, at h5-HT(1B) receptors expressed in CHO cells: (i) inverse agonist induced inhibition of Galpha(i3), and its reversal by S18127, reveals constitutive activation of this Galpha subunit; (ii) constitutive Galpha(i3) activation can be quantified by isotopic dilution [(35)S]GTPgammaS binding and (iii) decreasing NaCl concentrations enhances Galpha(i3

  16. Exploring the nature of the H-bonds between the human class II MHC protein, HLA-DR1 (DRB*0101) and the influenza virus hemagglutinin peptide, HA306-318, using the quantum theory of atoms in molecules.

    Science.gov (United States)

    Aray, Yosslen; Aguilera-García, Ricardo; Izquierdo, Daniel R

    2018-01-02

    The nature of the H-bonds between the human protein HLA-DR1 (DRB*0101) and the hemagglutinin peptide HA306-318 has been studied using the Quantum Theory of Atoms in Molecules for the first time. We have found four H-bond groups: one conventional CO··HN bond group and three nonconventional CO··HC, π··HC involving aromatic rings and HN··HC aliphatic groups. The calculated electron density at the determined H-bond critical points suggests the follow protein pocket binding trend: P1 (2,311) > P9 (1.109) > P4 (0.950) > P6 (0.553) > P7 (0.213) which agrees and reveal the nature of experimental findings, showing that P1 produces by a long way the strongest binding of the HLA-DR1 human protein molecule with the peptide backbone as consequence of the vast number of H-bonds in the P1 area and at the same time the largest specific binding of the peptide Tyr308 residue with aromatic residues located at the binding groove floor. The present results suggest the topological analysis of the electronic density as a valuable tool that allows a non-arbitrary partition of the pockets binding energy via the calculated electron density at the determined critical points.

  17. 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.

  18. Avian Influenza A (H5N1)

    Centers for Disease Control (CDC) Podcasts

    In this podcast, CDC's Dr. Tim Uyeki discusses H5N1, a subtype of influenza A virus. This highly pathogenic H5N1 virus doesn't usually infect people, although some rare infections with H5N1 viruses have occurred in humans. We need to use a comprehensive strategy to prevent the spread of H5N1 virus among birds, including having human health and animal health work closely together.

  19. Influenza A H5N1 immigration is filtered out at some international borders.

    Directory of Open Access Journals (Sweden)

    Robert G Wallace

    2008-02-01

    Full Text Available Geographic spread of highly pathogenic influenza A H5N1, the bird flu strain, appears a necessary condition for accelerating the evolution of a related human-to-human infection. As H5N1 spreads the virus diversifies in response to the variety of socioecological environments encountered, increasing the chance a human infection emerges. Genetic phylogenies have for the most part provided only qualitative evidence that localities differ in H5N1 diversity. For the first time H5N1 variation is quantified across geographic space.We constructed a statistical phylogeography of 481 H5N1 hemagglutinin genetic sequences from samples collected across 28 Eurasian and African localities through 2006. The MigraPhyla protocol showed southern China was a source of multiple H5N1 strains. Nested clade analysis indicated H5N1 was widely dispersed across southern China by both limited dispersal and long distance colonization. The UniFrac metric, a measure of shared phylogenetic history, grouped H5N1 from Indonesia, Japan, Thailand and Vietnam with those from southeastern Chinese provinces engaged in intensive international trade. Finally, H5N1's accumulative phylogenetic diversity was greatest in southern China and declined beyond. The gradient was interrupted by areas of greater and lesser phylogenetic dispersion, indicating H5N1 migration was restricted at some geopolitical borders. Thailand and Vietnam, just south of China, showed significant phylogenetic clustering, suggesting newly invasive H5N1 strains have been repeatedly filtered out at their northern borders even as both countries suffered recurring outbreaks of endemic strains. In contrast, Japan, while successful in controlling outbreaks, has been subjected to multiple introductions of the virus.The analysis demonstrates phylogenies can provide local health officials with more than hypotheses about relatedness. Pathogen dispersal, the functional relationships among disease ecologies across localities, and

  20. Avian Influenza A (H5N1)

    Centers for Disease Control (CDC) Podcasts

    2009-05-27

    In this podcast, CDC's Dr. Tim Uyeki discusses H5N1, a subtype of influenza A virus. This highly pathogenic H5N1 virus doesn't usually infect people, although some rare infections with H5N1 viruses have occurred in humans. We need to use a comprehensive strategy to prevent the spread of H5N1 virus among birds, including having human health and animal health work closely together.  Created: 5/27/2009 by Emerging Infectious Diseases.   Date Released: 5/27/2009.

  1. Immunogenicity and protective efficacy of a live attenuated H5N1 vaccine in nonhuman primates.

    Directory of Open Access Journals (Sweden)

    Shufang Fan

    2009-05-01

    Full Text Available The continued spread of highly pathogenic H5N1 influenza viruses among poultry and wild birds, together with the emergence of drug-resistant variants and the possibility of human-to-human transmission, has spurred attempts to develop an effective vaccine. Inactivated subvirion or whole-virion H5N1 vaccines have shown promising immunogenicity in clinical trials, but their ability to elicit protective immunity in unprimed human populations remains unknown. A cold-adapted, live attenuated vaccine with the hemagglutinin (HA and neuraminidase (NA genes of an H5N1 virus A/VN/1203/2004 (clade 1 was protective against the pulmonary replication of homologous and heterologous wild-type H5N1 viruses in mice and ferrets. In this study, we used reverse genetics to produce a cold-adapted, live attenuated H5N1 vaccine (AH/AAca that contains HA and NA genes from a recent H5N1 isolate, A/Anhui/2/05 virus (AH/05 (clade 2.3, and the backbone of the cold-adapted influenza H2N2 A/AnnArbor/6/60 virus (AAca. AH/AAca was attenuated in chickens, mice, and monkeys, and it induced robust neutralizing antibody responses as well as HA-specific CD4+ T cell immune responses in rhesus macaques immunized twice intranasally. Importantly, the vaccinated macaques were fully protected from challenge with either the homologous AH/05 virus or a heterologous H5N1 virus, A/bar-headed goose/Qinghai/3/05 (BHG/05; clade 2.2. These results demonstrate for the first time that a cold-adapted H5N1 vaccine can elicit protective immunity against highly pathogenic H5N1 virus infection in a nonhuman primate model and provide a compelling argument for further testing of double immunization with live attenuated H5N1 vaccines in human trials.

  2. 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

  3. 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...

  4. Characterization of a low pathogenic avian influenza H5N2 virus isolated from a turkey breeder flock in Manitoba, Canada.

    Science.gov (United States)

    Berhane, Y; Joseph, T; Kehler, H; Hisanaga, T; Embury-Hyatt, C; Diederich, S; McGreevy, K Hooper; Handel, K; Cottam-Birt, C; Pasick, J

    2014-03-01

    In November 2010, an outbreak of avian influenza (AI) due to the H5N2 subtype virus occurred in a turkey breeder farm in northern Manitoba, Canada. The only clinical signs observed were depression, decrease in food consumption, and loss of egg production. The hemagglutinin (HA) cleavage (HA(0)) site of the isolated H5N2 virus was PQRETR/GLF, consistent with low pathogenic AI viruses. The intravenous pathogenicity index of this virus was zero. Whole-genome sequencing of two isolates that originated from two different barns was performed, and both isolates had 100% identical protein sequence in PB2, HA, NP, M1, M2, NS1, and NS2. The remaining gene segments (PB1, PA, and NA) had a single amino-acid difference when compared with each other. The nucleotide and protein sequences of eight gene segments from both isolates showed 99 or greater identity with other AI viruses that have been circulating in free-living aquatic birds in Canada and the United States within the last 10 yr. Phylogenetic analysis of the HA and neuraminidase (NA) gene segments showed that these viruses are closely related to other H5 strains that have been isolated from Manitoba and other parts of Canada. Serologic testing of archived serum samples collected from these turkeys a week before the outbreak showed no evidence of AI infection. In addition, other farms that were located within 3 km radius from the infected farm and farms that had epidemiologic connection with the farm also tested negative for the presence of H5N2 AI virus or antibody. This indicates that the virus might have been introduced to the farm from wild aquatic birds only a short time before detection. Results of this study highlight the importance of early detection and the significance of ongoing Canada-wide surveillance of AI in domestic poultry as well as in wild aquatic birds/ducks.

  5. Detection of antibodies against H5 and H7 strains in birds: evaluation of influenza pseudovirus particle neutralization tests

    Directory of Open Access Journals (Sweden)

    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.

  6. 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.

  7. Role for migratory wild birds in the global spread of avian influenza H5N8

    Science.gov (United States)

    ,; Ip, Hon S.

    2016-01-01

    Avian influenza viruses affect both poultry production and public health. A subtype H5N8 (clade 2.3.4.4) virus, following an outbreak in poultry in South Korea in January 2014, rapidly spread worldwide in 2014–2015. Our analysis of H5N8 viral sequences, epidemiological investigations, waterfowl migration, and poultry trade showed that long-distance migratory birds can play a major role in the global spread of avian influenza viruses. Further, we found that the hemagglutinin of clade 2.3.4.4 virus was remarkably promiscuous, creating reassortants with multiple neuraminidase subtypes. Improving our understanding of the circumpolar circulation of avian influenza viruses in migratory waterfowl will help to provide early warning of threats from avian influenza to poultry, and potentially human, health.

  8. Use of the Chinchilla model to evaluate the vaccinogenic potential of the Moraxella catarrhalis filamentous hemagglutinin-like proteins MhaB1 and MhaB2.

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    Teresa L Shaffer

    Full Text Available Moraxella catarrhalis causes significant health problems, including 15-20% of otitis media cases in children and ~10% of respiratory infections in adults with chronic obstructive pulmonary disease. The lack of an efficacious vaccine, the rapid emergence of antibiotic resistance in clinical isolates, and high carriage rates reported in children are cause for concern. In addition, the effectiveness of conjugate vaccines at reducing the incidence of otitis media caused by Streptococcus pneumoniae and nontypeable Haemophilus influenzae suggest that M. catarrhalis infections may become even more prevalent. Hence, M. catarrhalis is an important and emerging cause of infectious disease for which the development of a vaccine is highly desirable. Studying the pathogenesis of M. catarrhalis and the testing of vaccine candidates have both been hindered by the lack of an animal model that mimics human colonization and infection. To address this, we intranasally infected chinchilla with M. catarrhalis to investigate colonization and examine the efficacy of a protein-based vaccine. The data reveal that infected chinchillas produce antibodies against antigens known to be major targets of the immune response in humans, thus establishing immune parallels between chinchillas and humans during M. catarrhalis infection. Our data also demonstrate that a mutant lacking expression of the adherence proteins MhaB1 and MhaB2 is impaired in its ability to colonize the chinchilla nasopharynx, and that immunization with a polypeptide shared by MhaB1 and MhaB2 elicits antibodies interfering with colonization. These findings underscore the importance of adherence proteins in colonization and emphasize the relevance of the chinchilla model to study M. catarrhalis-host interactions.

  9. 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....

  10. Pathogenicity of Highly Pathogenic Avian Influenza Virus H5N1 in Naturally Infected Poultry in Egypt.

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    Ibrahim Thabet Hagag

    Full Text Available Highly pathogenic avian influenza virus (HPAIV H5N1 has been endemic in Egypt since 2006, and there is increasing concern for its potential to become highly transmissible among humans. Infection by HPAIV H5N1 has been described in experimentally challenged birds. However, the pathogenicity of the H5N1 isolated in Egypt has never been reported in naturally infected chickens and ducks. Here we report a 2013 outbreak of HPAIV H5N1 in commercial poultry farms and backyards in Sharkia Province, Egypt. The main symptoms were ecchymosis on the shanks and feet, cyanosis of the comb and wattles, subcutaneous edema of the head and neck for chickens, and nervous signs (torticollis for ducks. Within 48-72 hrs of the onset of illness, the average mortality rates were 22.8-30% and 28.5-40% in vaccinated chickens and non-vaccinated ducks, respectively. Tissue samples of chickens and ducks were collected for analyses with cross-section immunohistochemistry and real-time RT-PCR for specific viral RNA transcripts. While viral RNA was detected in nearly all tissues and sera collected, viral nucleoprotein was detected almost ubiquitously in all tissues, including testis. Interestingly, viral antigen was also observed in endothelial cells of most organs in chickens, and clearly detected in the trachea and brain in particular. Viral nucleoprotein was also detected in mononuclear cells of various organs, especially pulmonary tissue. We performed phylogenetic analyses and compared the genomic sequences of the hemagglutinin (HA and nonstructural proteins (NS among the isolated viruses, the HPAIV circulated in Egypt in the past and currently, and some available vaccine strains. Further analysis of deduced amino acids of both HA and NS1 revealed that our isolates carried molecular determinants of HPAIV, including the multibasic amino acids (PQGERRRK/KR*GLF in the cleavage site in HA and glutamate at position 92 (D92E in NS1. This is the first report of the pathogenicity

  11. 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.

  12. Suboptimal protection against H5N1 highly pathogenic avian influenza viruses from Vietnam in ducks vaccinated with commercial poultry vaccines.

    Science.gov (United States)

    Cha, Ra Mi; Smith, Diane; Shepherd, Eric; Davis, C Todd; Donis, Ruben; Nguyen, Tung; Nguyen, Hoang Dang; Do, Hoa Thi; Inui, Ken; Suarez, David L; Swayne, David E; Pantin-Jackwood, Mary

    2013-10-09

    Domestic ducks are the second most abundant poultry species in many Asian countries including Vietnam, and play a critical role in the epizootiology of H5N1 highly pathogenic avian influenza (HPAI) [FAO]. In this study, we examined the protective efficacy in ducks of two commercial H5N1 vaccines widely used in Vietnam; Re-1 containing A/goose/Guangdong/1/1996 hemagglutinin (HA) clade 0 antigens, and Re-5 containing A/duck/Anhui/1/2006 HA clade 2.3.4 antigens. Ducks received two doses of either vaccine at 7 and at 14 or 21 days of age followed by challenge at 30 days of age with viruses belonging to the HA clades 1.1, 2.3.4.3, 2.3.2.1.A and 2.3.2.1.B isolated between 2008 and 2011 in Vietnam. Ducks vaccinated with the Re-1 vaccine were protected after infection with the two H5N1 HPAI viruses isolated in 2008 (HA clades 1.1 and 2.3.4.3) showing no mortality and limited virus shedding. The Re-1 and Re-5 vaccines conferred 90-100% protection against mortality after challenge with the 2010 H5N1 HPAI viruses (HA clade 2.3.2.1.A); but vaccinated ducks shed virus for more than 7 days after challenge. Similarly, the Re-1 and Re-5 vaccines only showed partial protection against the 2011 H5N1 HPAI viruses (HA clade 2.3.2.1.A and 2.3.2.1.B), with a high proportion of vaccinated ducks shedding virus for more than 10 days. Furthermore, 50% mortality was observed in ducks vaccinated with Re-1 and challenged with the 2.3.2.1.B virus. The HA proteins of the 2011 challenge viruses had the greatest number of amino acid differences from the two vaccines as compared to the viruses from 2008 and 2009, which correlates with the lesser protection observed with these viruses. These studies demonstrate the suboptimal protection conferred by the Re-1 and Re-5 commercial vaccines in ducks against H5N1 HPAI clade 2.3.2.1 viruses, and underscore the importance of monitoring vaccine efficacy in the control of H5N1 HPAI in ducks. Published by Elsevier Ltd.

  13. Challenge for One Health: Co-Circulation of Zoonotic H5N1 and H9N2 Avian Influenza Viruses in Egypt.

    Science.gov (United States)

    Kim, Shin-Hee

    2018-03-09

    Highly pathogenic avian influenza (HPAI) H5N1 viruses are currently endemic in poultry in Egypt. Eradication of the viruses has been unsuccessful due to improper application of vaccine-based control strategies among other preventive measures. The viruses have evolved rapidly with increased bird-to-human transmission efficacy, thus affecting both animal and public health. Subsequent spread of potentially zoonotic low pathogenic avian influenza (LPAI) H9N2 in poultry has also hindered efficient control of avian influenza. The H5N1 viruses acquired enhanced bird-to-human transmissibility by (1) altering amino acids in hemagglutinin (HA) that enable binding affinity to human-type receptors, (2) loss of the glycosylation site and 130 loop in the HA protein and (3) mutation of E627K in the PB2 protein to enhance viral replication in mammalian hosts. The receptor binding site of HA of Egyptian H9N2 viruses has been shown to contain the Q234L substitution along with a H191 mutation, which can increase human-like receptor specificity. Therefore, co-circulation of H5N1 and H9N2 viruses in poultry farming and live bird markets has increased the risk of human exposure, resulting in complication of the epidemiological situation and raising a concern for potential emergence of a new influenza A virus pandemic. For efficient control of infection and transmission, the efficacy of vaccine and vaccination needs to be improved with a comprehensive control strategy, including enhanced biosecurity, education, surveillance, rapid diagnosis and culling of infected poultry.

  14. Challenge for One Health: Co-Circulation of Zoonotic H5N1 and H9N2 Avian Influenza Viruses in Egypt

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    Shin-Hee Kim

    2018-03-01

    Full Text Available Highly pathogenic avian influenza (HPAI H5N1 viruses are currently endemic in poultry in Egypt. Eradication of the viruses has been unsuccessful due to improper application of vaccine-based control strategies among other preventive measures. The viruses have evolved rapidly with increased bird-to-human transmission efficacy, thus affecting both animal and public health. Subsequent spread of potentially zoonotic low pathogenic avian influenza (LPAI H9N2 in poultry has also hindered efficient control of avian influenza. The H5N1 viruses acquired enhanced bird-to-human transmissibility by (1 altering amino acids in hemagglutinin (HA that enable binding affinity to human-type receptors, (2 loss of the glycosylation site and 130 loop in the HA protein and (3 mutation of E627K in the PB2 protein to enhance viral replication in mammalian hosts. The receptor binding site of HA of Egyptian H9N2 viruses has been shown to contain the Q234L substitution along with a H191 mutation, which can increase human-like receptor specificity. Therefore, co-circulation of H5N1 and H9N2 viruses in poultry farming and live bird markets has increased the risk of human exposure, resulting in complication of the epidemiological situation and raising a concern for potential emergence of a new influenza A virus pandemic. For efficient control of infection and transmission, the efficacy of vaccine and vaccination needs to be improved with a comprehensive control strategy, including enhanced biosecurity, education, surveillance, rapid diagnosis and culling of infected poultry.

  15. Avian influenza A (H5N1)

    NARCIS (Netherlands)

    de Jong, Menno D.; Hien, Tran Tinh

    2006-01-01

    Since their reemergence in 2003, highly pathogenic avian influenza A (H5N1) viruses have reached endemic levels among poultry in several southeast Asian countries and have caused a still increasing number of more than 100 reported human infections with high mortality. These developments have ignited

  16. Human Clade 2.3.4.4 A/H5N6 Influenza Virus Lacks Mammalian Adaptation Markers and Does Not Transmit via the Airborne Route between Ferrets.

    Science.gov (United States)

    Herfst, Sander; Mok, Chris K P; van den Brand, Judith M A; van der Vliet, Stefan; Rosu, Miruna E; Spronken, Monique I; Yang, Zifeng; de Meulder, Dennis; Lexmond, Pascal; Bestebroer, Theo M; Peiris, J S Malik; Fouchier, Ron A M; Richard, Mathilde

    2018-01-01

    Since their emergence in 1997, A/H5N1 influenza viruses of the A/goose/Guangdong/1/96 lineage have diversified in multiple genetic and antigenic clades upon continued circulation in poultry in several countries in Eurasia and Africa. Since 2009, reassortant viruses carrying clade 2.3.4.4 hemagglutinin (HA) and internal and neuraminidase (NA) genes of influenza A viruses of different avian origin have been detected, yielding various HA-NA combinations, such as A/H5N1, A/H5N2, A/H5N3, A/H5N5, A/H5N6, and A/H5N8. Previous studies reported on the low pathogenicity and lack of airborne transmission of A/H5N2 and A/H5N8 viruses in the ferret model. However, although A/H5N6 viruses are the only clade 2.3.4.4 viruses that crossed the species barrier and infected humans, the risk they pose for human health remains poorly characterized. Here, the characterization of A/H5N6 A/Guangzhou/39715/2014 virus in vitro and in ferrets is described. This A/H5N6 virus possessed high polymerase activity, mediated by the E627K substitution in the PB2 protein, which corresponds to only one biological trait out of the three that were previously shown to confer airborne transmissibility to A/H5N1 viruses between ferrets. This might explain its lack of airborne transmission between ferrets. After intranasal inoculation, A/H5N6 virus replicated to high titers in the respiratory tracts of ferrets and was excreted for at least 6 days. Moreover, A/H5N6 virus caused severe pneumonia in ferrets upon intratracheal inoculation. Thus, A/H5N6 virus causes a more severe disease in ferrets than previously investigated clade 2.3.4.4 viruses, but our results demonstrate that the risk from airborne spread is currently low. IMPORTANCE Avian influenza A viruses are a threat to human health, as they cross the species barrier and infect humans occasionally, often with severe outcome. The antigenic and genetic diversity of A/H5 viruses from the A/goose/Guangdong/1/96 lineage is increasing, due to continued

  17. Experimental and Field Results Regarding Immunity Induced by a Recombinant Turkey Herpesvirus H5 Vector Vaccine Against H5N1 and Other H5 Highly Pathogenic Avian Influenza Virus Challenges.

    Science.gov (United States)

    Gardin, Yannick; Palya, Vilmos; Dorsey, Kristi Moore; El-Attrache, John; Bonfante, Francesco; Wit, Sjaak de; Kapczynski, Darrell; Kilany, Walid Hamdy; Rauw, Fabienne; Steensels, Mieke; Soejoedono, Retno D

    2016-05-01

    Vaccination against H5N1 highly pathogenic avian influenza (AI) virus (HPAIV) is one of the possible complementary means available for affected countries to control AI when the disease has become, or with a high risk of becoming, endemic. Efficacy of the vaccination against AI relies essentially, but not exclusively, on the capacity of the vaccine to induce immunity against the targeted virus (which is prone to undergo antigenic variations), as well as its capacity to overcome interference with maternal immunity transmitted by immunized breeding hens to their progeny. This property of the vaccine is a prerequisite for its administration at the hatchery, which assures higher and more reliable vaccine coverage of the populations than vaccination at the farm. A recombinant vector vaccine (Vectormune® AI), based on turkey herpesvirus expressing the hemagglutinin gene of an H5N1 HPAIV as an insert, has been used in several experiments conducted in different research laboratories, as well as in controlled field trials. The results have demonstrated a high degree of homologous and cross protection against different genetic clades of the H5N1 HPAIV. Furthermore, vaccine-induced immunity was not impaired by the presence of passive immunity, but on the contrary, cumulated with it for improved early protection. The demonstrated levels of protection against the different challenge viruses exhibited variations in terms of postchallenge mortality, as well as challenge virus shedding. The data presented here highlight the advantages of this vaccine as a useful and reliable tool to complement biosecurity and sanitary policies for better controlling the disease due to HPAIV of H5 subtypes, when the vaccination is applied as a control measure.

  18. Avian influenza A virus subtype H5N2 in a red-lored Amazon parrot.

    Science.gov (United States)

    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.

  19. 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)

  20. A molecular and antigenic survey of H5N1 highly pathogenic avian influenza virus isolates from smallholder duck farms in Central Java, Indonesia during 2007-2008

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    Junaidi Akhmad

    2011-09-01

    Full Text Available Abstract Background Indonesia is one of the countries most severely affected by H5N1 highly pathogenic avian influenza (HPAI virus in terms of poultry and human health. However, there is little information on the diversity of H5N1 viruses circulating in backyard farms, where chickens and ducks often intermingle. In this study, H5N1 virus infection occurring in 96 smallholder duck farms in central Java, Indonesia from 2007-2008 was investigated and the molecular and antigenic characteristics of H5N1 viruses isolated from these farms were analysed. Results All 84 characterised viruses belonged to H5N1 clade 2.1 with three virus sublineages being identified: clade 2.1.1 (1, clade 2.1.3 (80, and IDN/6/05-like viruses (3 that did not belong to any of the present clades. All three clades were found in ducks, while only clade 2.1.3 was isolated from chickens. There were no significant amino acid mutations of the hemagglutinin (HA and neuraminidase (NA sites of the viruses, including the receptor binding, glycosylation, antigenic and catalytic sites and NA inhibitor targets. All the viruses had polybasic amino acids at the HA cleavage site. No evidence of major antigenic variants was detected. Based on the HA gene, identical virus variants could be found on different farms across the study sites and multiple genetic variants could be isolated from HPAI outbreaks simultaneously or at different time points from single farms. HPAI virus was isolated from both ducks and chickens; however, the proportion of surviving duck cases was considerably higher than in chickens. Conclusions The 2.1.3 clade was the most common lineage found in this study. All the viruses had sequence characteristic of HPAI, but negligible variations in other recognized amino acids at the HA and NA proteins which determine virus phenotypes. Multiple genetic variants appeared to be circulating simultaneously within poultry communities. The high proportion of live duck cases compared to

  1. A molecular and antigenic survey of H5N1 highly pathogenic avian influenza virus isolates from smallholder duck farms in Central Java, Indonesia during 2007-2008.

    Science.gov (United States)

    Wibawa, Hendra; Henning, Joerg; Wong, Frank; Selleck, Paul; Junaidi, Akhmad; Bingham, John; Daniels, Peter; Meers, Joanne

    2011-09-07

    Indonesia is one of the countries most severely affected by H5N1 highly pathogenic avian influenza (HPAI) virus in terms of poultry and human health. However, there is little information on the diversity of H5N1 viruses circulating in backyard farms, where chickens and ducks often intermingle. In this study, H5N1 virus infection occurring in 96 smallholder duck farms in central Java, Indonesia from 2007-2008 was investigated and the molecular and antigenic characteristics of H5N1 viruses isolated from these farms were analysed. All 84 characterised viruses belonged to H5N1 clade 2.1 with three virus sublineages being identified: clade 2.1.1 (1), clade 2.1.3 (80), and IDN/6/05-like viruses (3) that did not belong to any of the present clades. All three clades were found in ducks, while only clade 2.1.3 was isolated from chickens. There were no significant amino acid mutations of the hemagglutinin (HA) and neuraminidase (NA) sites of the viruses, including the receptor binding, glycosylation, antigenic and catalytic sites and NA inhibitor targets. All the viruses had polybasic amino acids at the HA cleavage site. No evidence of major antigenic variants was detected. Based on the HA gene, identical virus variants could be found on different farms across the study sites and multiple genetic variants could be isolated from HPAI outbreaks simultaneously or at different time points from single farms. HPAI virus was isolated from both ducks and chickens; however, the proportion of surviving duck cases was considerably higher than in chickens. The 2.1.3 clade was the most common lineage found in this study. All the viruses had sequence characteristic of HPAI, but negligible variations in other recognized amino acids at the HA and NA proteins which determine virus phenotypes. Multiple genetic variants appeared to be circulating simultaneously within poultry communities. The high proportion of live duck cases compared to chickens over the study period suggests that ducks are

  2. 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.

  3. Isolation of avian influenza H5N1 virus from vaccinated commercial layer flock in Egypt

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    El-Zoghby Elham F

    2012-11-01

    Full Text Available Abstract Background Uninterrupted transmission of highly pathogenic avian influenza virus (HPAIV H5N1 of clade 2.2.1 in Egypt since 2006 resulted in establishment of two main genetic clusters. The 2.2.1/C group where all recent human and majority of backyard origin viruses clustered together, meanwhile the majority of viruses derived from vaccinated poultry in commercial farms grouped in 2.2.1.1 clade. Findings In the present investigation, an HPAIV H5N1 was isolated from twenty weeks old layers chickens that were vaccinated with a homologous H5N1 vaccine at 1, 7 and 16 weeks old. At twenty weeks of age, birds showed cyanosis of comb and wattle, decrease in egg production and up to 27% mortality. Examined serum samples showed low antibody titer in HI test (Log2 3.2± 4.2. The hemagglutinin (HA and neuraminidase (NA genes of the isolated virus were closely related to viruses in 2.2.1/C group isolated from poultry in live bird market (LBM and backyards or from infected people. Conspicuous mutations in the HA and NA genes including a deletion within the receptor binding domain in the HA globular head region were observed. Conclusions Despite repeated vaccination of layer chickens using a homologous H5N1 vaccine, infection with HPAIV H5N1 resulted in significant morbidity and mortality. In endemic countries like Egypt, rigorous control measures including enforcement of biosecurity, culling of infected birds and constant update of vaccine virus strains are highly required to prevent circulation of HPAIV H5N1 between backyard birds, commercial poultry, LBM and humans.

  4. 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.

  5. 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.

  6. 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

  7. Does GaH5 exist?

    Science.gov (United States)

    Speakman, Lucas D.; Turney, Justin M.; Schaefer, Henry F.

    2005-11-01

    The existence or nonexistence of GaH5 has been widely discussed [N. M. Mitzel, Angew. Chem. Int. Ed. 42, 3856 (2003)]. Seven possible structures for gallium pentahydride have been systematically investigated using ab initio electronic structure theory. Structures and vibrational frequencies have been determined employing self-consistent field, coupled cluster including all single and double excitations (CCSD), and CCSD with perturbative triples levels of theory, with at least three correlation-consistent polarized-valence-(cc-pVXZ and aug-cc-pVXZ) type basis sets. The X˜A'1 state for GaH5 is predicted to be weakly bound complex 1 between gallane and molecular hydrogen, with Cs symmetry. The dissociation energy corresponding to GaH5→GaH3+H2 is predicted to be De=2.05kcalmol-1. The H-H stretching fundamental is predicted to be v =4060cm-1, compared to the tentatively assigned experimental feature of Wang and Andrews [J. Phys. Chem. A 107, 11371 (2003)] at 4087cm-1. A second Cs structure 2 with nearly equal energy is predicted to be a transition state, corresponding to a 90° rotation of the H2 bond. Thus the rotation of the hydrogen molecule is essentially free. However, hydrogen scrambling through the C2v structure 3 seems unlikely, as the activation barrier for scrambling is at least 30kcalmol-1 higher in energy than that for the dissociation of GaH5 to GaH3 and H2. Two additional structures consisting of GaH3 with a dihydrogen bond perpendicular to gallane (C3v structure 4) and an in-plane dihydrogen bond [Cs(III) structure 5] were also examined. A C3v symmetry second-order saddle point has nearly the same energy as the GaH3+H2 dissociation limit, while the Cs(III) structure 5 is a transition structure to the C3v structure. The C4v structure 6 and the D3h structure 7 are much higher in energy than GaH3+H2 by 88 and 103kcalmol-1, respectively.

  8. Characterisation of a highly pathogenic H5N1 clade 2.3.2 influenza virus isolated from swans in Shanghai, China.

    Science.gov (United States)

    Zhao, Guo; Zhong, Lei; Lu, Xinlun; Hu, Jiao; Gu, Xiaobing; Kai, Yan; Song, Qingqing; Sun, Qing; Liu, Jinbao; Peng, Daxin; Wang, Xiaoquan; Liu, Xiaowen; Liu, Xiufan

    2012-02-01

    In spring 2009, one strain of H5N1 clade 2.3.2 virus was isolated from wild swans in Shanghai, indicating the importance of the wild swan in the ecology of this highly pathogenic avian influenza virus (HPAIV) in Eastern China. Pathogenicity experiments conducted in this study indicated that the virus was highly pathogenic for chickens but lowly pathogenic for mammalian hosts, as evidenced by reduced infection of mice. The analysis of complete genome sequences and genetic evolution showed that A/Swan/Shanghai/10/09 (SW/SH/09) may be derived from the strain A/silky chicken/Shantou/475/2004 (CK/ST/04), which is homologous to the influenza viruses isolated from chicken, duck, pika, little egret, swan, mandarin duck and bar-headed goose in China Hunan, China Qinghai, Mongolia, Russia, Japan, Korea, Laos and Hong Kong during 2007-2011, indicating that the virus has retro-infected diverse wild birds from chicken, and significant spread of the virus is still ongoing through overlapping migratory flyways. On the basis of the molecular analysis, we also found that there was a deletion of the glycosylation site (NSS) in amino acid 156 of the hemagglutinin (HA) protein when compared with that of the other Clade 2.3.2 viruses isolated between 2007 and 2011. More importantly, the sequence analysis of SW/SH/09 virus displayed the drug-resistant mutations on the matrix protein (M2) and neuraminidase (NA) genes.

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

    Directory of Open Access Journals (Sweden)

    Raden Wasito

    2015-05-01

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

  10. Protection against H5N1 Highly Pathogenic Avian and Pandemic (H1N1) 2009 Influenza Virus Infection in Cynomolgus Monkeys by an Inactivated H5N1 Whole Particle Vaccine

    Science.gov (United States)

    Nakayama, Misako; Shichinohe, Shintaro; Itoh, Yasushi; Ishigaki, Hirohito; Kitano, Mitsutaka; Arikata, Masahiko; Pham, Van Loi; Ishida, Hideaki; Kitagawa, Naoko; Okamatsu, Masatoshi; Sakoda, Yoshihiro; Ichikawa, Takaya; Tsuchiya, Hideaki; Nakamura, Shinichiro; Le, Quynh Mai; Ito, Mutsumi; Kawaoka, Yoshihiro; Kida, Hiroshi; Ogasawara, Kazumasa

    2013-01-01

    H5N1 highly pathogenic avian influenza virus (HPAIV) infection has been reported in poultry and humans with expanding clade designations. Therefore, a vaccine that induces immunity against a broad spectrum of H5N1 viruses is preferable for pandemic preparedness. We established a second H5N1 vaccine candidate, A/duck/Hokkaido/Vac-3/2007 (Vac-3), in our virus library and examined the efficacy of inactivated whole particles of this strain against two clades of H5N1 HPAIV strains that caused severe morbidity in cynomolgus macaques. Virus propagation in vaccinated macaques infected with either of the H5N1 HPAIV strains was prevented compared with that in unvaccinated macaques. This vaccine also prevented propagation of a pandemic (H1N1) 2009 virus in macaques. In the vaccinated macaques, neutralization activity, which was mainly shown by anti-hemagglutinin antibody, against H5N1 HPAIVs in plasma was detected, but that against H1N1 virus was not detected. However, neuraminidase inhibition activity in plasma and T-lymphocyte responses in lymph nodes against H1N1 virus were detected. Therefore, cross-clade and heterosubtypic protective immunity in macaques consisted of humoral and cellular immunity induced by vaccination with Vac-3. PMID:24376571

  11. Protection against H5N1 highly pathogenic avian and pandemic (H1N1 2009 influenza virus infection in cynomolgus monkeys by an inactivated H5N1 whole particle vaccine.

    Directory of Open Access Journals (Sweden)

    Misako Nakayama

    Full Text Available H5N1 highly pathogenic avian influenza virus (HPAIV infection has been reported in poultry and humans with expanding clade designations. Therefore, a vaccine that induces immunity against a broad spectrum of H5N1 viruses is preferable for pandemic preparedness. We established a second H5N1 vaccine candidate, A/duck/Hokkaido/Vac-3/2007 (Vac-3, in our virus library and examined the efficacy of inactivated whole particles of this strain against two clades of H5N1 HPAIV strains that caused severe morbidity in cynomolgus macaques. Virus propagation in vaccinated macaques infected with either of the H5N1 HPAIV strains was prevented compared with that in unvaccinated macaques. This vaccine also prevented propagation of a pandemic (H1N1 2009 virus in macaques. In the vaccinated macaques, neutralization activity, which was mainly shown by anti-hemagglutinin antibody, against H5N1 HPAIVs in plasma was detected, but that against H1N1 virus was not detected. However, neuraminidase inhibition activity in plasma and T-lymphocyte responses in lymph nodes against H1N1 virus were detected. Therefore, cross-clade and heterosubtypic protective immunity in macaques consisted of humoral and cellular immunity induced by vaccination with Vac-3.

  12. Influenza A virus H5-specific antibodies in mute swans (Cygnus olor) in the USA.

    Science.gov (United States)

    Kistler, Whitney M; Stallknecht, David E; Lebarbenchon, Camille; Pedersen, Kerri; Marks, David R; Mickley, Randy; DeLiberto, Thomas J; Yabsley, Michael J

    2015-04-01

    The use of serologic assays for influenza A virus (IAV) surveillance in wild birds has increased because of the availability of commercial enzyme-linked immunosorbent assays (ELISAs). Recently, an H5-specific blocking ELISA (bELISA) was shown to reliably detect H5-specific antibodies to low- and high-pathogenic H5 viruses in experimentally infected waterfowl. Mute Swans (Cygnus olor) were frequently associated with highly pathogenic H5N1 outbreaks in Europe and may have a similar role if highly pathogenic H5N1 is introduced into North America. We measured the prevalence of antibodies to the nucleoprotein and H5 protein in Mute Swans using three serologic assays. We collected 340 serum samples from Mute Swans in Michigan, New Jersey, New York, and Rhode Island, US. We detected antibodies to the IAV nucleoprotein in 66.2% (225/340) of the samples. We detected H5-specific antibodies in 62.9% (214/340) and 18.8% (64/340) using a modified H5 bELISA protocol and hemagglutination inhibition (HI) assay, respectively. The modified H5 bELISA protocol detected significantly more positive samples than did the manufacturer's protocol. We also tested 46 samples using virus neutralization. Neutralization results had high agreement with the modified H5 bELISA protocol and detected a higher prevalence than did the HI assay. These results indicate that North American Mute Swans have high nucleoprotein and H5 antibody prevalences.

  13. A commercial ELISA detects high levels of human H5 antibody but cross-reacts with influenza A antibodies.

    Science.gov (United States)

    Stelzer-Braid, Sacha; Wong, Bruce; Robertson, Peter; Lynch, Garry W; Laurie, Karen; Shaw, Robert; Barr, Ian; Selleck, Paul W; Baleriola, Cristina; Escott, Ros; Katsoulotos, Gregory; Rawlinson, William D

    2008-10-01

    Commercial serological assays to determine influenza A H5N1 infection are available, although the accuracy and reproducibility of these are not reported in detail. This study aimed to assess the validity of a commercial ELISA H5 hemagglutinin (HA) antibody kit. A commercial ELISA for detection of antibodies towards influenza A H5 HA was evaluated using human sera from vaccinated individuals. The ELISA was used to screen 304 sera with elevated influenza A complement fixation titres collected between the period 1995-2007. The ELISA was found to be accurate for sera with high levels of anti-H5 antibodies, and would be useful in clinical settings where a rapid result is required. Thirteen of the stored sera were positive using the ELISA, but were confirmed as negative for H5N1 exposure using further serological tests. Absorption studies suggested that antibodies towards seasonal H3N2 and H1N1 influenza may cross-react with H5 antigen, giving false positive results with the ELISA.

  14. 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.

  15. The histone H5 variant in Xenopus laevis

    NARCIS (Netherlands)

    Moorman, A. F.; de Boer, P. A.; Linders, M. T.; Charles, R.

    1984-01-01

    The presumptive histone H5 of Xenopus laevis has been characterized by SDS and acid-urea-Triton polyacrylamide gel electrophoresis and compared with chicken histone H5. Chicken H5 has a lower electrophoretic mobility compared to that of Xenopus H5 in both gel systems. It is shown, using a polyclonal

  16. 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....

  17. Structural and antigenic variation among diverse clade 2 H5N1 viruses.

    Directory of Open Access Journals (Sweden)

    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.

  18. Physical and functional interactions between ZIP kinase and UbcH5

    International Nuclear Information System (INIS)

    Ohbayashi, Norihiko; Okada, Katsuya; Kawakami, Shiho; Togi, Sumihito; Sato, Noriko; Ikeda, Osamu; Kamitani, Shinya; Muromoto, Ryuta; Sekine, Yuichi; Kawai, Taro; Akira, Shizuo; Matsuda, Tadashi

    2008-01-01

    Zipper-interacting protein kinase (ZIPK) is a widely expressed serine/threonine kinase that has been implicated in cell death and transcriptional regulation, but its mechanism of regulation remains unknown. In our previous study, we showed that leukemia inhibitory factor stimulated threonine-265 phosphorylation of ZIPK, thereby leading to phosphorylation and activation of signal transducer and activator of transcription 3. Here, we identified UbcH5c as a novel ZIPK-binding partner by yeast two-hybrid screening. Importantly, we found that UbcH5c induced ubiquitination of ZIPK. Small-interfering RNA-mediated reduction of endogenous UbcH5 expression decreased ZIPK ubiquitination. Furthermore, coexpression of UbcH5c with ZIPK influenced promyelocytic leukemia protein nuclear body (PML-NB) formation. These results suggest that UbcH5 regulates ZIPK accumulation in PML-NBs by interacting with ZIPK and stimulating its ubiquitination

  19. Easy H5Peasy: Interactive Teaching with H5P Technology (workshop)

    OpenAIRE

    Aizpurua, Michelle De

    2017-01-01

    This 90 minute workshop was for the Heartbeat of Teaching symposium, part of the Educational Designer's Making a Difference series for Monash University. Hosted by the Faculty of Medicine, Nursing and Health Sciences. Abstract: Using new technologies in teaching can be intimidating. New programs have steep learning curves and it can be hard to know where to start. H5P however is a quick and easy way to develop interactive activities for online environments, such as Moodle, or for us...

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

    Directory of Open Access Journals (Sweden)

    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.

  1. 26 CFR 1.642(h)-5 - Example.

    Science.gov (United States)

    2010-04-01

    ... 26 Internal Revenue 8 2010-04-01 2010-04-01 false Example. 1.642(h)-5 Section 1.642(h)-5 Internal... TAXES Estates, Trusts, and Beneficiaries § 1.642(h)-5 Example. The application of section 642(h) may be illustrated by the following example: Example. (a) A decedent dies January 31, 1954, leaving a will which...

  2. Theoretical study of the ionization of B2H5

    International Nuclear Information System (INIS)

    Curtiss, L.A.; Pople, J.A.

    1989-01-01

    Ab initio molecular orbital calculations at the G1 level of theory have been carried out on neutral B 2 H 5 radical, doubly bridged B 2 H + 5 cation, and the first triplet excited state of B 2 H + 5 . Singly bridged B 2 H 5 is 4.0 kcal/mol (without zero-point energies) more stable than doubly bridged B 2 H 5 . Based on this work and previous theoretical work on triply bridged B 2 H + 5 , ionization potentials (vertical and adiabatic) are determined for B 2 H 5 . The adiabatic ionization potentials of the two B 2 H 5 structures are 6.94 eV (singly bridged) and 7.53 eV (doubly bridged). A very large difference is found between the vertical and adiabatic ionization potentials (3.37 eV) of the singly bridged B 2 H 5 structure. The first triplet state of B 2 H + 5 is found to be 4.55 eV higher in energy than the lowest energy B 2 H + 5 cation (triply bridged). The results of this theoretical study support the interpretation of Ruscic, Schwarz, and Berkowitz of their recent photoionization measurements on B 2 H 5

  3. 42 CFR 52h.5 - Conflict of interest.

    Science.gov (United States)

    2010-10-01

    ... 42 Public Health 1 2010-10-01 2010-10-01 false Conflict of interest. 52h.5 Section 52h.5 Public... RESEARCH GRANT APPLICATIONS AND RESEARCH AND DEVELOPMENT CONTRACT PROJECTS § 52h.5 Conflict of interest. (a) This section applies only to conflicts of interest involving members of peer review groups. This...

  4. An Impedance Aptasensor with Microfluidic Chips for Specific Detection of H5N1 Avian Influenza Virus

    Directory of Open Access Journals (Sweden)

    Jacob Lum

    2015-07-01

    Full Text Available In this research a DNA aptamer, which was selected through SELEX (systematic evolution of ligands by exponential enrichment to be specific against the H5N1 subtype of the avian influenza virus (AIV, was used as an alternative reagent to monoclonal antibodies in an impedance biosensor utilizing a microfluidics flow cell and an interdigitated microelectrode for the specific detection of H5N1 AIV. The gold surface of the interdigitated microelectrode embedded in a microfluidics flow cell was modified using streptavidin. The biotinylated aptamer against H5N1 was then immobilized on the electrode surface using biotin–streptavidin binding. The target virus was captured on the microelectrode surface, causing an increase in impedance magnitude. The aptasensor had a detection time of 30 min with a detection limit of 0.0128 hemagglutinin units (HAU. Scanning electron microscopy confirmed the binding of the target virus onto the electrode surface. The DNA aptamer was specific to H5N1 and had no cross-reaction to other subtypes of AIV (e.g., H1N1, H2N2, H7N2. The newly developed aptasensor offers a portable, rapid, low-cost alternative to current methods with the same sensitivity and specificity.

  5. Outbreaks of avian influenza A (H5N2), (H5N8), and (H5N1) among birds--United States, December 2014-January 2015.

    Science.gov (United States)

    Jhung, Michael A; Nelson, Deborah I

    2015-02-06

    During December 15, 2014-January 16, 2015, the U.S. Department of Agriculture received 14 reports of birds infected with Asian-origin, highly pathogenic avian influenza A (HPAI) (H5N2), (H5N8), and (H5N1) viruses. These reports represent the first reported infections with these viruses in U.S. wild or domestic birds. Although these viruses are not known to have caused disease in humans, their appearance in North America might increase the likelihood of human infection in the United States. Human infection with other avian influenza viruses, such as HPAI (H5N1) and (H5N6) viruses and (H7N9) virus, has been associated with severe, sometimes fatal, disease, usually following contact with poultry.

  6. Structural differences between the avian and human H7N9 hemagglutinin proteins are attributable to modifications in salt bridge formation: a computational study with implications in viral evolution.

    Science.gov (United States)

    Cueno, Marni E; Imai, Kenichi; Tamura, Muneaki; Ochiai, Kuniyasu

    2013-01-01

    Influenza A hemagglutinin (HA) is a homotrimeric glycoprotein composed of a fibrous globular stem supporting a globular head containing three sialic acid binding sites responsible for infection. The H7N9 strain has consistently infected an avian host, however, the novel 2013 strain is now capable of infecting a human host which would imply that the HA in both strains structurally differ. A better understanding of the structural differences between the avian and human H7N9 strains may shed light into viral evolution and transmissibility. In this study, we elucidated the structural differences between the avian and human H7N9 strains. Throughout the study, we generated HA homology models, verified the quality of each model, superimposed HA homology models to determine structural differences, and, likewise, elucidated the probable cause for these structural differences. We detected two different types of structural differences between the novel H7N9 human and representative avian strains, wherein, one type (Pattern-1) showed three non-overlapping regions while the other type (Pattern-2) showed only one non-overlapping region. In addition, we found that superimposed HA homology models exhibiting Pattern-1 contain three non-overlapping regions designated as: Region-1 (S1571-A1601); Region-3 (R2621-S2651); and Region-4 (S2701-D2811), whereas, superimposed HA homology models showing Pattern-2 only contain one non-overlapping region designated as Region-2 (S1371-S1451). We attributed the two patterns we observed to either the presence of salt bridges involving the E1141 residue or absence of the R1411:D771 salt bridge. Interestingly, comparison between the human H7N7 and H7N9 HA homology models showed high structural similarity. We propose that the putative absence of the R1411:D771 salt bridge coupled with the putative presence of the E1141:R2621 and E1141:K2641 salt bridges found in the 2013 H7N9 HA homology model is associated to human-type receptor binding. This

  7. Structural differences between the avian and human H7N9 hemagglutinin proteins are attributable to modifications in salt bridge formation: a computational study with implications in viral evolution.

    Directory of Open Access Journals (Sweden)

    Marni E Cueno

    Full Text Available Influenza A hemagglutinin (HA is a homotrimeric glycoprotein composed of a fibrous globular stem supporting a globular head containing three sialic acid binding sites responsible for infection. The H7N9 strain has consistently infected an avian host, however, the novel 2013 strain is now capable of infecting a human host which would imply that the HA in both strains structurally differ. A better understanding of the structural differences between the avian and human H7N9 strains may shed light into viral evolution and transmissibility. In this study, we elucidated the structural differences between the avian and human H7N9 strains. Throughout the study, we generated HA homology models, verified the quality of each model, superimposed HA homology models to determine structural differences, and, likewise, elucidated the probable cause for these structural differences. We detected two different types of structural differences between the novel H7N9 human and representative avian strains, wherein, one type (Pattern-1 showed three non-overlapping regions while the other type (Pattern-2 showed only one non-overlapping region. In addition, we found that superimposed HA homology models exhibiting Pattern-1 contain three non-overlapping regions designated as: Region-1 (S1571-A1601; Region-3 (R2621-S2651; and Region-4 (S2701-D2811, whereas, superimposed HA homology models showing Pattern-2 only contain one non-overlapping region designated as Region-2 (S1371-S1451. We attributed the two patterns we observed to either the presence of salt bridges involving the E1141 residue or absence of the R1411:D771 salt bridge. Interestingly, comparison between the human H7N7 and H7N9 HA homology models showed high structural similarity. We propose that the putative absence of the R1411:D771 salt bridge coupled with the putative presence of the E1141:R2621 and E1141:K2641 salt bridges found in the 2013 H7N9 HA homology model is associated to human-type receptor binding

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  10. 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...

  11. Reduced experimental infectivity and transmissibility of intercontinental H5 (H5N8 and H5N2) compared to Eurasian H5N1 highly pathogenic avian influenza viruses for chickens, turkeys, and Japanese quail

    Science.gov (United States)

    H5N1 high pathogenicity avian influenza (HPAI) virus (HPAIV) emerged in 1996 in Guangdong China and has since spread to infect and cause deaths in wild birds, poultry and humans in over 63 countries in Asia, Europe and Africa; and more recently a reassortant H5N8 clade 2.3.4.4 HPAI virus has spread ...

  12. Characterization of H5N6 highly pathogenic avian influenza viruses isolated from wild and captive birds in the winter season of 2016-2017 in Northern Japan.

    Science.gov (United States)

    Hiono, Takahiro; Okamatsu, Masatoshi; Matsuno, Keita; Haga, Atsushi; Iwata, Ritsuko; Nguyen, Lam Thanh; Suzuki, Mizuho; Kikutani, Yuto; Kida, Hiroshi; Onuma, Manabu; Sakoda, Yoshihiro

    2017-09-01

    On 15 November 2016, a black swan that had died in a zoo in Akita prefecture, northern Japan, was strongly suspected to have highly pathogenic avian influenza (HPAI); an HPAI virus (HPAIV) belonging to the H5N6 subtype was isolated from specimens taken from the bird. After the initial report, 230 cases of HPAI caused by H5N6 viruses from wild birds, captive birds, and domestic poultry farms were reported throughout the country during the winter season. In the present study, 66 H5N6 HPAIVs isolated from northern Japan were further characterized. Phylogenetic analysis of the hemagglutinin gene showed that the H5N6 viruses isolated in northern Japan clustered into Group C of Clade 2.3.4.4 together with other isolates collected in Japan, Korea and Taiwan during the winter season of 2016-2017. The antigenicity of the Japanese H5N6 isolate differed slightly from that of HPAIVs isolated previously in Japan and China. The virus exhibited high pathogenicity and a high replication capacity in chickens, whereas virus growth was slightly lower in ducks compared with that of an H5N8 HPAIV isolate collected in Japan in 2014. Comprehensive analyses of Japanese isolates, including those from central, western, and southern Japan, as well as rapid publication of this information are essential for facilitating greater control of HPAIVs. © 2017 The Societies and John Wiley & Sons Australia, Ltd.

  13. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  15. Labelling of histone H5 and its interaction with DNA. 1. Histone H5 labelling with fluorescein isothiocyanate.

    Science.gov (United States)

    Favazza, M; Lerho, M; Houssier, C

    1990-06-01

    Histone H5 has been labelled with fluorescein isothiocyanate (FITC) with particular attention to the reaction conditions (pH, reaction time and input FITC/H5 molar ratio) and to the complete elimination of non-covalently bound dye. We preferred to use reaction conditions which yielded non-specific uniform labelling rather than specific alpha-NH2 terminal labelling, in order to obtain higher sensitivity in further studies dealing with the detection of perturbation at the binding sites of H5 on DNA. FITC-labelled H5 was further characterized by absorption and circular dichroism spectroscopy, and the fluorescein probe titrated in the 4-8 pH range. The structural integrity of H5 was found to be preserved after labelling. The positive electrostatic potential of the environment in which the FITC probe is embedded in the arginine/lysine-rich tails of H5 is believed to be responsible for the drop of pK of 1 unit found for H5-FITC as compared to free FITC. For the globular part of H5, the pK of covalently-bound FITC was only slightly lowered; this is a consequence of the much lower content in positively-charged amino-acid side chains in this region.

  16. 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

  17. Isolation and Characterization of Avian Influenza Viruses, Including Highly Pathogenic H5N1, from Poultry in Live Bird Markets in Hanoi, Vietnam, in 2001

    Science.gov (United States)

    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

  18. The cellular distribution of histone H5 in embryonic and adult tissues of Xenopus laevis and chicken

    NARCIS (Netherlands)

    Moorman, A. F.; de Boer, P. A.; Lamers, W. H.; Charles, R.

    1986-01-01

    The cellular distribution of histone H5 in embryonic and adult tissues of Xenopus laevis and chicken has been established with monoclonal antibodies to histone H5. Both in Xenopus and in chicken, the protein has presumably a more widespread cellular distribution than hitherto expected but is absent

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

    Science.gov (United States)

    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.

  20. Identification of Key Residues in Virulent Canine Distemper Virus Hemagglutinin That Control CD150/SLAM-Binding Activity▿

    Science.gov (United States)

    Zipperle, Ljerka; Langedijk, Johannes P. M.; Örvell, Claes; Vandevelde, Marc; Zurbriggen, Andreas; Plattet, Philippe

    2010-01-01

    Morbillivirus cell entry is controlled by hemagglutinin (H), an envelope-anchored viral glycoprotein determining interaction with multiple host cell surface receptors. Subsequent to virus-receptor attachment, H is thought to transduce a signal triggering the viral fusion glycoprotein, which in turn drives virus-cell fusion activity. Cell entry through the universal morbillivirus receptor CD150/SLAM was reported to depend on two nearby microdomains located within the hemagglutinin. Here, we provide evidence that three key residues in the virulent canine distemper virus A75/17 H protein (Y525, D526, and R529), clustering at the rim of a large recessed groove created by β-propeller blades 4 and 5, control SLAM-binding activity without drastically modulating protein surface expression or SLAM-independent F triggering. PMID:20631152

  1. Genomic and Phylogenetic Characterization of Novel, Recombinant H5N2 Avian Influenza Virus Strains Isolated from Vaccinated Chickens with Clinical Symptoms in China

    Directory of Open Access Journals (Sweden)

    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.

  2. 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.

  3. Heterologous Prime-Boost Vaccination Using an AS03B-Adjuvanted Influenza A(H5N1) Vaccine in Infants and Children <3 Years of Age

    Science.gov (United States)

    Nolan, Terry; Izurieta, Patricia; Lee, Bee-Wah; Chan, Poh Chong; Marshall, Helen; Booy, Robert; Drame, Mamadou; Vaughn, David W.

    2014-01-01

    Background. Protecting young children from pandemic influenza should also reduce transmission to susceptible adults, including pregnant women. Methods. An open study assessed immunogenicity and reactogenicity of a heterologous booster dose of A/turkey/Turkey/1/2005(H5N1)-AS03B (AS03B is an Adjuvant System containing α-tocopherol and squalene in an oil-in-water emulsion [5.93 mg tocopherol]) in infants and children aged 6 to < 36 months that was given 6 months following 2-dose primary vaccination with A/Indonesia/05/2005(H5N1)-AS03B. Vaccines contained 1.9 µg of hemagglutinin antigen and AS03B. Hemagglutinin inhibition (HI) responses, microneutralization titers, and antineuraminidase antibody levels were assessed for 6 months following the booster vaccination. Results. For each age stratum (defined on the basis of the subject's age at first vaccination as 6 to < 12 months, 12 to < 24 months, and 24 to < 36 months) and overall (n = 113), European influenza vaccine licensure criteria were fulfilled for responses to A/turkey/Turkey/1/2005(H5N1) 10 days following the booster vaccination. Local pain and fever increased with consecutive doses. Anamnestic immune responses were demonstrated for HI, neutralizing, and antineuraminidase antibodies against vaccine-homologous/heterologous strains. Antibody responses to vaccine-homologous/heterologous strains persisted in all children 6 months following the booster vaccination. Conclusions. Prevaccination of young children with a clade 2 strain influenza A(H5N1) AS03-adjuvanted vaccine followed by heterologous booster vaccination boosted immune responses to the homologous strain and a related clade, with persistence for at least 6 months. The results support a prime-boost vaccination approach in young children for pandemic influenza preparedness. Clinical Trials Registration. NCT01323946. PMID:24973461

  4. Unusually High Mortality in Waterfowl Caused by Highly Pathogenic Avian Influenza A(H5N1) in Bangladesh

    Science.gov (United States)

    Haider, N.; Sturm-Ramirez, K.; Khan, S. U.; Rahman, M. Z.; Sarkar, S.; Poh, M. K.; Shivaprasad, H. L.; Kalam, M. A.; Paul, S. K.; Karmakar, P. C.; Balish, A.; Chakraborty, A.; Mamun, A. A.; Mikolon, A. B.; Davis, C. T.; Rahman, M.; Donis, R. O.; Heffelfinger, J. D.; Luby, S. P.; Zeidner, N.

    2015-01-01

    Summary Mortality in ducks and geese caused by highly pathogenic avian influenza A (H5N1) infection had not been previously identified in Bangladesh. In June–July 2011, we investigated mortality in ducks, geese and chickens with suspected H5N1 infection in a north-eastern district of the country to identify the aetiologic agent and extent of the outbreak and identify possible associated human infections. We surveyed households and farms with affected poultry flocks in six villages in Netrokona district and collected cloacal and oropharyngeal swabs from sick birds and tissue samples from dead poultry. We conducted a survey in three of these villages to identify suspected human influenza-like illness cases and collected nasopharyngeal and throat swabs. We tested all swabs by real-time RT-PCR, sequenced cultured viruses, and examined tissue samples by histopathology and immunohistochemistry to detect and characterize influenza virus infection. In the six villages, among the 240 surveyed households and 11 small-scale farms, 61% (1789/2930) of chickens, 47% (4816/10 184) of ducks and 73% (358/493) of geese died within 14 days preceding the investigation. Of 70 sick poultry swabbed, 80% (56/70) had detectable RNA for influenza A/H5, including 89% (49/55) of ducks, 40% (2/5) of geese and 50% (5/10) of chickens. We isolated virus from six of 25 samples; sequence analysis of the hemagglutinin and neuraminidase gene of these six isolates indicated clade 2.3.2.1a of H5N1 virus. Histopathological changes and immunohistochemistry staining of avian influenza viral antigens were recognized in the brain, pancreas and intestines of ducks and chickens. We identified ten human cases showing signs compatible with influenza-like illness; four were positive for influenza A/H3; however, none were positive for influenza A/H5. The recently introduced H5N1 clade 2.3.2.1a virus caused unusually high mortality in ducks and geese. Heightened surveillance in poultry is warranted to guide

  5. Unusually High Mortality in Waterfowl Caused by Highly Pathogenic Avian Influenza A(H5N1) in Bangladesh.

    Science.gov (United States)

    Haider, N; Sturm-Ramirez, K; Khan, S U; Rahman, M Z; Sarkar, S; Poh, M K; Shivaprasad, H L; Kalam, M A; Paul, S K; Karmakar, P C; Balish, A; Chakraborty, A; Mamun, A A; Mikolon, A B; Davis, C T; Rahman, M; Donis, R O; Heffelfinger, J D; Luby, S P; Zeidner, N

    2017-02-01

    Mortality in ducks and geese caused by highly pathogenic avian influenza A(H5N1) infection had not been previously identified in Bangladesh. In June-July 2011, we investigated mortality in ducks, geese and chickens with suspected H5N1 infection in a north-eastern district of the country to identify the aetiologic agent and extent of the outbreak and identify possible associated human infections. We surveyed households and farms with affected poultry flocks in six villages in Netrokona district and collected cloacal and oropharyngeal swabs from sick birds and tissue samples from dead poultry. We conducted a survey in three of these villages to identify suspected human influenza-like illness cases and collected nasopharyngeal and throat swabs. We tested all swabs by real-time RT-PCR, sequenced cultured viruses, and examined tissue samples by histopathology and immunohistochemistry to detect and characterize influenza virus infection. In the six villages, among the 240 surveyed households and 11 small-scale farms, 61% (1789/2930) of chickens, 47% (4816/10 184) of ducks and 73% (358/493) of geese died within 14 days preceding the investigation. Of 70 sick poultry swabbed, 80% (56/70) had detectable RNA for influenza A/H5, including 89% (49/55) of ducks, 40% (2/5) of geese and 50% (5/10) of chickens. We isolated virus from six of 25 samples; sequence analysis of the hemagglutinin and neuraminidase gene of these six isolates indicated clade 2.3.2.1a of H5N1 virus. Histopathological changes and immunohistochemistry staining of avian influenza viral antigens were recognized in the brain, pancreas and intestines of ducks and chickens. We identified ten human cases showing signs compatible with influenza-like illness; four were positive for influenza A/H3; however, none were positive for influenza A/H5. The recently introduced H5N1 clade 2.3.2.1a virus caused unusually high mortality in ducks and geese. Heightened surveillance in poultry is warranted to guide appropriate

  6. Enhanced Human-Type Receptor Binding by Ferret-Transmissible H5N1 with a K193T Mutation.

    Science.gov (United States)

    Peng, Wenjie; Bouwman, Kim M; McBride, Ryan; Grant, Oliver C; Woods, Robert J; Verheije, Monique H; Paulson, James C; de Vries, Robert P

    2018-05-15

    All human influenza pandemics have originated from avian influenza viruses. Although multiple changes are needed for an avian virus to be able to transmit between humans, binding to human-type receptors is essential. Several research groups have reported mutations in H5N1 viruses that exhibit specificity for human-type receptors and promote respiratory droplet transmission between ferrets. Upon detailed analysis, we have found that these mutants exhibit significant differences in fine receptor specificity compared to human H1N1 and H3N2 and retain avian-type receptor binding. We have recently shown that human influenza viruses preferentially bind to α2-6-sialylated branched N-linked glycans, where the sialic acids on each branch can bind to receptor sites on two protomers of the same hemagglutinin (HA) trimer. In this binding mode, the glycan projects over the 190 helix at the top of the receptor-binding pocket, which in H5N1 would create a stearic clash with lysine at position 193. Thus, we hypothesized that a K193T mutation would improve binding to branched N-linked receptors. Indeed, the addition of the K193T mutation to the H5 HA of a respiratory-droplet-transmissible virus dramatically improves both binding to human trachea epithelial cells and specificity for extended α2-6-sialylated N-linked glycans recognized by human influenza viruses. IMPORTANCE Infections by avian H5N1 viruses are associated with a high mortality rate in several species, including humans. Fortunately, H5N1 viruses do not transmit between humans because they do not bind to human-type receptors. In 2012, three seminal papers have shown how these viruses can be engineered to transmit between ferrets, the human model for influenza virus infection. Receptor binding, among others, was changed, and the viruses now bind to human-type receptors. Receptor specificity was still markedly different compared to that of human influenza viruses. Here we report an additional mutation in ferret

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

    Science.gov (United States)

    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.

  8. The deprotonation energies of BH5 and AlH5: Comparisons to GaH5

    International Nuclear Information System (INIS)

    Speakman, Lucas D.; Turney, Justin M.; Schaefer, Henry F.

    2007-01-01

    Hypercoordinate boron is most unusual, leading to considerable theoretical and experimental research on the parent BH 5 molecule. The deprotonation energies of BH 5 and the related molecules AlH 5 and GaH 5 have been of particular interest. Here the energy differences for XH 5 ->XH 4 - +H(X=BandAl) are computed to be 332.4 and 326.3kcalmol -1 , respectively, with an aug-cc-pVQZ basis set at the CCSD(T) level of theory. Vibrational frequencies for BH 4 - and AlH 4 - are also reported as 1098, 1210, 2263, and 2284cm -1 and 760, 779, 1658, and 1745cm -1 , respectively, again at the CCSD(T) aug-cc-pVQZ level of theory. Comparisons with the valence isoelectronic GaH 5 molecule are made

  9. 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

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

    OpenAIRE

    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 H5Nx 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 ha...

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

    Directory of Open Access Journals (Sweden)

    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.

  12. Risk factors for exposure to influenza a viruses, including subtype H5 viruses, in Thai free-grazing ducks.

    Science.gov (United States)

    Beaudoin, A L; Kitikoon, P; Schreiner, P J; Singer, R S; Sasipreeyajan, J; Amonsin, A; Gramer, M R; Pakinsee, S; Bender, J B

    2014-08-01

    Free-grazing ducks (FGD) have been associated with highly pathogenic avian influenza (HPAI) H5N1 outbreaks and may be a viral reservoir. In July-August 2010, we assessed influenza exposure of Thai FGD and risk factors thereof. Serum from 6254 ducks was analysed with enzyme-linked immunosorbent assay (ELISA) to detect antibodies to influenza A nucleoprotein (NP), and haemagglutinin H5 protein. Eighty-five per cent (5305 ducks) were seropositive for influenza A. Of the NP-seropositive sera tested with H5 assays (n = 1423), 553 (39%) were H5 ELISA positive and 57 (4%) suspect. Twelve per cent (74 of 610) of H5 ELISA-positive/suspect ducks had H5 titres ≥ 1 : 20 by haemagglutination inhibition. Risk factors for influenza A seropositivity include older age, poultry contact, flock visitors and older purchase age. Study flocks had H5 virus exposure as recently as March 2010, but no HPAI H5N1 outbreaks have been identified in Thailand since 2008, highlighting a need for rigorous FGD surveillance. © 2012 Blackwell Verlag GmbH.

  13. Laser Ablation Experiments on the Tamdakht H5 Chondrite

    Science.gov (United States)

    White, Susan M.; Stern, Eric

    2017-01-01

    High-powered lasers were used to induce ablation and to form fusion crusts in the lab on Tamdakht H5 chondrites and basalt. These ground tests were undertaken to improve our understanding, and ultimately improve our abilty to model and predict, meteoroid ablation during atmospheric entry. The infrared fiber laser at the LHMEL facilty, operated in the continuous wave (i.e. non-pulsed) mode, provided radiation surface heat flux at levels similar to meteor entry for these tests. Results are presented from the first round of testing on samples of Tamdakht H5 ordinary chondrite which were ex-posed to entry-relevant heating rates between 2 and 10 kWcm2.

  14. Hemagglutinin-specific neutralization of subacute sclerosing panencephalitis viruses.

    Directory of Open Access Journals (Sweden)

    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

  15. 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.

  16. 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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  17. Infectivity, transmission and pathogenicity of H5 highly pathogenic avian influenza clade 2.3.4.4 (H5N8 and H5N2) United States index viruses in Pekin ducks and Chinese geese

    Science.gov (United States)

    In late 2014, a H5N8 highly pathogenic avian influenza (HPAI) virus, clade 2.3.4.4, spread by migratory birds into North America mixing with low pathogenicity AI viruses to produce a H5N2 HPAI virus. The H5N8 and H5N2 HPAI viruses were detected initially in wild waterfowl and backyard birds, and lat...

  18. Characterization of a non-pathogenic H5N1 influenza virus isolated from a migratory duck flying from Siberia in Hokkaido, Japan, in October 2009

    Directory of Open Access Journals (Sweden)

    Okamatsu Masatoshi

    2011-02-01

    Full Text Available Abstract Background Infection with H5N1 highly pathogenic avian influenza viruses (HPAIVs of domestic poultry and wild birds has spread to more than 60 countries in Eurasia and Africa. It is concerned that HPAIVs may be perpetuated in the lakes in Siberia where migratory water birds nest in summer. To monitor whether HPAIVs circulate in migratory water birds, intensive surveillance of avian influenza has been performed in Mongolia and Japan in autumn each year. Until 2008, there had not been any H5N1 viruses isolated from migratory water birds that flew from their nesting lakes in Siberia. In autumn 2009, A/mallard/Hokkaido/24/09 (H5N1 (Mal/Hok/24/09 was isolated from a fecal sample of a mallard (Anas platyrhynchos that flew from Siberia to Hokkaido, Japan. The isolate was assessed for pathogenicity in chickens, domestic ducks, and quails and analyzed antigenically and phylogenetically. Results No clinical signs were observed in chickens inoculated intravenously with Mal/Hok/24/09 (H5N1. There was no viral replication in chickens inoculated intranasally with the isolate. None of the domestic ducks and quails inoculated intranasally with the isolate showed any clinical signs. There were no multiple basic amino acid residues at the cleavage site of the hemagglutinin (HA of the isolate. Each gene of Mal/Hok/24/09 (H5N1 is phylogenetically closely related to that of influenza viruses isolated from migratory water birds that flew from their nesting lakes in autumn. Additionally, the antigenicity of the HA of the isolate was similar to that of the viruses isolated from migratory water birds in Hokkaido that flew from their northern territory in autumn and different from those of HPAIVs isolated from birds found dead in China, Mongolia, and Japan on the way back to their northern territory in spring. Conclusion Mal/Hok/24/09 (H5N1 is a non-pathogenic avian influenza virus for chickens, domestic ducks, and quails, and is antigenically and genetically

  19. 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

    DEFF Research Database (Denmark)

    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...

  20. Highly Pathogenic Avian Influenza A(H5N1) Viruses at the Animal-Human Interface in Vietnam, 2003-2010.

    Science.gov (United States)

    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.

  1. Role of domestic ducks in the emergence of a new genotype of highly pathogenic H5N1 avian influenza A viruses in Bangladesh.

    Science.gov (United States)

    Barman, Subrata; Marinova-Petkova, Atanaska; Hasan, M Kamrul; Akhtar, Sharmin; El-Shesheny, Rabeh; Turner, Jasmine Cm; Franks, John; Walker, David; Seiler, Jon; Friedman, Kimberly; Kercher, Lisa; Jeevan, Trushar; Darnell, Daniel; Kayali, Ghazi; Jones-Engel, Lisa; McKenzie, Pamela; Krauss, Scott; Webby, Richard J; Webster, Robert G; Feeroz, Mohammed M

    2017-08-09

    Highly pathogenic avian influenza H5N1 viruses were first isolated in Bangladesh in February 2007. Subsequently, clades 2.2.2, 2.3.4.2 and 2.3.2.1a were identified in Bangladesh, and our previous surveillance data revealed that by the end of 2014, the circulating viruses exclusively comprised clade 2.3.2.1a. We recently determined the status of circulating avian influenza viruses in Bangladesh by conducting surveillance of live poultry markets and waterfowl in wetland areas from February 2015 through February 2016. Until April 2015, clade 2.3.2.1a persisted without any change in genotype. However, in June 2015, we identified a new genotype of H5N1 viruses, clade 2.3.2.1a, which quickly became predominant. These newly emerged H5N1 viruses contained the hemagglutinin, neuraminidase and matrix genes of circulating 2.3.2.1a Bangladeshi H5N1 viruses and five other genes of low pathogenic Eurasian-lineage avian influenza A viruses. Some of these internal genes were closely related to those of low pathogenic viruses isolated from ducks in free-range farms and wild birds in a wetland region of northeastern Bangladesh, where commercially raised domestic ducks have frequent contact with migratory birds. These findings indicate that migratory birds of the Central Asian flyway and domestic ducks in the free-range farms in Tanguar haor-like wetlands played an important role in the emergence of this novel genotype of highly pathogenic H5N1 viruses.

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

    Science.gov (United States)

    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

  3. Beardsley vs. Faith: Physical diversity among H5 chondrites

    International Nuclear Information System (INIS)

    Zbik, M.; Lang, B.

    1984-01-01

    The microstructure and some physico-mechanical properties of Beardsley and Faith, both H5 chondrites, were compared. Although the bulk sample porosities are respectively 9.1% and 14.5%, suggesting lower consolidation of Faith, the effective porosities are 5.63% and 1.79% - much higher for Beardsley. The material of Faith, characterized by vugs, vesicles and closed channels - abundant but all sized below 0.01 μm - is of higher strength than that of Beardsley, where joints, fissures, open channels and microcracks are sized from 0.1 to 10 μm. The higher elastic properties of Faith are shown by the velocity of the compressional wave of 6.66 km s -1 against that of 5.12 km s -1 for Beardsley. An example of physical diversity among chondrites to the same chemical-petrologic type is therefore demonstrated. (orig.)

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

    Science.gov (United States)

    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.

  5. Roll of hemagglutinin gene in the biology of avian inflenza virus

    Directory of Open Access Journals (Sweden)

    Masoud Soltanialvar

    2016-06-01

    Full Text Available The hemagglutinin (HA, the major envelope glycoprotein of influenza, plays an important role during the early stage of infection, and changes in the HA gene prior to the emergence of pathogenic avian influenza viruses. The HA protein controls viral entry through membrane fusion of the viral envelope with the host cell membrane and allows the genetic information released to initiate new virus synthesis. Sharp antigenic variation of HA remains the critical challenge to the development of effective vaccines. Therefore, we highlight the role of HA in need of review: structure of HA, the fusion process and the HA receptor binding specificity in interspecies transmission and the impact of multiple mutations at antigenic sites and host antibodies to the parental virus, and the host susceptibility to productive infection by the drift strains.

  6. Comparing introduction to Europe of highly pathogenic avian influenza viruses A(H5N8) in 2014 and A(H5N1) in 2005

    NARCIS (Netherlands)

    Adlhoch, C.; Gossner, C.; Koch, G.; Brown, I.; Bouwstra, R.J.; Verdonck, F.; Penttinen, P.; Harder, T.

    2014-01-01

    Since the beginning of November 2014, nine outbreaks of highly pathogenic avian influenza virus (HPAIV) A(H5N8) in poultry have been detected in four European countries. In this report, similarities and differences between the modes of introduction of HPAIV A(H5N1) and A(H5N8) into Europe are

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

    Directory of Open Access Journals (Sweden)

    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.

  8. Avian influenza H5N1 transmission in households, Indonesia.

    Directory of Open Access Journals (Sweden)

    Tjandra Y Aditama

    Full Text Available BACKGROUND: Disease transmission patterns are needed to inform public health interventions, but remain largely unknown for avian influenza H5N1 virus infections. A recent study on the 139 outbreaks detected in Indonesia between 2005 and 2009 found that the type of exposure to sources of H5N1 virus for both the index case and their household members impacted the risk of additional cases in the household. This study describes the disease transmission patterns in those outbreak households. METHODOLOGY/PRINCIPAL FINDINGS: We compared cases (n = 177 and contacts (n = 496 in the 113 sporadic and 26 cluster outbreaks detected between July 2005 and July 2009 to estimate attack rates and disease intervals. We used final size household models to fit transmission parameters to data on household size, cases and blood-related household contacts to assess the relative contribution of zoonotic and human-to-human transmission of the virus, as well as the reproduction number for human virus transmission. The overall household attack rate was 18.3% and secondary attack rate was 5.5%. Secondary attack rate remained stable as household size increased. The mean interval between onset of subsequent cases in outbreaks was 5.6 days. The transmission model found that human transmission was very rare, with a reproduction number between 0.1 and 0.25, and the upper confidence bounds below 0.4. Transmission model fit was best when the denominator population was restricted to blood-related household contacts of index cases. CONCLUSIONS/SIGNIFICANCE: The study only found strong support for human transmission of the virus when a single large cluster was included in the transmission model. The reproduction number was well below the threshold for sustained transmission. This study provides baseline information on the transmission dynamics for the current zoonotic virus and can be used to detect and define signatures of a virus with increasing capacity for human

  9. Possible impact of global warming on the evolution of hemagglutinins from influenza a viruses.

    Science.gov (United States)

    Yan, Shaomin; Wu, Guang

    2011-02-01

    To determine if global warming has an impact on the evolution of hemagglutinins from influenza A viruses, because both global warming and influenza pandemics/epidemics threaten the world. 4 706 hemagglutinins from influenza A viruses sampled from 1956 to 2009 were converted to a time-series to show their evolutionary process and compared with the global, northern hemisphere and southern hemisphere temperatures, to determine if their trends run in similar or opposite directions. Point-to-point comparisons between temperature and quantified hemagglutinins were performed for all species and for the major prevailing species. The comparisons show that the trends for both hemagglutinin evolution and temperature change run in a similar direction. Global warming has a consistent and progressive impact on the hemagglutinin evolution of influenza A viruses.

  10. H5N1-SeroDetect EIA and rapid test: a novel differential diagnostic assay for serodiagnosis of H5N1 infections and surveillance.

    Science.gov (United States)

    Khurana, Surender; Sasono, Pretty; Fox, Annette; Nguyen, Van Kinh; Le, Quynh Mai; Pham, Quang Thai; Nguyen, Tran Hien; Nguyen, Thanh Liem; Horby, Peter; Golding, Hana

    2011-12-01

    Continuing evolution of highly pathogenic (HP) H5N1 influenza viruses in wild birds with transmission to domestic poultry and humans poses a pandemic threat. There is an urgent need for a simple and rapid serological diagnostic assay which can differentiate between antibodies to seasonal and H5N1 strains and that could provide surveillance tools not dependent on virus isolation and nucleic acid technologies. Here we describe the establishment of H5N1 SeroDetect enzyme-linked immunosorbent assay (ELISA) and rapid test assays based on three peptides in HA2 (488-516), PB1-F2 (2-75), and M2e (2-24) that are highly conserved within H5N1 strains. These peptides were identified by antibody repertoire analyses of H5N1 influenza survivors in Vietnam using whole-genome-fragment phage display libraries (GFPDLs). To date, both platforms have demonstrated high levels of sensitivity and specificity in detecting H5N1 infections (clade 1 and clade 2.3.4) in Vietnamese patients as early as 7 days and up to several years postinfection. H5N1 virus-uninfected individuals in Vietnam and the United States, including subjects vaccinated with seasonal influenza vaccines or with confirmed seasonal virus infections, did not react in the H5N1-SeroDetect assays. Moreover, sera from individuals vaccinated with H5N1 subunit vaccine with moderate anti-H5N1 neutralizing antibody titers did not react positively in the H5N1-SeroDetect ELISA or rapid test assays. The simple H5N1-SeroDetect ELISA and rapid tests could provide an important tool for large-scale surveillance for potential exposure to HP H5N1 strains in both humans and birds.

  11. Efficacy of single dose of a bivalent vaccine containing inactivated Newcastle disease virus and reassortant highly pathogenic avian influenza H5N1 virus against lethal HPAI and NDV infection in chickens.

    Directory of Open Access Journals (Sweden)

    Dong-Hun Lee

    Full Text Available Highly pathogenic avian influenza (HPAI and Newcastle disease (ND are 2 devastating diseases of poultry, which cause great economic losses to the poultry industry. In the present study, we developed a bivalent vaccine containing antigens of inactivated ND and reassortant HPAI H5N1 viruses as a candidate poultry vaccine, and we evaluated its immunogenicity and protective efficacy in specific pathogen-free chickens. The 6:2 reassortant H5N1 vaccine strain containing the surface genes of the A/Chicken/Korea/ES/2003(H5N1 virus was successfully generated by reverse genetics. A polybasic cleavage site of the hemagglutinin segment was replaced by a monobasic cleavage site. We characterized the reverse genetics-derived reassortant HPAI H5N1 clade 2.5 vaccine strain by evaluating its growth kinetics in eggs, minimum effective dose in chickens, and cross-clade immunogenicity against HPAI clade 1 and 2. The bivalent vaccine was prepared by emulsifying inactivated ND (La Sota strain and reassortant HPAI viruses with Montanide ISA 70 adjuvant. A single immunization with this vaccine induced high levels of hemagglutination-inhibiting antibody titers and protected chickens against a lethal challenge with the wild-type HPAI and ND viruses. Our results demonstrate that the bivalent, inactivated vaccine developed in this study is a promising approach for the control of both HPAI H5N1 and ND viral infections.

  12. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    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.

  15. Theoretical characterizations of novel C2H5O+ reactions

    Science.gov (United States)

    Hudson, Charles E.; McAdoo, David J.

    2004-03-01

    Assorted reactions of C2H5O+ isomers are characterized by theory, including tracing their courses by means of intrinsic reaction coordinate computations. We establish that CH3CH=OH+ eliminates methane by transferring H from oxygen to a methyl hydrogen and then to the CC bond to produce CHO++CH4. This adds to the limited knowledge of the involvement of hypervalent structures in the reactions of cations in the gas phase. Second, we characterized the course of CH3CH=OH+-->H3O++HC[triple bond; length as m-dash]CH. In this dissociation, H first migrates from the methyl to the oxygen to give O-protonated vinyl alcohol, a stable intermediate. Then the H2O swings outward to over the middle of the CC bond while one of the two hydrogens on the non-O-bearing carbon revolves to between the oxygen and the two carbons, leading to formation of a [H3O+ HC[triple bond; length as m-dash]CH] complex. This complex contains sufficient energy to dissociate its partners because a high barrier is crossed in its formation. Third, we found that methane elimination from CH3O+=CH2 involves stretching of the CH3---O bond and then rotation of the methyl so that a methyl hydrogen is pointed directly toward the oxygen. This reaction is completed by further rotation of the methyl to abstract a methylene hydrogen to the opposite side of the methyl from that initially bonded to oxygen. This clearly establishes that this dissociation takes place through an ion-neutral complex. Each of the reaction coordinates for the three preceding reactions traverses a novel bonding stage involving H, evidence that such are not unusual in gas phase ion chemistry. Finally, we showed that in the rearrangement CH3O+=CH2-->CH2=O+CH3, before Ht is transferred CH2 rotates around the C=C bond from being in the skeletal plane to being perpendicular to it, and Ht remains in the skeletal plane throughout its transfer. This pathway appears to balance avoiding an orbital symmetry-forbidden suprafacial transition state with

  16. Efficacy of two H5N9-inactivated vaccines against challenge with a recent H5N1 highly pathogenic avian influenza isolate from a chicken in Thailand.

    Science.gov (United States)

    Bublot, Michel; Le Gros, François-Xavier; Nieddu, Daniela; Pritchard, Nikki; Mickle, Thomas R; Swayne, David E

    2007-03-01

    The objective of this study was to compare the efficacy of two avian influenza (AI) H5-inactivated vaccines containing either an American (A/turkey/Wisconsin/68 H5N9; H5N9-WI) or a Eurasian isolate (A/chicken/Italy/22A/98 H5N9; H5N9-It). Three-week-old specific pathogen-free chickens were vaccinated once and challenged 3 wk later with a H5N1 highly pathogenic AI (HPAI) virus isolated from a chicken in Thailand in 2004. All unvaccinated challenged birds died within 2 days, whereas 90% and 100% of chickens vaccinated with H5N9-WI and H5N9-It, respectively, were protected against morbidity and mortality. Both vaccines prevented cloacal shedding and significantly reduced oral shedding of the challenge HPAI virus. Additional chickens (vaccinated or unvaccinated) were placed in contact with the directly challenged birds 18 hr after challenge. All unvaccinated chickens in contact with unvaccinated challenged birds died within 3 days after contact, whereas unvaccinated chickens in contact with vaccinated challenged birds either showed a significantly delayed mortality or did not become infected. All vaccinated contacts were protected against clinical signs, and most chickens did not shed detectable amount of HPAI virus. Altogether, these data indicate that both vaccines protected very well against morbidity and mortality and reduced or prevented shedding induced by direct or contact exposure to Asian H5N1 HPAI virus.

  17. Comparing introduction to Europe of highly pathogenic avian influenza viruses A(H5N8) in 2014 and A(H5N1) in 2005.

    Science.gov (United States)

    Adlhoch, C; Gossner, C; Koch, G; Brown, I; Bouwstra, R; Verdonck, F; Penttinen, P; Harder, T

    2014-12-18

    Since the beginning of November 2014, nine outbreaks of highly pathogenic avian influenza virus (HPAIV) A(H5N8) in poultry have been detected in four European countries. In this report, similarities and differences between the modes of introduction of HPAIV A(H5N1) and A(H5N8) into Europe are described. Experiences from outbreaks of A(H5N1) in Europe demonstrated that early detection to control HPAIV in poultry has proven pivotal to minimise the risk of zoonotic transmission and prevention of human cases.

  18. Human mesenchymal stromal cells reduce influenza A H5N1-associated acute lung injury in vitro and in vivo.

    Science.gov (United States)

    Chan, Michael C W; Kuok, Denise I T; Leung, Connie Y H; Hui, Kenrie P Y; Valkenburg, Sophie A; Lau, Eric H Y; Nicholls, John M; Fang, Xiaohui; Guan, Yi; Lee, Jae W; Chan, Renee W Y; Webster, Robert G; Matthay, Michael A; Peiris, J S Malik

    2016-03-29

    Influenza can cause acute lung injury. Because immune responses often play a role, antivirals may not ensure a successful outcome. To identify pathogenic mechanisms and potential adjunctive therapeutic options, we compared the extent to which avian influenza A/H5N1 virus and seasonal influenza A/H1N1 virus impair alveolar fluid clearance and protein permeability in an in vitro model of acute lung injury, defined the role of virus-induced soluble mediators in these injury effects, and demonstrated that the effects are prevented or reduced by bone marrow-derived multipotent mesenchymal stromal cells. We verified the in vivo relevance of these findings in mice experimentally infected with influenza A/H5N1. We found that, in vitro, the alveolar epithelium's protein permeability and fluid clearance were dysregulated by soluble immune mediators released upon infection with avian (A/Hong Kong/483/97, H5N1) but not seasonal (A/Hong Kong/54/98, H1N1) influenza virus. The reduced alveolar fluid transport associated with down-regulation of sodium and chloride transporters was prevented or reduced by coculture with mesenchymal stromal cells. In vivo, treatment of aged H5N1-infected mice with mesenchymal stromal cells increased their likelihood of survival. We conclude that mesenchymal stromal cells significantly reduce the impairment of alveolar fluid clearance induced by A/H5N1 infection in vitro and prevent or reduce A/H5N1-associated acute lung injury in vivo. This potential adjunctive therapy for severe influenza-induced lung disease warrants rapid clinical investigation.

  19. Protective Efficacy of Recombinant Turkey Herpes Virus (rHVT-H5) and Inactivated H5N1 Vaccines in Commercial Mulard Ducks against the Highly Pathogenic Avian Influenza (HPAI) H5N1 Clade 2.2.1 Virus.

    Science.gov (United States)

    Kilany, Walid H; Safwat, Marwa; Mohammed, Samy M; Salim, Abdullah; Fasina, Folorunso Oludayo; Fasanmi, Olubunmi G; Shalaby, Azhar G; Dauphin, Gwenaelle; Hassan, Mohammed K; Lubroth, Juan; Jobre, Yilma M

    2016-01-01

    In Egypt, ducks kept for commercial purposes constitute the second highest poultry population, at 150 million ducks/year. Hence, ducks play an important role in the introduction and transmission of avian influenza (AI) in the Egyptian poultry population. Attempts to control outbreaks include the use of vaccines, which have varying levels of efficacy and failure. To date, the effects of vaccine efficacy has rarely been determined in ducks. In this study, we evaluated the protective efficacy of a live recombinant vector vaccine based on a turkey Herpes Virus (HVT) expressing the H5 gene from a clade 2.2 H5N1 HPAIV strain (A/Swan/Hungary/499/2006) (rHVT-H5) and a bivalent inactivated H5N1 vaccine prepared from clade 2.2.1 and 2.2.1.1 H5N1 seeds in Mulard ducks. A 0.3ml/dose subcutaneous injection of rHVT-H5 vaccine was administered to one-day-old ducklings (D1) and another 0.5ml/dose subcutaneous injection of the inactivated MEFLUVAC was administered at 7 days (D7). Four separate challenge experiments were conducted at Days 21, 28, 35 and 42, in which all the vaccinated ducks were challenged with 106EID50/duck of H5N1 HPAI virus (A/chicken/Egypt/128s/2012(H5N1) (clade 2.2.1) via intranasal inoculation. Maternal-derived antibody regression and post-vaccination antibody immune responses were monitored weekly. Ducks vaccinated at 21, 28, 35 and 42 days with the rHVT-H5 and MEFLUVAC vaccines were protected against mortality (80%, 80%, 90% and 90%) and (50%, 70%, 80% and 90%) respectively, against challenges with the H5N1 HPAI virus. The amount of viral shedding and shedding rates were lower in the rHVT-H5 vaccine groups than in the MEFLUVAC groups only in the first two challenge experiments. However, the non-vaccinated groups shed significantly more of the virus than the vaccinated groups. Both rHVT-H5 and MEFLUVAC provide early protection, and rHVT-H5 vaccine in particular provides protection against HPAI challenge.

  20. Production of inactivated influenza H5N1 vaccines from MDCK cells in serum-free medium.

    Directory of Open Access Journals (Sweden)

    Alan Yung-Chih Hu

    Full Text Available BACKGROUND: Highly pathogenic influenza viruses pose a constant threat which could lead to a global pandemic. Vaccination remains the principal measure to reduce morbidity and mortality from such pandemics. The availability and surging demand for pandemic vaccines needs to be addressed in the preparedness plans. This study presents an improved high-yield manufacturing process for the inactivated influenza H5N1 vaccines using Madin-Darby canine kidney (MDCK cells grown in a serum-free (SF medium microcarrier cell culture system. PRINCIPAL FINDING: The current study has evaluated the performance of cell adaptation switched from serum-containing (SC medium to several commercial SF media. The selected SF medium was further evaluated in various bioreactor culture systems for process scale-up evaluation. No significant difference was found in the cell growth in different sizes of bioreactors studied. In the 7.5 L bioreactor runs, the cell concentration reached to 2.3 × 10(6 cells/mL after 5 days. The maximum virus titers of 1024 Hemagglutinin (HA units/50 µL and 7.1 ± 0.3 × 10(8 pfu/mL were obtained after 3 days infection. The concentration of HA antigen as determined by SRID was found to be 14.1 µg/mL which was higher than those obtained from the SC medium. A mouse immunogenicity study showed that the formalin-inactivated purified SF vaccine candidate formulated with alum adjuvant could induce protective level of virus neutralization titers similar to those obtained from the SC medium. In addition, the H5N1 viruses produced from either SC or SF media showed the same antigenic reactivity with the NIBRG14 standard antisera. CONCLUSIONS: The advantages of this SF cell-based manufacturing process could reduce the animal serum contamination, the cost and lot-to-lot variation of SC medium production. This study provides useful information to manufacturers that are planning to use SF medium for cell-based influenza vaccine production.

  1. Uridine 3H-5 and leucine 3H-5 uptake in Planarian cells Polycelis tenuis (Iijima) cultivated in vitro

    International Nuclear Information System (INIS)

    Franquinet, Raphael; Le Moigne, Albert; Lender, Theodore

    1975-01-01

    RNA and protein synthesis in planarian cells cultivated in vitro was studied by histoautoradiography. In the non-differentiated cells, uptake of precursor is intense from the beginning of the culture, and sensitive to addition of trophic factor known for their activating effect on mitosis and regeneration. On the contrary the rate of incorporation in differentiated cells is low and uniform, independently of the differents factors added to the medium [fr

  2. 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)

  3. Exploring the early stages of the pH-induced conformational change of influenza hemagglutinin.

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  5. Unique Structural Features of Influenza Virus H15 Hemagglutinin

    Energy Technology Data Exchange (ETDEWEB)

    Tzarum, Netanel; McBride, Ryan; Nycholat, Corwin M.; Peng, Wenjie; Paulson, James C.; Wilson, Ian A. (Scripps)

    2017-04-12

    Influenza A H15 viruses are members of a subgroup (H7-H10-H15) of group 2 hemagglutinin (HA) subtypes that include H7N9 and H10N8 viruses that were isolated from humans during 2013. The isolation of avian H15 viruses is, however, quite rare and, until recently, geographically restricted to wild shorebirds and waterfowl in Australia. The HAs of H15 viruses contain an insertion in the 150-loop (loop beginning at position 150) of the receptor-binding site common to this subgroup and a unique insertion in the 260-loop compared to any other subtype. Here, we show that the H15 HA has a high preference for avian receptor analogs by glycan array analyses. The H15 HA crystal structure reveals that it is structurally closest to H7N9 HA, but the head domain of the H15 trimer is wider than all other HAs due to a tilt and opening of the HA1 subunits of the head domain. The extended 150-loop of the H15 HA retains the conserved conformation as in H7 and H10 HAs. Furthermore, the elongated 260-loop increases the exposed HA surface and can contribute to antigenic variation in H15 HAs. Since avian-origin H15 HA viruses have been shown to cause enhanced disease in mammalian models, further characterization and immune surveillance of H15 viruses are warranted.

    IMPORTANCEIn the last 2 decades, an apparent increase has been reported for cases of human infection by emerging avian influenza A virus subtypes, including H7N9 and H10N8 viruses isolated during 2013. H15 is the other member of the subgroup of influenza A virus group 2 hemagglutinins (HAs) that also include H7 and H10. H15 viruses have been restricted to Australia, but recent isolation of H15 viruses in western Siberia suggests that they could be spread more globally via the avian flyways that converge and emanate from this region. Here we report on characterization of the three-dimensional structure and receptor specificity of the H15 hemagglutinin, revealing distinct features and specificities that can

  6. Rapid detection of the avian influenza virus H5N1 subtype in Egypt ...

    African Journals Online (AJOL)

    The unprecedented spread of highly pathogenic avian influenza virus subtype H5N1 in Egypt ... Effective diagnosis and control management are needed to control the disease. ... Reconstituted clinical samples consisting of H5 AIVs mixed with ...

  7. Rapid detection of the avian influenza virus H5N1 subtype in Egypt

    African Journals Online (AJOL)

    Dr

    highly pathogenic avian influenza virus subtype H5N1 in Egypt is threatening poultry and ... Key words: Avian influenza virus, H5N1, fluorescent antibody enzyme-linked immunosorbent assay (ELISA) ..... poultry and is potentially zoonotic.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

  10. Novel Eurasian highly pathogenic influenza A H5 viruses in wild birds, Washington, USA

    Science.gov (United States)

    Ip, Hon S.; Kim Torchetti, Mia; Crespo, Rocio; Kohrs, Paul; DeBruyn, Paul; Mansfield, Kristin G.; Baszler, Timothy; Badcoe, Lyndon; Bodenstein, Barbara L.; Shearn-Bochsler, Valerie I.; Killian, Mary Lea; Pederson, Janice C.; Hines, Nichole; Gidlewski, Thomas; DeLiberto, Thomas; Sleeman, Jonathan M.

    2015-01-01

    Novel Eurasian lineage avian influenza A(H5N8) virus has spread rapidly and globally since January 2014. In December 2014, H5N8 and reassortant H5N2 viruses were detected in wild birds in Washington, USA, and subsequently in backyard birds. When they infect commercial poultry, these highly pathogenic viruses pose substantial trade issues.

  11. Novel Eurasian highly pathogenic avian influenza A H5 viruses in wild birds, Washington, USA, 2014.

    Science.gov (United States)

    Ip, Hon S; Torchetti, Mia Kim; Crespo, Rocio; Kohrs, Paul; DeBruyn, Paul; Mansfield, Kristin G; Baszler, Timothy; Badcoe, Lyndon; Bodenstein, Barbara; Shearn-Bochsler, Valerie; Killian, Mary Lea; Pedersen, Janice C; Hines, Nichole; Gidlewski, Thomas; DeLiberto, Thomas; Sleeman, Jonathan M

    2015-05-01

    Novel Eurasian lineage avian influenza A(H5N8) virus has spread rapidly and globally since January 2014. In December 2014, H5N8 and reassortant H5N2 viruses were detected in wild birds in Washington, USA, and subsequently in backyard birds. When they infect commercial poultry, these highly pathogenic viruses pose substantial trade issues.

  12. Potent Inhibitors against Newcastle Disease Virus Hemagglutinin-Neuraminidase.

    Science.gov (United States)

    Rota, Paola; La Rocca, Paolo; Piccoli, Marco; Montefiori, Marco; Cirillo, Federica; Olsen, Lars; Orioli, Marica; Allevi, Pietro; Anastasia, Luigi

    2018-02-06

    Neuraminidase activity is essential for the infection and propagation of paramyxoviruses, including human parainfluenza viruses (hPIVs) and the Newcastle disease virus (NDV). Thus, many inhibitors have been developed based on the 2-deoxy-2,3-didehydro-d-N-acetylneuraminic acid inhibitor (DANA) backbone. Along this line, herein we report a series of neuraminidase inhibitors, having C4 (p-toluenesulfonamido and azido substituents) and C5 (N-perfluorinated chains) modifications to the DANA backbone, resulting in compounds with 5- to 15-fold greater potency than the currently most active compound, the N-trifluoroacetyl derivative of DANA (FANA), toward the NDV hemagglutinin-neuraminidase (NDV-HN). Remarkably, these inhibitors were found to be essentially inactive against the human sialidase NEU3, which is present on the outer layer of the cell membrane and is highly affected by the current NDV inhibitor FANA. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Selective labelling of 5-HT{sub 7} receptor recognition sites in rat brain using [{sup 3}H]5-carboxamidotryptamine

    Energy Technology Data Exchange (ETDEWEB)

    Stowe, R.L.; Barnes, N.M. [Department of Pharmacology, The Medical School, University of Birmingham, Edgbaston, Birmingham B15 2TT (United Kingdom)

    1998-12-01

    The aim of the present study was to establish a radioligand binding assay to selectively label the native 5-HT{sub 7} receptor expressed in rat brain. In rat whole brain (minus cerebellum and striatum) homogenate, ({+-})-pindolol (10 {mu}M)-insensitive [{sup 3}H]5-CT ([{sup 3}H]5-carboxamidotryptamine; 0.5 nM) specific binding (defined by 5-HT, 10 {mu}M) displayed a pharmacological profile similar to the recombinant 5-HT{sub 7} receptor, although the Hill coefficients for competition curves generated by methiothepin, ritanserin, sumatriptan, clozapine and pimozide were significantly less than unity. In homogenates of rat hypothalamus, ({+-})-pindolol (10 {mu}M)-insensitive [{sup 3}H]5-CT recognition sites also resembled, pharmacologically, the 5-HT{sub 7} receptor, although pimozide still generated Hill coefficients significantly less than unity. Subsequent studies were performed in the additional presence of WAY100635 (100 nM) to prevent [{sup 3}H]5-CT binding to residual, possibly, 5-HT{sub 1A} sites. Competition for this [{sup 3}H]5-CT binding indicated the labelling in whole rat brain homogenate of a homogenous population of sites with the pharmacological profile of the 5-HT{sub 7} receptor. Saturation studies also indicated that ({+-})-pindolol (10 {mu}M)/WAY 100635 (100 nM)-insensitive [{sup 3}H]5-CT binding to homogenates of whole rat brain was saturable and to an apparently homogenous population of sites which were labelled with nanomolar affinity (B{sub max}=33.2{+-}0.7 fmol mg{sup -1} protein, pK{sub d}=8.78{+-}0.05, mean{+-}S.E.M., n=3). The development of this 5-HT{sub 7} receptor binding assay will aid investigation of the rat native 5-HT{sub 7} receptor. (Copyright (c) 1998 Elsevier Science B.V., Amsterdam. All rights reserved.)

  14. Selective labelling of 5-HT7 receptor recognition sites in rat brain using [3H]5-carboxamidotryptamine

    International Nuclear Information System (INIS)

    Stowe, R.L.; Barnes, N.M.

    1998-01-01

    The aim of the present study was to establish a radioligand binding assay to selectively label the native 5-HT 7 receptor expressed in rat brain. In rat whole brain (minus cerebellum and striatum) homogenate, (±)-pindolol (10 μM)-insensitive [ 3 H]5-CT ([ 3 H]5-carboxamidotryptamine; 0.5 nM) specific binding (defined by 5-HT, 10 μM) displayed a pharmacological profile similar to the recombinant 5-HT 7 receptor, although the Hill coefficients for competition curves generated by methiothepin, ritanserin, sumatriptan, clozapine and pimozide were significantly less than unity. In homogenates of rat hypothalamus, (±)-pindolol (10 μM)-insensitive [ 3 H]5-CT recognition sites also resembled, pharmacologically, the 5-HT 7 receptor, although pimozide still generated Hill coefficients significantly less than unity. Subsequent studies were performed in the additional presence of WAY100635 (100 nM) to prevent [ 3 H]5-CT binding to residual, possibly, 5-HT 1A sites. Competition for this [ 3 H]5-CT binding indicated the labelling in whole rat brain homogenate of a homogenous population of sites with the pharmacological profile of the 5-HT 7 receptor. Saturation studies also indicated that (±)-pindolol (10 μM)/WAY 100635 (100 nM)-insensitive [ 3 H]5-CT binding to homogenates of whole rat brain was saturable and to an apparently homogenous population of sites which were labelled with nanomolar affinity (B max =33.2±0.7 fmol mg -1 protein, pK d =8.78±0.05, mean±S.E.M., n=3). The development of this 5-HT 7 receptor binding assay will aid investigation of the rat native 5-HT 7 receptor. (Copyright (c) 1998 Elsevier Science B.V., Amsterdam. All rights reserved.)

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

    Science.gov (United States)

    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

  16. Technology transfer and scale-up of the Flublok recombinant hemagglutinin (HA) influenza vaccine manufacturing process.

    Science.gov (United States)

    Buckland, Barry; Boulanger, Robert; Fino, Mireli; Srivastava, Indresh; Holtz, Kathy; Khramtsov, Nikolai; McPherson, Clifton; Meghrous, Jamal; Kubera, Paul; Cox, Manon M J

    2014-09-22

    Multiple different hemagglutinin (HA) protein antigens have been reproducibly manufactured at the 650L scale by Protein Sciences Corporation (PSC) based on an insect cell culture with baculovirus infection. Significantly, these HA protein antigens were produced by the same Universal Manufacturing process as described in the biological license application (BLA) for the first recombinant influenza vaccine approved by the FDA (Flublok). The technology is uniquely designed so that a change in vaccine composition can be readily accommodated from one HA protein antigen to another one. Here we present a vaccine candidate to combat the recently emerged H7N9 virus as an example starting with the genetic sequence for the required HA, creation of the baculovirus and ending with purified protein antigen (or vaccine component) at the 10L scale accomplished within 38 days under GMP conditions. The same process performance is being achieved at the 2L, 10L, 100L, 650L and 2500L scale. An illustration is given of how the technology was transferred from the benchmark 650L scale facility to a retrofitted microbial facility at the 2500L scale within 100 days which includes the time for facility engineering changes. The successful development, technology transfer and scale-up of the Flublok process has major implications for being ready to make vaccine rapidly on a worldwide scale as a defense against pandemic influenza. The technology described does not have the same vulnerability to mutations in the egg adapted strain, and resulting loss in vaccine efficacy, faced by egg based manufacture. Copyright © 2014 Elsevier Ltd. All rights reserved.

  17. Distinct Pathogenesis of Hong Kong-Origin H5N1 Viruses in Mice Compared to That of Other Highly Pathogenic H5 Avian Influenza Viruses

    OpenAIRE

    Dybing, Jody K.; Schultz-Cherry, Stacey; Swayne, David E.; Suarez, David L.; Perdue, Michael L.

    2000-01-01

    In 1997, an outbreak of virulent H5N1 avian influenza virus occurred in poultry in Hong Kong (HK) and was linked to a direct transmission to humans. The factors associated with transmission of avian influenza virus to mammals are not fully understood, and the potential risk of other highly virulent avian influenza A viruses infecting and causing disease in mammals is not known. In this study, two avian and one human HK-origin H5N1 virus along with four additional highly pathogenic H5 avian in...

  18. 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.

  19. 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

  20. Produksi IgY Antivirus Avian Influenza H5N1 dan Prospek Pemanfaatannya dalam Pengebalan Pasif

    Directory of Open Access Journals (Sweden)

    I Wayan Teguh Wibawan

    2009-09-01

    Full Text Available Immunoglobulin Y (IgY in yolk has been shown in several studies to prevent both bacterial and viralinfections. This research was conducted to find evidence that IgY specific against avian influenza virus(AIV of H5N1 subtype can be produced in a large quantity in egg yolk. Laying hens were vaccinated withAI killed-vaccine (IPB-Shigeta. The IgY was purified using affinity chromatograpy technique, and anti-H5activity was measured using a standard haemaglutination inhibition (HI and agar gel immunodifusion.The concentration of IgY was calculated, and the protein pattern was detected using polyacrilamid gel(AGID electrophoresis (PAGE. Anti H5 antibody as high as 27 – 29 HI units was detected and produce aspecific line of precipitation in AGID. The concentration of IgY was 7.89 mg/ml. Purified specific IgY consistof 6 main protein bands with molecular weights ranging from 35 to 225 kD. These proteins were sensitiveto heat treatment (75oC for 30 minutes, to acid condition (pH2 as well as the pepsin and trypsin. Theseresults indicated the possibility of using specific IgY for passive immunisation to prevent AIV infection oras immunotherapeutic applications for AI treatment in humans.

  1. Pathogenicity and Transmission of H5 and H7 Highly Pathogenic Avian Influenza Viruses in Mallards

    Science.gov (United States)

    Costa-Hurtado, Mar; Shepherd, Eric; DeJesus, Eric; Smith, Diane; Spackman, Erica; Kapczynski, Darrell R.; Suarez, David L.; Stallknecht, David E.; Swayne, David E.

    2016-01-01

    ABSTRACT Wild aquatic birds have been associated with the intercontinental spread of H5 subtype highly pathogenic avian influenza (HPAI) viruses of the A/goose/Guangdong/1/96 (Gs/GD) lineage during 2005, 2010, and 2014, but dispersion by wild waterfowl has not been implicated with spread of other HPAI viruses. To better understand why Gs/GD H5 HPAI viruses infect and transmit more efficiently in waterfowl than other HPAI viruses, groups of mallard ducks were challenged with one of 14 different H5 and H7 HPAI viruses, including a Gs/GD lineage H5N1 (clade 2.2) virus from Mongolia, part of the 2005 dispersion, and the H5N8 and H5N2 index HPAI viruses (clade 2.3.4.4) from the United States, part of the 2014 dispersion. All virus-inoculated ducks and contact exposed ducks became infected and shed moderate to high titers of the viruses, with the exception that mallards were resistant to Ck/Pennsylvania/83 and Ck/Queretaro/95 H5N2 HPAI virus infection. Clinical signs were only observed in ducks challenged with the H5N1 2005 virus, which all died, and with the H5N8 and H5N2 2014 viruses, which had decreased weight gain and fever. These three viruses were also shed in higher titers by the ducks, which could facilitate virus transmission and spread. This study highlights the possible role of wild waterfowl in the spread of HPAI viruses. IMPORTANCE The spread of H5 subtype highly pathogenic avian influenza (HPAI) viruses of the Gs/GD lineage by migratory waterfowl is a serious concern for animal and public health. H5 and H7 HPAI viruses are considered to be adapted to gallinaceous species (chickens, turkeys, quail, etc.) and less likely to infect and transmit in wild ducks. In order to understand why this is different with certain Gs/GD lineage H5 HPAI viruses, we compared the pathogenicity and transmission of several H5 and H7 HPAI viruses from previous poultry outbreaks to Gs/GD lineage H5 viruses, including H5N1 (clade 2.2), H5N8 and H5N2 (clade 2.3.4.4) viruses, in

  2. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  4. Transforming growth factor-β: activation by neuraminidase and role in highly pathogenic H5N1 influenza pathogenesis.

    Directory of Open Access Journals (Sweden)

    Christina M Carlson

    2010-10-01

    Full Text Available Transforming growth factor-beta (TGF-β, a multifunctional cytokine regulating several immunologic processes, is expressed by virtually all cells as a biologically inactive molecule termed latent TGF-β (LTGF-β. We have previously shown that TGF-β activity increases during influenza virus infection in mice and suggested that the neuraminidase (NA protein mediates this activation. In the current study, we determined the mechanism of activation of LTGF-β by NA from the influenza virus A/Gray Teal/Australia/2/1979 by mobility shift and enzyme inhibition assays. We also investigated whether exogenous TGF-β administered via a replication-deficient adenovirus vector provides protection from H5N1 influenza pathogenesis and whether depletion of TGF-β during virus infection increases morbidity in mice. We found that both the influenza and bacterial NA activate LTGF-β by removing sialic acid motifs from LTGF-β, each NA being specific for the sialic acid linkages cleaved. Further, NA likely activates LTGF-β primarily via its enzymatic activity, but proteases might also play a role in this process. Several influenza A virus subtypes (H1N1, H1N2, H3N2, H5N9, H6N1, and H7N3 except the highly pathogenic H5N1 strains activated LTGF-β in vitro and in vivo. Addition of exogenous TGF-β to H5N1 influenza virus-infected mice delayed mortality and reduced viral titers whereas neutralization of TGF-β during H5N1 and pandemic 2009 H1N1 infection increased morbidity. Together, these data show that microbe-associated NAs can directly activate LTGF-β and that TGF-β plays a pivotal role protecting the host from influenza pathogenesis.

  5. Ascorbic acid prevents nonreceptor specific binding of [3H]-5-hydroxytryptamine to bovine cerebral cortex membranes

    International Nuclear Information System (INIS)

    Hamblin, M.W.; Adriaenssens, P.I.; Ariani, K.; Cawthon, R.M.; Stratford, C.A.; Tan, G.L.; Ciaranello, R.D.

    1987-01-01

    [ 3 H]-5-Hydroxytryptamine ([ 3 H]-5-HT) decomposes rapidly when exposed to air in solution at physiological pH if antioxidants are not present. The decomposition products appear to bind to two saturable sites on brain membranes (apparent Kd values = 1-2 and 100-1000 nM). This binding mimics ''specific'' ligand/receptor binding in that it is inhibited by 10 microM unlabeled 5-HT. This inhibition is not competitive, but rather is due to the prevention of [ 3 H]-5-HT breakdown by excess unlabeled 5-HT. Unlike genuine ligand/receptor binding, the binding of [ 3 H]-5-HT breakdown products is essentially irreversible and does not display a tissue distribution consistent with binding to authentic 5-HT receptors. [ 3 H]-5-HT decomposition can be eliminated by the inclusion of 0.05 to 5 mM ascorbic acid. At these concentrations ascorbic acid is not deleterious to reversible [ 3 H]-5-HT binding. When [ 3 H] 5-HT exposure to air occurs in the presence of brain membranes, the apparent antioxidant activity of brain membranes themselves affords protection against [ 3 H]-5-HT degradation equal to ascorbic acid. This protection is effective below final [ 3 H]-5-HT concentrations of 10 nM. Above 10 nM [ 3 H]-5-HT, addition of ascorbic acid or other antioxidants is necessary to avoid the occurrence of additional low affinity (apparent Kd = 15-2000 nM) binding sites that are specific but nonetheless irreversible. When care is taken to limit [ 3 H]-5-HT oxidation, the only reversible and saturable specific binding sites observed are of the 5-HT1 high affinity (Kd = 1-2 nM) type. Radioligand oxidation artifacts may be involved in previous reports of low affinity (Kd = 15-250 nM) [ 3 H]-5-HT binding sites in brain membrane preparations

  6. First introduction of highly pathogenic H5NI avian influenza A viruses in wild and domestic birds in Denmark, Northern Europe

    DEFF Research Database (Denmark)

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

    2007-01-01

    Background: Since 2005 highly pathogenic ( HP) avian influenza A H5N1 viruses have spread from Asia to Africa and Europe infecting poultry, humans and wild birds. HP H5N1 virus was isolated in Denmark for the first time in March 2006. A total of 44 wild birds were found positive for the HP H5N1...... infection. In addition, one case was reported in a backyard poultry flock. Results: Full-genome characterisation of nine isolates revealed that the Danish H5N1 viruses were highly similar to German H5N1 isolates in all genes from the same time period. The haemagglutinin gene grouped phylogenetically in H5...... clade 2 subclade 2 and closest relatives besides the German isolates were isolates from Croatia in 2005, Nigeria and Niger in 2006 and isolates from Astrakhan in Russia 2006. The German and Danish isolates shared unique substitutions in the NA, PB1 and NS2 proteins. Conclusion: The first case of HP H5N1...

  7. Emerging highly pathogenic H5 avian influenza viruses in France during winter 2015/16: phylogenetic analyses and markers for zoonotic potential.

    Science.gov (United States)

    Briand, François-Xavier; Schmitz, Audrey; Ogor, Katell; Le Prioux, Aurélie; Guillou-Cloarec, Cécile; Guillemoto, Carole; Allée, Chantal; Le Bras, Marie-Odile; Hirchaud, Edouard; Quenault, Hélène; Touzain, Fabrice; Cherbonnel-Pansart, Martine; Lemaitre, Evelyne; Courtillon, Céline; Gares, Hélène; Daniel, Patrick; Fediaevsky, Alexandre; Massin, Pascale; Blanchard, Yannick; Eterradossi, Nicolas; van der Werf, Sylvie; Jestin, Véronique; Niqueux, Eric

    2017-03-02

    Several new highly pathogenic (HP) H5 avian influenza virus (AIV) have been detected in poultry farms from south-western France since November 2015, among which an HP H5N1. The zoonotic potential and origin of these AIVs immediately became matters of concern. One virus of each subtype H5N1 (150169a), H5N2 (150233) and H5N9 (150236) was characterised. All proved highly pathogenic for poultry as demonstrated molecularly by the presence of a polybasic cleavage site in their HA protein - with a sequence (HQRRKR/GLF) previously unknown among avian H5 HPAI viruses - or experimentally by the in vivo demonstration of an intravenous pathogenicity index of 2.9 for the H5N1 HP isolate. Phylogenetic analyses based on the full genomes obtained by NGS confirmed that the eight viral segments of the three isolates were all part of avian Eurasian phylogenetic lineage but differed from the Gs/Gd/1/96-like lineage. The study of the genetic characteristics at specific amino acid positions relevant for modulating the adaptation to and the virulence for mammals showed that presently, these viruses possess most molecular features characteristic of AIV and lack some major characteristics required for efficient respiratory transmission to or between humans. The three isolates are therefore predicted to have no significant pandemic potential. This article is copyright of The Authors, 2017.

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

    Directory of Open Access Journals (Sweden)

    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.

  9. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  11. Gnarled-trunk evolutionary model of influenza A virus hemagglutinin.

    Directory of Open Access Journals (Sweden)

    Kimihito Ito

    Full Text Available Human influenza A viruses undergo antigenic changes with gradual accumulation of amino acid substitutions on the hemagglutinin (HA molecule. A strong antigenic mismatch between vaccine and epidemic strains often requires the replacement of influenza vaccines worldwide. To establish a practical model enabling us to predict the future direction of the influenza virus evolution, relative distances of amino acid sequences among past epidemic strains were analyzed by multidimensional scaling (MDS. We found that human influenza viruses have evolved along a gnarled evolutionary pathway with an approximately constant curvature in the MDS-constructed 3D space. The gnarled pathway indicated that evolution on the trunk favored multiple substitutions at the same amino acid positions on HA. The constant curvature was reasonably explained by assuming that the rate of amino acid substitutions varied from one position to another according to a gamma distribution. Furthermore, we utilized the estimated parameters of the gamma distribution to predict the amino acid substitutions on HA in subsequent years. Retrospective prediction tests for 12 years from 1997 to 2009 showed that 70% of actual amino acid substitutions were correctly predicted, and that 45% of predicted amino acid substitutions have been actually observed. Although it remains unsolved how to predict the exact timing of antigenic changes, the present results suggest that our model may have the potential to recognize emerging epidemic strains.

  12. Characterization of the influenza A H5N1 viruses of the 2008-09 outbreaks in India reveals a third introduction and possible endemicity.

    Directory of Open Access Journals (Sweden)

    Alok K Chakrabarti

    Full Text Available Widespread infection of highly pathogenic avian influenza A H5N1 was reported from backyard and commercial poultry in West Bengal (WB, an eastern state of India in early 2008. Infection gradually spread to Tripura, Assam and Sikkim, the northeastern states, with 70 outbreaks reported between January 2008 and May 2009. Whole genome sequence analysis of three isolates from WB, one isolate from Tripura along with the analysis of hemagglutinin (HA and neuraminidase (NA genes of 17 other isolates was performed during this study. In the HA gene phylogenetic tree, all the 2008-09 Indian isolates belonged to EMA3 sublineage of clade 2.2. The closest phylogenetic relationship was found to be with the 2007-09 isolates from Bangladesh and not with the earlier 2006 and 2007 Indian isolates implying a third introduction into the country. The receptor-binding pocket of HA1 of two isolates from WB showed S221P mutation, one of the markers predicted to be associated with human receptor specificity. Two substitutions E119A (2 isolates of WB and N294S (2 other isolates of WB known to confer resistance to NA inhibitors were observed in the active site of neuraminidase. Several additional mutations were observed within the 2008-09 Indian isolates indicating genetic diversification. Overall, the study is indicative of a possible endemicity in the eastern and northeastern parts of the country, demanding active surveillance specifically in view of the critical mutations that have been observed in the influenza A H5N1 viruses.

  13. 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

  14. [Construction of a recombinant HVT virus expressing the HA gene of avian influenza virus H5N1 via Rde/ET recombination system].

    Science.gov (United States)

    Lan, Desong; Shi, Xingming; Wang, Yunfeng; Liu, Changjun; Wang, Mei; Cui, Hongyu; Tian, Guobin; Li, Jisong; Tong, Guangzhi

    2009-01-01

    In recent years,manipulation of large herpesvirus genomes has been facilitated by using bacterial artificial chromosome (BAC) vectors. We have previously reported the construction of the BAC clones (HVT BACs) of herpesvirus of turkey (HVT). With these BAC clones in hand,we manipulated the genome of HVT by utilizing Red/ET recombination system, and developed a biologically safe live vaccine based on the HVT BACs. In this two-step approach, we first transformed the plasmid pRedET into the DH10B competent cells that carried the HVT BACs,and added inducer L-arabinose into the cells. We prepared the cells into competent cells and electroporated the linear rpsL-neo counter-selection/selection cassette flanked by the 50 bp long homology arms into the cells. So the functional cassette was inserted into the U(S)2 locus. Only colonies carrying the modified BAC would survive Kanamycin selection on the agar plates. The successful integration of the rpsL-neo cassette was monitored by PCR and Streptomycin selection, for the insertion of rpsL-neo cassette cells will become Streptomycin sensitive. Secondly, in the same way, we replaced the rpsL-neo cassette with the hemagglutinin (HA) gene of (HPAIV) A/Goose/ Guangdong/1/96(H5N1) flanked by the same homology arms. Only colonies which lost the rpsL-neo cassette will grow on Streptomycin containing plates. Finally, we obtained many colonies of which the HA gene of the AIV was inserted into the U(S)2 locus to be modified of HVT. And we reconstituted one recombinant virus from transfecting one of these BAC clones DNA into chick embryo fibroblasts (CEFs). We achieved one rescued recombinant virus which designated as rHVT-HA3. The H5 subtype HA gene expression in this recombinant virus rHVT-HA3 was confirmed by immunofluorescence assay.

  15. Theoretical investigation of molecular hydrogen reactions with active centres in B6H5- and AlB6H5- clusters

    International Nuclear Information System (INIS)

    Mebel', A.M.; Charkin, O.P.

    1991-01-01

    Nonempirical calculations of sections of potential surface (PS) along the shortest way of reaction of hydrogen interaction with different active centres in AlB 5 H 5 - cluster were conducted. Mechanisms of reactions of valent-saturated hydrides (BH, AlH) and clusters (B 6 H 5 - , AlB 5 H 5 - ) with molecular hydrogen are correlated. Qualitative model enabling to form an opinion about the presence or the absence of barrier on PS of the shortest way of reactions of breaking σ-bond of H-H type on the active centre of cluster, is suggested. The model is based on analysis of the character of canonical MO reagents and products

  16. Subclinical avian influenza A(H5N1) virus infection in human, Vietnam

    NARCIS (Netherlands)

    Le, Mai Quynh; Horby, Peter; Fox, Annette; Nguyen, Hien Tran; Le Nguyen, Hang Khanh; Hoang, Phuong Mai Vu; Nguyen, Khanh Cong; de Jong, Menno D.; Jeeninga, Rienk E.; Rogier van Doorn, H.; Farrar, Jeremy; Wertheim, Heiman F. L.

    2013-01-01

    Laboratory-confirmed cases of subclinical infection with avian influenza A(H5N1) virus in humans are rare, and the true number of these cases is unknown. We describe the identification of a laboratory-confirmed subclinical case in a woman during an influenza A(H5N1) contact investigation in northern

  17. Virulence of H5N1 Influenza Virus in Cattle Egrets (Bubulcus Ibis)

    DEFF Research Database (Denmark)

    Phuong, Do Quy; Dung, Nguyen Tien; Jørgensen, Poul Henrik

    2011-01-01

    for insect control in households. In this study, six Cattle Egrets were experimentally infected intranasally with highly pathogenic avian influenza (AI) A/duck/Vietnam/40D/04 (H5N1) to investigate a possible epidemiologic role for Cattle Egrets in outbreaks of H5N1 AI in Vietnam. The Cattle Egrets were...

  18. Fragmentations and rearrangements of metastable [C2H5OS]+ ions

    NARCIS (Netherlands)

    de Vries, Marcel; Oudman, D; Weringa, WD

    1992-01-01

    Several [C2H5OS]+ ions with different structures were generated from the appropriate precursors and their metastable ion spectra were determined. Deuterium labelled analogues of some of the [C2H5OS]+ ions were used to elucidate the nature of the observed fragmentations and their mechanisms.

  19. Development of a dual-protective live attenuated vaccine against H5N1 and H9N2 avian influenza viruses by modifying the NS1 gene.

    Science.gov (United States)

    Choi, Eun-hye; Song, Min-Suk; Park, Su-Jin; Pascua, Philippe Noriel Q; Baek, Yun Hee; Kwon, Hyeok-il; Kim, Eun-Ha; Kim, Semi; Jang, Hyung-Kwan; Poo, Haryoung; Kim, Chul-Joong; Choi, Young Ki

    2015-07-01

    An increasing number of outbreaks of avian influenza H5N1 and H9N2 viruses in poultry have caused serious economic losses and raised concerns for human health due to the risk of zoonotic transmission. However, licensed H5N1 and H9N2 vaccines for animals and humans have not been developed. Thus, to develop a dual H5N1 and H9N2 live-attenuated influenza vaccine (LAIV), the HA and NA genes from a virulent mouse-adapted avian H5N2 (A/WB/Korea/ma81/06) virus and a recently isolated chicken H9N2 (A/CK/Korea/116/06) virus, respectively, were introduced into the A/Puerto Rico/8/34 backbone expressing truncated NS1 proteins (NS1-73, NS1-86, NS1-101, NS1-122) but still possessing a full-length NS gene. Two H5N2/NS1-LAIV viruses (H5N2/NS1-86 and H5N2/NS1-101) were highly attenuated compared with the full-length and remaining H5N2/NS-LAIV viruses in a mouse model. Furthermore, viruses containing NS1 modifications were found to induce more IFN-β activation than viruses with full-length NS1 proteins and were correspondingly attenuated in mice. Intranasal vaccination with a single dose (10(4.0) PFU/ml) of these viruses completely protected mice from a lethal challenge with the homologous A/WB/Korea/ma81/06 (H5N2), heterologous highly pathogenic A/EM/Korea/W149/06 (H5N1), and heterosubtypic highly virulent mouse-adapted H9N2 viruses. This study clearly demonstrates that the modified H5N2/NS1-LAIV viruses attenuated through the introduction of mutations in the NS1 coding region display characteristics that are desirable for live attenuated vaccines and hold potential as vaccine candidates for mammalian hosts.

  20. Highly Pathogenic H5N1 Influenza A Virus Strains Provoke Heterogeneous IFN-α/β Responses That Distinctively Affect Viral Propagation in Human Cells

    Science.gov (United States)

    Matthaei, Markus; Budt, Matthias; Wolff, Thorsten

    2013-01-01

    The fatal transmissions of highly pathogenic avian influenza A viruses (IAV) of the H5N1 subtype to humans and high titer replication in the respiratory tract indicate that these pathogens can overcome the bird-to-human species barrier. While type I interferons (IFN-α/β) are well described to contribute to the species barrier of many zoonotic viruses, current data to the role of these antiviral cytokines during human H5N1 IAV infections is limited and contradictory. We hypothesized an important role for the IFN system in limiting productive infection of avian H5N1 strains in human cells. Hence, we examined IFN-α/β gene activation by different avian and human H5N1 isolates, if the IFN-α/β response restricts H5N1 growth and whether the different strains were equally capable to regulate the IFN-α/β system via their IFN-antagonistic NS1 proteins. Two human H5N1 isolates and a seasonal H3N2 strain propagated efficiently in human respiratory cells and induced little IFN-β, whereas three purely avian H5N1 strains were attenuated for replication and provoked higher IFN secretion. Replication of avian viruses was significantly enhanced on interferon-deficient cells, and exogenous IFN potently limited the growth of all strains in human cells. Moreover, IFN-α/β activation by all strains depended on retinoic acid-inducible gene I excluding principal differences in receptor activation between the different viruses. Interestingly, all H5N1 NS1 proteins suppressed IFN-α/β induction comparably well to the NS1 of seasonal IAV. Thus, our study shows that H5N1 strains are heterogeneous in their capacity to activate human cells in an NS1-independent manner. Our findings also suggest that H5N1 viruses need to acquire adaptive changes to circumvent strong IFN-α/β activation in human host cells. Since no single amino acid polymorphism could be associated with a respective high- or low induction phenotype we propose that the necessary adaptations to overcome the human IFN

  1. Highly pathogenic H5N1 influenza A virus strains provoke heterogeneous IFN-α/β responses that distinctively affect viral propagation in human cells.

    Directory of Open Access Journals (Sweden)

    Markus Matthaei

    Full Text Available The fatal transmissions of highly pathogenic avian influenza A viruses (IAV of the H5N1 subtype to humans and high titer replication in the respiratory tract indicate that these pathogens can overcome the bird-to-human species barrier. While type I interferons (IFN-α/β are well described to contribute to the species barrier of many zoonotic viruses, current data to the role of these antiviral cytokines during human H5N1 IAV infections is limited and contradictory. We hypothesized an important role for the IFN system in limiting productive infection of avian H5N1 strains in human cells. Hence, we examined IFN-α/β gene activation by different avian and human H5N1 isolates, if the IFN-α/β response restricts H5N1 growth and whether the different strains were equally capable to regulate the IFN-α/β system via their IFN-antagonistic NS1 proteins. Two human H5N1 isolates and a seasonal H3N2 strain propagated efficiently in human respiratory cells and induced little IFN-β, whereas three purely avian H5N1 strains were attenuated for replication and provoked higher IFN secretion. Replication of avian viruses was significantly enhanced on interferon-deficient cells, and exogenous IFN potently limited the growth of all strains in human cells. Moreover, IFN-α/β activation by all strains depended on retinoic acid-inducible gene I excluding principal differences in receptor activation between the different viruses. Interestingly, all H5N1 NS1 proteins suppressed IFN-α/β induction comparably well to the NS1 of seasonal IAV. Thus, our study shows that H5N1 strains are heterogeneous in their capacity to activate human cells in an NS1-independent manner. Our findings also suggest that H5N1 viruses need to acquire adaptive changes to circumvent strong IFN-α/β activation in human host cells. Since no single amino acid polymorphism could be associated with a respective high- or low induction phenotype we propose that the necessary adaptations to

  2. Transmission of highly pathogenic avian influenza H5N1 virus in Pekin ducks is significantly reduced by a genetically distant H5N2 vaccine

    NARCIS (Netherlands)

    Goot, van der J.A.; Boven, van M.; Stegeman, A.; Water, van de S.G.P.; Jong, de M.C.M.; Koch, G.

    2008-01-01

    Domestic ducks play an important role in the epidemiology of H5N1 avian influenza. Although it is known that vaccines that have a high homology with the challenge virus are able to prevent infection in ducks, little is yet known about the ability of genetically more distant vaccines in preventing

  3. Mucosal immunity induced by adenovirus-based H5N1 HPAI vaccine confers protection against a lethal H5N2 avian influenza virus challenge

    International Nuclear Information System (INIS)

    Park, Ki Seok; Lee, Jiyeung; Ahn, So Shin; Byun, Young-Ho; Seong, Baik Lin; Baek, Yun Hee; Song, Min-Suk; Choi, Young Ki; Na, Yun Jeong; Hwang, Inhwan; Sung, Young Chul; Lee, Chang Geun

    2009-01-01

    Development of effective vaccines against highly pathogenic avian influenza (HPAI) H5N1 viruses is a global public health priority. Considering the difficulty in predicting HPAI H5N1 pandemic strains, one strategy used in their design includes the development of formulations with the capacity of eliciting broad cross-protective immunity against multiple viral antigens. To this end we constructed a replication-defective recombinant adenovirus-based avian influenza virus vaccine (rAdv-AI) expressing the codon-optimized M2eX-HA-hCD40L and the M1-M2 fusion genes from HPAI H5N1 human isolate. Although there were no significant differences in the systemic immune responses observed between the intramuscular prime-intramuscular boost regimen (IM/IM) and the intranasal prime-intramuscular boost regimen (IN/IM), IN/IM induced more potent CD8 + T cell and antibody responses at mucosal sites than the IM/IM vaccination, resulting in more effective protection against lethal H5N2 avian influenza (AI) virus challenge. These findings suggest that the strategies used to induce multi-antigen-targeted mucosal immunity, such as IN/IM delivery of rAdv-AI, may be a promising approach for developing broad protective vaccines that may be more effective against the new HPAI pandemic strains.

  4. A unique influenza A (H5N1 virus causing a focal poultry outbreak in 2007 in Manipur, India

    Directory of Open Access Journals (Sweden)

    Raut Satish

    2009-02-01

    Full Text Available Abstract Background A focal H5N1 outbreak in poultry was reported from Manipur, a north-eastern state, of India, in 2007. The aim of this study was to genetically characterize the Manipur isolate to understand the relationship with other H5N1 isolates and to trace the possible source of introduction of the virus into the country. Results Characterization of the complete genome revealed that the virus belonged to clade 2.2. It was distinctly different from viruses of the three EMA sublineages of clade 2.2 but related to isolates from wild migratory waterfowl from Russia, China and Mongolia. The HA gene, had the cleavage site GERRRRKR, earlier reported in whooper swan isolates from Mongolia in 2005. A stop codon at position 29 in the PB1-F2 protein could have implications on the replication efficiency. The acquisition of polymorphisms as seen in recent isolates of 2005–07 from distinct geographical regions suggests the possibility of transportation of H5N1 viruses through migratory birds. Conclusion Considering that all eight genes of the earlier Indian isolates belonged to the EMA3 sublineage and similar strains have not been reported from neighbouring countries of the subcontinent, it appears that the virus may have been introduced independently.

  5. DC-SIGN mediates avian H5N1 influenza virus infection in cis and in trans

    International Nuclear Information System (INIS)

    Wang, S.-F.; Huang, Jason C.; Lee, Y.-M.; Liu, S.-J.; Chan, Yu-Jiun; Chau, Y.-P.; Chong, P.; Chen, Y.-M.A.

    2008-01-01

    DC-SIGN, a C-type lectin receptor expressed in dendritic cells (DCs), has been identified as a receptor for human immunodeficiency virus type 1, hepatitis C virus, Ebola virus, cytomegalovirus, dengue virus, and the SARS coronavirus. We used H5N1 pseudotyped and reverse-genetics (RG) virus particles to study their ability to bind with DC-SIGN. Electronic microscopy and functional assay results indicate that pseudotyped viruses containing both HA and NA proteins express hemagglutination and are capable of infecting cells expressing α-2,3-linked sialic acid receptors. Results from a capture assay show that DC-SIGN-expressing cells (including B-THP-1/DC-SIGN and T-THP-1/DC-SIGN) and peripheral blood dendritic cells are capable of transferring H5N1 pseudotyped and RG virus particles to target cells; this action can be blocked by anti-DC-SIGN monoclonal antibodies. In summary, (a) DC-SIGN acts as a capture or attachment molecule for avian H5N1 virus, and (b) DC-SIGN mediates infections in cis and in trans

  6. Molecular Evolution and Emergence of H5N6 Avian Influenza Virus in Central China.

    Science.gov (United States)

    Du, Yingying; Chen, Mingyue; Yang, Jiayun; Jia, Yane; Han, Shufang; Holmes, Edward C; Cui, Jie

    2017-06-15

    H5N6 avian influenza virus (AIV) has posed a potential threat to public health since its emergence in China in 2013. To understand the evolution and emergence of H5N6 AIV in the avian population, we performed molecular surveillance of live poultry markets (LPMs) in Wugang Prefecture, Hunan Province, in central China, during 2014 and 2015. Wugang Prefecture is located on the Eastern Asian-Australian migratory bird flyway, and a human death due to an H5N6 virus was reported in the prefecture on 21 November 2016. In total, we sampled and sequenced the complete genomes of 175 H5N6 AIVs. Notably, our analysis revealed that H5N6 AIVs contain at least six genotypes arising from segment reassortment, including a rare variant that possesses an HA gene derived from H5N1 clade 2.3.2 and a novel NP gene that has its origins with H7N3 viruses. In addition, phylogenetic analysis revealed that genetically similar H5N6 AIVs tend to cluster according to their geographic regions of origin. These results help to reveal the evolutionary behavior of influenza viruses prior to their emergence in humans. IMPORTANCE The newly emerged H5N6 influenza A virus has caused more than 10 human deaths in China since 2013. In November 2016, a human death due to an H5N6 virus, in Wugang Prefecture, Hunan Province, was confirmed by the WHO. To better understand the evolution and emergence of H5N6 viruses, we surveyed live poultry markets (LPMs) in Wugang Prefecture before the reported human death, with a focus on revealing the diversity and genomic origins of H5N6 in birds during 2014 and 2015. In general, H5N6 viruses in this region were most closely related to H5N1 clade 2.3.4.4, with the exception of one virus with an HA gene derived from clade 2.3.2 such that it represents a novel reassortant. Clearly, the ongoing surveillance of LPMs is central to monitoring the emergence of pathogenic influenza viruses. Copyright © 2017 American Society for Microbiology.

  7. Recognizing true H5N1 infections in humans during confirmed outbreaks.

    Science.gov (United States)

    Zaman, Mukhtiar; Gasimov, Viktor; Oner, Ahmet Faik; Dogan, Nazim; Adisasmito, Wiku; Coker, Richard; Bamgboye, Ebun L; Chan, Paul K S; Hanshaoworakul, Wanna; Lee, Nelson; Phommasack, Bounlay; Touch, Sok; Tsang, Owen; Swenson, Anna; Toovey, Stephen; Dreyer, Nancy Ann

    2014-02-13

    The goal of this study was to evaluate whether any characteristics that are evident at presentation for urgent medical attention could be used to differentiate cases of H5N1 in the absence of viral testing. Information about exposure to poultry, clinical signs and symptoms, treatments, and outcomes was abstracted from existing data in the global avian influenza registry (www.avianfluregistry.org) using standardized data collection tools for documented and possible cases of H5N1 infection who presented for medical attention between 2005-2011 during known H5N1 outbreaks in Azerbaijan, Indonesia, Pakistan and Turkey. Demography, exposure to poultry, and presenting symptoms were compared, with only the common symptoms of fever and headache presenting significantly more frequently in confirmed H5N1 cases than in possible cases. Reported exposure to  infected humans was also more common in confirmed cases. In contrast, unexplained respiratory illness, sore throat, excess sputum production, and rhinorrhea were more frequent in possible cases. Overall, oseltamivir treatment showed a survival benefit, with the greatest benefit shown in H5N1 cases who were treated within two days of symptom onset (51% reduction in case fatality). Since prompt treatment with antivirals conferred a strong survival benefit for H5N1 cases, presumptive antiviral treatment should be considered for all possible cases presenting during an outbreak of H5N1 as a potentially life-saving measure.

  8. Viremia associated with fatal outcomes in ferrets infected with avian H5N1 influenza virus.

    Directory of Open Access Journals (Sweden)

    Xue Wang

    Full Text Available Avian H5N1 influenza viruses cause severe disease and high mortality in infected humans. However, tissue tropism and underlying pathogenesis of H5N1 virus infection in humans needs further investigation. The objective of this work was to study viremia, tissue tropism and disease pathogenesis of H5N1 virus infection in the susceptible ferret animal model. To evaluate the relationship of morbidity and mortality with virus loads, we performed studies in ferrets infected with the H5N1 strain A/VN/1203/04 to assess clinical signs after infection and virus load in lung, brain, ileum, nasal turbinate, nasal wash, and blood. We observed that H5N1 infection in ferrets is characterized by high virus load in the brain and and low levels in the ileum using real-time PCR. In addition, viral RNA was frequently detected in blood one or two days before death and associated with symptoms of diarrhea. Our observations further substantiate pathogenicity of H5N1 and further indicate that viremia may be a bio-marker for fatal outcomes in H5N1 infection.

  9. Isolation and characterization of highly pathogenic avian influenza virus subtype H5N1 from donkeys

    Science.gov (United States)

    2010-01-01

    Background The highly pathogenic H5N1 is a major avian pathogen that crosses species barriers and seriously affects humans as well as some mammals. It mutates in an intensified manner and is considered a potential candidate for the possible next pandemic with all the catastrophic consequences. Methods Nasal swabs were collected from donkeys suffered from respiratory distress. The virus was isolated from the pooled nasal swabs in specific pathogen free embryonated chicken eggs (SPF-ECE). Reverse transcriptase polymerase chain reaction (RT-PCR) and sequencing of both haemagglutingin and neuraminidase were performed. H5 seroconversion was screened using haemagglutination inhibition (HI) assay on 105 donkey serum samples. Results We demonstrated that H5N1 jumped from poultry to another mammalian host; donkeys. Phylogenetic analysis showed that the virus clustered within the lineage of H5N1 from Egypt, closely related to 2009 isolates. It harboured few genetic changes compared to the closely related viruses from avian and humans. The neuraminidase lacks oseltamivir resistant mutations. Interestingly, HI screening for antibodies to H5 haemagglutinins in donkeys revealed high exposure rate. Conclusions These findings extend the host range of the H5N1 influenza virus, possess implications for influenza virus epidemiology and highlight the need for the systematic surveillance of H5N1 in animals in the vicinity of backyard poultry units especially in endemic areas. PMID:20398268

  10. Isolation and characterization of highly pathogenic avian influenza virus subtype H5N1 from donkeys

    Directory of Open Access Journals (Sweden)

    Abdel-Ghany Ahmad E

    2010-04-01

    Full Text Available Abstract Background The highly pathogenic H5N1 is a major avian pathogen that crosses species barriers and seriously affects humans as well as some mammals. It mutates in an intensified manner and is considered a potential candidate for the possible next pandemic with all the catastrophic consequences. Methods Nasal swabs were collected from donkeys suffered from respiratory distress. The virus was isolated from the pooled nasal swabs in specific pathogen free embryonated chicken eggs (SPF-ECE. Reverse transcriptase polymerase chain reaction (RT-PCR and sequencing of both haemagglutingin and neuraminidase were performed. H5 seroconversion was screened using haemagglutination inhibition (HI assay on 105 donkey serum samples. Results We demonstrated that H5N1 jumped from poultry to another mammalian host; donkeys. Phylogenetic analysis showed that the virus clustered within the lineage of H5N1 from Egypt, closely related to 2009 isolates. It harboured few genetic changes compared to the closely related viruses from avian and humans. The neuraminidase lacks oseltamivir resistant mutations. Interestingly, HI screening for antibodies to H5 haemagglutinins in donkeys revealed high exposure rate. Conclusions These findings extend the host range of the H5N1 influenza virus, possess implications for influenza virus epidemiology and highlight the need for the systematic surveillance of H5N1 in animals in the vicinity of backyard poultry units especially in endemic areas.

  11. Endothelial cell tropism is a determinant of H5N1 pathogenesis in mammalian species.

    Directory of Open Access Journals (Sweden)

    Smanla Tundup

    2017-03-01

    Full Text Available The cellular and molecular mechanisms underpinning the unusually high virulence of highly pathogenic avian influenza H5N1 viruses in mammalian species remains unknown. Here, we investigated if the cell tropism of H5N1 virus is a determinant of enhanced virulence in mammalian species. We engineered H5N1 viruses with restricted cell tropism through the exploitation of cell type-specific microRNA expression by incorporating microRNA target sites into the viral genome. Restriction of H5N1 replication in endothelial cells via miR-126 ameliorated disease symptoms, prevented systemic viral spread and limited mortality, despite showing similar levels of peak viral replication in the lungs as compared to control virus-infected mice. Similarly, restriction of H5N1 replication in endothelial cells resulted in ameliorated disease symptoms and decreased viral spread in ferrets. Our studies demonstrate that H5N1 infection of endothelial cells results in excessive production of cytokines and reduces endothelial barrier integrity in the lungs, which culminates in vascular leakage and viral pneumonia. Importantly, our studies suggest a need for a combinational therapy that targets viral components, suppresses host immune responses, and improves endothelial barrier integrity for the treatment of highly pathogenic H5N1 virus infections.

  12. Broadly-reactive human monoclonal antibodies elicited following pandemic H1N1 influenza virus exposure protect mice from highly pathogenic H5N1 challenge.

    Science.gov (United States)

    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

  13. Isolation and molecular characterization of an H5N1 swine influenza virus in China in 2015.

    Science.gov (United States)

    Wu, Haibo; Yang, Fan; Lu, Rufeng; Xu, Lihua; Liu, Fumin; Peng, Xiuming; Wu, Nanping

    2018-03-01

    In 2015, an H5N1 influenza virus was isolated from a pig in Zhejiang Province, Eastern China. This strain was characterized by whole-genome sequencing with subsequent phylogenetic analysis. Phylogenetic analysis showed that all segments from this strain belonged to clade 2.3.2 and that it had received its genes from poultry influenza viruses in China. A Glu627Lys mutation associated with pathogenicity was observed in the PB2 protein. This strain was moderately pathogenic in mice and was able to replicate without prior adaptation. These results suggest that active surveillance of swine influenza should be used as an early warning system for influenza outbreaks in mammals.

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

    Science.gov (United States)

    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

  15. Spatio-temporal dynamics of global H5N1 outbreaks match bird migration patterns

    Directory of Open Access Journals (Sweden)

    Yali Si

    2009-11-01

    Full Text Available The global spread of highly pathogenic avian influenza H5N1 in poultry, wild birds and humans, poses a significant pandemic threat and a serious public health risk. An efficient surveillance and disease control system relies on the understanding of the dispersion patterns and spreading mechanisms of the virus. A space-time cluster analysis of H5N1 outbreaks was used to identify spatio-temporal patterns at a global scale and over an extended period of time. Potential mechanisms explaining the spread of the H5N1 virus, and the role of wild birds, were analyzed. Between December 2003 and December 2006, three global epidemic phases of H5N1 influenza were identified. These H5N1 outbreaks showed a clear seasonal pattern, with a high density of outbreaks in winter and early spring (i.e., October to March. In phase I and II only the East Asia Australian flyway was affected. During phase III, the H5N1 viruses started to appear in four other flyways: the Central Asian flyway, the Black Sea Mediterranean flyway, the East Atlantic flyway and the East Africa West Asian flyway. Six disease cluster patterns along these flyways were found to be associated with the seasonal migration of wild birds. The spread of the H5N1 virus, as demonstrated by the space-time clusters, was associated with the patterns of migration of wild birds. Wild birds may therefore play an important role in the spread of H5N1 over long distances. Disease clusters were also detected at sites where wild birds are known to overwinter and at times when migratory birds were present. This leads to the suggestion that wild birds may also be involved in spreading the H5N1 virus over short distances.

  16. Influence of virus strain and antigen mass on efficacy of H5 avian influenza inactivated vaccines.

    Science.gov (United States)

    Swayne, D E; Beck, J R; Garcia, M; Stone, H D

    1999-06-01

    The influence of vaccine strain and antigen mass on the ability of inactivated avian influenza (AI) viruses to protect chicks from a lethal, highly pathogenic (HP) AI virus challenge was studied. Groups of 4-week-old chickens were immunized with inactivated vaccines containing one of 10 haemagglutinin subtype H5 AI viruses, one heterologous H7 AI virus or normal allantoic fluid (sham), and challenged 3 weeks later by intra-nasal inoculation with a HP H5 chicken-origin AI virus. All 10 H5 vaccines provided good protection from clinical signs and death, and produced positive serological reactions on agar gel immunodiffusion and haemagglutination inhibition tests. In experiment 1, challenge virus was recovered from the oropharynx of 80% of chickens in the H5 vaccine group. In five H5 vaccine groups, challenge virus was not recovered from the cloaca of chickens. In the other five H5 vaccine groups, the number of chickens with detection of challenge virus from the cloaca was lower than in the sham group (P turkey/Wisconsin/68 (H5N9) was the best vaccine candidate of the H5 strains tested (PD50= 0.006 μg AI antigen). These data demonstrate that chickens vaccinated with inactivated H5 whole virus AI vaccines were protected from clinical signs and death, but usage of vaccine generally did not prevent infection by the challenge virus, as indicated by recovery of virus from the oropharynx. Vaccine use reduced cloacal detection rates, and quantity of virus shed from the cloaca and oropharynx in some vaccine groups, which would potentially reduce environmental contamination and disease transmission in the field.

  17. An M2e-based multiple antigenic peptide vaccine protects mice from lethal challenge with divergent H5N1 influenza viruses

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    Chan Chris CS

    2010-01-01

    Full Text Available Abstract Background A growing concern has raised regarding the pandemic potential of the highly pathogenic avian influenza (HPAI H5N1 viruses. Consequently, there is an urgent need to develop an effective and safe vaccine against the divergent H5N1 influenza viruses. In the present study, we designed a tetra-branched multiple antigenic peptide (MAP-based vaccine, designated M2e-MAP, which contains the sequence overlapping the highly conserved extracellular domain of matrix protein 2 (M2e of a HPAI H5N1 virus, and investigated its immune responses and cross-protection against different clades of H5N1 viruses. Results Our results showed that M2e-MAP vaccine induced strong M2e-specific IgG antibody responses following 3-dose immunization of mice with M2e-MAP in the presence of Freunds' or aluminium (alum adjuvant. M2e-MAP vaccination limited viral replication and attenuated histopathological damage in the challenged mouse lungs. The M2e-MAP-based vaccine protected immunized mice against both clade1: VN/1194 and clade2.3.4: SZ/406H H5N1 virus challenge, being able to counteract weight lost and elevate survival rate following lethal challenge of H5N1 viruses. Conclusions These results suggest that M2e-MAP presenting M2e of H5N1 virus has a great potential to be developed into an effective subunit vaccine for the prevention of infection by a broad spectrum of HPAI H5N1 viruses.

  18. The Madin-Darby canine kidney cell culture derived influenza A/H5N1 vaccine: a phase I trial in Taiwan.

    Science.gov (United States)

    Pan, Sung-Ching; Kung, Hsiang-Chi; Kao, Tsui-Mai; Wu, Hsio; Dong, Shao-Xing; Hu, Mei-Hua; Chou, Ai-Hsiang; Chong, Pele; Hsieh, Szu-Min; Chang, Shan-Chwen

    2013-12-01

    Avian H5N1 influenza has caused human infections globally and has a high mortality rate. Rapid production of effective vaccines is needed. A phase 1, randomized, observer-blinded clinical trial was conducted to examine the safety and immunogenicity of an inactivated whole virion vaccine against the influenza A/H5N1 virus produced from the Madin-Darby canine kidney (MDCK) cell line. Participants were randomized to four groups and administered two intramuscular doses of vaccine containing 3 μg hemagglutinin (HA), 3 μg HA with 300 μg aluminum phosphate (AlPO4), 6 μg HA, and 6 μg HA with 300 μg AlPO4, respectively, at two visits, 21 days apart. Serum hemagglutination inhibition (HAI) and neutralizing antibody levels were determined at baseline and on Days 21 and 42. Sixty healthy individuals were enrolled. The neutralization assay showed a significant immune response in the 6 μg with ALPO4 group on Day 42 compared to pre-vaccination levels (11.32±9.77 vs. 4.00±0, p=0.02). The adjuvant effect in neutralization assay was also significant on Day 42 in the 6 μg group (4.52±1.94 without adjuvant vs. 11.32±9.77 with adjuvant, p=0.02). HAI assay also showed an aluminum adjuvant-induced increasing trend in HAI geometric mean titer on Day 42 in the 3 μg and 6 μg groups (6.02 versus 8.20, p=0.05 and 5.74 versus 8.21, p=0.14). The most frequent adverse event was local pain (20% to 60%). There were no vaccine-related severe adverse effects. MDCK cell line-derived H5N1 vaccine was well tolerated. It is necessary to investigate further the immunogenicity of higher antigen doses and the role of aluminum adjuvant in augmenting the effect of the vaccine. Copyright © 2012. Published by Elsevier B.V.

  19. Evidence from lateral mobility studies for dynamic interactions of a mutant influenza hemagglutinin with coated pits.

    Science.gov (United States)

    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

  20. Using knowledge fusion to analyze avian influenza H5N1 in East and Southeast Asia.

    Directory of Open Access Journals (Sweden)

    Erjia Ge

    Full Text Available Highly pathogenic avian influenza (HPAI H5N1, a disease associated with high rates of mortality in infected human populations, poses a serious threat to public health in many parts of the world. This article reports findings from a study aimed at improving our understanding of the spatial pattern of the highly pathogenic avian influenza, H5N1, risk in East-Southeast Asia where the disease is both persistent and devastating. Though many disciplines have made important contributions to our understanding of H5N1, it remains a challenge to integrate knowledge from different disciplines. This study applies genetic analysis that identifies the evolution of the H5N1 virus in space and time, epidemiological analysis that determines socio-ecological factors associated with H5N1 occurrence, and statistical analysis that identifies outbreak clusters, and then applies a methodology to formally integrate the findings of the three sets of methodologies. The present study is novel in two respects. First it makes the initiative attempt to use genetic sequences and space-time data to create a space-time phylogenetic tree to estimate and map the virus' ability to spread. Second, by integrating the results we are able to generate insights into the space-time occurrence and spread of H5N1 that we believe have a higher level of corroboration than is possible when analysis is based on only one methodology. Our research identifies links between the occurrence of H5N1 by area and a set of socio-ecological factors including altitude, population density, poultry density, and the shortest path distances to inland water, coastlines, migrating routes, railways, and roads. This study seeks to lay a solid foundation for the interdisciplinary study of this and other influenza outbreaks. It will provide substantive information for containing H5N1 outbreaks.

  1. Using knowledge fusion to analyze avian influenza H5N1 in East and Southeast Asia.

    Science.gov (United States)

    Ge, Erjia; Haining, Robert; Li, Chi Pang; Yu, Zuguo; Waye, Miu Yee; Chu, Ka Hou; Leung, Yee

    2012-01-01

    Highly pathogenic avian influenza (HPAI) H5N1, a disease associated with high rates of mortality in infected human populations, poses a serious threat to public health in many parts of the world. This article reports findings from a study aimed at improving our understanding of the spatial pattern of the highly pathogenic avian influenza, H5N1, risk in East-Southeast Asia where the disease is both persistent and devastating. Though many disciplines have made important contributions to our understanding of H5N1, it remains a challenge to integrate knowledge from different disciplines. This study applies genetic analysis that identifies the evolution of the H5N1 virus in space and time, epidemiological analysis that determines socio-ecological factors associated with H5N1 occurrence, and statistical analysis that identifies outbreak clusters, and then applies a methodology to formally integrate the findings of the three sets of methodologies. The present study is novel in two respects. First it makes the initiative attempt to use genetic sequences and space-time data to create a space-time phylogenetic tree to estimate and map the virus' ability to spread. Second, by integrating the results we are able to generate insights into the space-time occurrence and spread of H5N1 that we believe have a higher level of corroboration than is possible when analysis is based on only one methodology. Our research identifies links between the occurrence of H5N1 by area and a set of socio-ecological factors including altitude, population density, poultry density, and the shortest path distances to inland water, coastlines, migrating routes, railways, and roads. This study seeks to lay a solid foundation for the interdisciplinary study of this and other influenza outbreaks. It will provide substantive information for containing H5N1 outbreaks.

  2. Inefficient Transmission of H5N1 Influenza Viruses in a Ferret Contact Model▿

    OpenAIRE

    Yen, Hui-Ling; Lipatov, Aleksandr S.; Ilyushina, Natalia A.; Govorkova, Elena A.; Franks, John; Yilmaz, Neziha; Douglas, Alan; Hay, Alan; Krauss, Scott; Rehg, Jerold E.; Hoffmann, Erich; Webster, Robert G.

    2007-01-01

    The abilities to infect and transmit efficiently among humans are essential for a novel influenza A virus to cause a pandemic. To evaluate the pandemic potential of widely disseminated H5N1 influenza viruses, a ferret contact model using experimental groups comprised of one inoculated ferret and two contact ferrets was used to study the transmissibility of four human H5N1 viruses isolated from 2003 to 2006. The effects of viral pathogenicity and receptor binding specificity (affinity to synth...

  3. Effect of acute and chronic tramadol on [3H]-5-HT uptake in rat cortical synaptosomes

    OpenAIRE

    Giusti, Pietro; Buriani, Alessandro; Cima, Lorenzo; Lipartiti, Maria

    1997-01-01

    Tramadol hydrochloride is a centrally acting opioid analgesic, the efficacy and potency of which is only five to ten times lower than that of morphine. Opioid, as well as non-opioid mechanisms, may participate in the analgesic activity of tramadol.[3H]-5-hydroxytryptamine (5-HT) uptake in rat isolated cortical synaptosomes was studied in the presence of tramadol, desipramine, fluoxetine, methadone and morphine. Methadone and tramadol inhibited synaptosomal [3H]-5-HT uptake with apparent Kis o...

  4. Amino Acid Substitutions Associated with Avian H5N6 Influenza A Virus Adaptation to Mice

    Directory of Open Access Journals (Sweden)

    Chunmao Zhang

    2017-09-01

    Full Text Available At least 15 cases of human beings infected with H5N6 have been reported since 2014, of which at least nine were fatal. The highly pathogenic avian H5N6 influenza virus may pose a serious threat to both public health and the poultry industry. However, the molecular features promoting the adaptation of avian H5N6 influenza viruses to mammalian hosts is not well understood. Here, we sequentially passaged an avian H5N6 influenza A virus (A/Northern Shoveler/Ningxia/488-53/2015 10 times in mice to identify the adaptive amino acid substitutions that confer enhanced virulence to H5N6 in mammals. The 1st and 10th passages of the mouse-adapted H5N6 viruses were named P1 and P10, respectively. P1 and P10 displayed higher pathogenicity in mice than their parent strain. P10 showed significantly higher replication capability in vivo and could be detected in the brains of mice, whereas P1 displayed higher replication efficiency in their lungs but was not detectable in the brain. Similar to its parent strain, P10 remained no transmissible between guinea pigs. Using genome sequencing and alignment, multiple amino acid substitutions, including PB2 E627K, PB2 T23I, PA T97I, and HA R239H, were found in the adaptation of H5N6 to mice. In summary, we identified amino acid changes that are associated with H5N6 adaptation to mice.

  5. Avian Influenza Virus (H5N1): a Threat to Human Health

    OpenAIRE

    Peiris, J. S. Malik; de Jong, Menno D.; Guan, Yi

    2007-01-01

    Pandemic influenza virus has its origins in avian influenza viruses. The highly pathogenic avian influenza virus subtype H5N1 is already panzootic in poultry, with attendant economic consequences. It continues to cross species barriers to infect humans and other mammals, often with fatal outcomes. Therefore, H5N1 virus has rightly received attention as a potential pandemic threat. However, it is noted that the pandemics of 1957 and 1968 did not arise from highly pathogenic influenza viruses, ...

  6. Opposite replication polarities of transcribed and nontranscribed histone H5 genes

    International Nuclear Information System (INIS)

    Trempe, J.P.; Lindstrom, Y.I.; Leffak, M.

    1988-01-01

    The authors used an in vitro nuclear runoff replication assay to analyze the direction of replication of the active and inactive histone H5 genes in avian cells. In embryonic erythrocytes the transcribed histone H5 gene displayed sensitivity to endogenous nuclease cleavage. In contrast, this gene was insensitive to endogenous nuclease digestion under the same conditions in nuclei of the lymphoblastoid cell line MSB-1, and histone H5 gene transcripts were not detectable by dot-blot analysis of MSB-1 cell RNA. When nuclei were isolated from embryonic erythrocyctes and incubated with bromodeoxyuridine triphosphate, runoff replication from endogenous nuclease cleavage sites led to a relative enrichment for fragments near the 3' end of the histone H5 gene in the density-labeled DNA. In nuclei of MSB-1 cells or chicken embryo fibroblasts, however, runoff replication from restriction enzyme-cut sites (or induced endogenous nuclease-cut sites in MSB-1 nuclei) led to a relative enrichment for fragments near the 5' end of the H5 gene in dense DNA. Based on the enhanced incorporation of bromodeoxyuridine into origin-distal regions of DNA during the in vitro runoff replication assay, the authors conclude that the active histone H5 gene in embryonic erythrocytes is preferentially replicated in the transcriptional direction from an origin in the 5'-flanking DNA, whereas its inactive counterparts in MSB-1 cells and chicken embryo fibroblasts are preferentially replicated in the opposite direction

  7. Estimation of transmission parameters of H5N1 avian influenza virus in chickens.

    Directory of Open Access Journals (Sweden)

    Annemarie Bouma

    2009-01-01

    Full Text Available Despite considerable research efforts, little is yet known about key epidemiological parameters of H5N1 highly pathogenic influenza viruses in their avian hosts. Here we show how these parameters can be estimated using a limited number of birds in experimental transmission studies. Our quantitative estimates, based on Bayesian methods of inference, reveal that (i the period of latency of H5N1 influenza virus in unvaccinated chickens is short (mean: 0.24 days; 95% credible interval: 0.099-0.48 days; (ii the infectious period of H5N1 virus in unvaccinated chickens is approximately 2 days (mean: 2.1 days; 95%CI: 1.8-2.3 days; (iii the reproduction number of H5N1 virus in unvaccinated chickens need not be high (mean: 1.6; 95%CI: 0.90-2.5, although the virus is expected to spread rapidly because it has a short generation interval in unvaccinated chickens (mean: 1.3 days; 95%CI: 1.0-1.5 days; and (iv vaccination with genetically and antigenically distant H5N2 vaccines can effectively halt transmission. Simulations based on the estimated parameters indicate that herd immunity may be obtained if at least 80% of chickens in a flock are vaccinated. We discuss the implications for the control of H5N1 avian influenza virus in areas where it is endemic.

  8. Epidemiology of avian influenza H5N1 virus in Egypt and its zoonotic potential

    Directory of Open Access Journals (Sweden)

    Nahed Hamed Ghoneim

    2014-09-01

    Full Text Available Objective: To investigate the epidemiology of avian influenza H5N1 virus in domestic poultry and its zoonotic potential in Egypt. Methods: Tracheal swabs were collected from two hundred and forty three domestic poultry (chickens, ducks and geese from commercial farms and backyards, and thirty two blood samples from unvaccinated chickens. Fifty two throat swabs and twenty blood samples were collected from persons who are in contact with diseased and/or infected birds. Tracheal and throat swabs were examined for the presence of avian influenza virus H5N1 genome by real-time RT-PCR whereas blood samples were tested by competitive ELISA for the presence of avian influenza virus H5 antibodies. Results: The overall prevalence of H5N1 in the examined birds was 5.3% while the prevalence rates among different poultry species were 9%, 4.7% and 0% for ducks, chicken and geese respectively. Moreover, we detected H5 antibodies in 12.5% of the examined backyard chickens. All examined humans were negative for both viral RNA and antibodies. Conclusions: Our findings highlight the broad circulation of H5N1 virus among poultry in Egypt whereas it still has a limited zoonotic potential so far.

  9. Peptide displacement of [3H]5-hydroxytryptamine binding to bovine cortical membranes

    International Nuclear Information System (INIS)

    Takeuchi, Y.; Root-Bernstein, R.S.; Shih, J.C.

    1990-01-01

    Chemical studies have demonstrated that peptides such as the encephalitogenic (EAE) peptide of myelin basic protein (MBP) and luteinizing hormone-releasing hormone (LHRH) can bind serotonin (5-hydroxytryptamine, 5-HT) in vitro. The present research was undertaken to determine whether such binding interferes with 5-HT binding to its 5-HT1 receptors on bovine cerebral cortical membranes. EAE peptide and LHRH displaced [ 3 H]5-HT with IC50s of 4.0 x 10(-4) and 1.8 x 10(-3) M respectively. MBP itself also showed apparent displacing ability with an IC50 of 6.0 x 10(-5) M, though it also caused aggregation of cortical membranes that might have interfered with normal receptor binding. These results support previous suggestions that the tryptophan peptide region of MBP may act as a 5-HT receptor in the neural system. We also tested the effects of muramyl dipeptide (N-acetyl-muramyl-L-Ala-D-isoGln, MD), a bacterial cell-wall breakdown product that acts as a slow-wave sleep promoter, binds to LHRH and EAE peptide, and competes for 5-HT binding sites on macrophages. It showed no significant displacement of 5-HT binding to cortical membranes (IC50 greater than 10(-1) M), but its D-Ala analogue did (IC50 = 1.7 x 10(-3) M). Thus, it seems likely that the 5-HT-related effects of naturally occurring muramyl peptides are physiologically limited by receptor types

  10. Phase II, randomized, open, controlled study of AS03-adjuvanted H5N1 pre-pandemic influenza vaccine in children aged 3 to 9 years: follow-up of safety and immunogenicity persistence at 24 months post-vaccination.

    Science.gov (United States)

    Díez-Domingo, Javier; Baldó, José-María; Planelles-Catarino, Maria Victoria; Garcés-Sánchez, María; Ubeda, Isabel; Jubert-Rosich, Angels; Marès, Josep; Garcia-Corbeira, Pilar; Moris, Philippe; Teko, Maurice; Vanden Abeele, Carline; Gillard, Paul

    2015-03-01

    An AS03-adjuvanted H5N1 influenza vaccine elicited broad and persistent immune responses with an acceptable safety profile up to 6 months following the first vaccination in children aged 3-9 years. In this follow-up of the Phase II study, we report immunogenicity persistence and safety at 24 months post-vaccination in children aged 3-9 years. The randomized, open-label study assessed two doses of H5N1 A/Vietnam/1194/2004 influenza vaccine (1·9 μg or 3·75 μg hemagglutinin antigen) formulated with AS03A or AS03B (11·89 mg or 5·93 mg tocopherol, respectively). Control groups received seasonal trivalent influenza vaccine. Safety was assessed prospectively and included potential immune-mediated diseases (pIMDs). Immunogenicity was assessed by hemagglutination-inhibition assay 12 and 24 months after vaccination; cross-reactivity and cell-mediated responses were also assessed. (NCT00502593). The safety population included 405 children. Over 24 months, five events fulfilled the criteria for pIMDs, of which four occurred in H5N1 vaccine recipients, including uveitis (n = 1) and autoimmune hepatitis (n = 1), which were considered to be vaccine-related. Overall, safety profiles of the vaccines were clinically acceptable. Humoral immune responses at 12 and 24 months were reduced versus those observed after the second dose of vaccine, although still within the range of those observed after the first dose. Persistence of cell-mediated immunity was strong, and CD4(+) T cells with a TH 1 profile were observed. Two doses of an AS03-adjuvanted H5N1 influenza vaccine in children showed low but persistent humoral immune responses and a strong persistence of cell-mediated immunity, with clinically acceptable safety profiles up to 24 months following first vaccination. © 2014 The Authors. Influenza and Other Respiratory Viruses Published by John Wiley & Sons Ltd.

  11. Prophylactic and therapeutic efficacy of human monoclonal antibodies against H5N1 influenza.

    Directory of Open Access Journals (Sweden)

    Cameron P Simmons

    2007-05-01

    Full Text Available New prophylactic and therapeutic strategies to combat human infections with highly pathogenic avian influenza (HPAI H5N1 viruses are needed. We generated neutralizing anti-H5N1 human monoclonal antibodies (mAbs and tested their efficacy for prophylaxis and therapy in a murine model of infection.Using Epstein-Barr virus we immortalized memory B cells from Vietnamese adults who had recovered from infections with HPAI H5N1 viruses. Supernatants from B cell lines were screened in a virus neutralization assay. B cell lines secreting neutralizing antibodies were cloned and the mAbs purified. The cross-reactivity of these antibodies for different strains of H5N1 was tested in vitro by neutralization assays, and their prophylactic and therapeutic efficacy in vivo was tested in mice. In vitro, mAbs FLA3.14 and FLD20.19 neutralized both Clade I and Clade II H5N1 viruses, whilst FLA5.10 and FLD21.140 neutralized Clade I viruses only. In vivo, FLA3.14 and FLA5.10 conferred protection from lethality in mice challenged with A/Vietnam/1203/04 (H5N1 in a dose-dependent manner. mAb prophylaxis provided a statistically significant reduction in pulmonary virus titer, reduced associated inflammation in the lungs, and restricted extrapulmonary dissemination of the virus. Therapeutic doses of FLA3.14, FLA5.10, FLD20.19, and FLD21.140 provided robust protection from lethality at least up to 72 h postinfection with A/Vietnam/1203/04 (H5N1. mAbs FLA3.14, FLD21.140 and FLD20.19, but not FLA5.10, were also therapeutically active in vivo against the Clade II virus A/Indonesia/5/2005 (H5N1.These studies provide proof of concept that fully human mAbs with neutralizing activity can be rapidly generated from the peripheral blood of convalescent patients and that these mAbs are effective for the prevention and treatment of H5N1 infection in a mouse model. A panel of neutralizing, cross-reactive mAbs might be useful for prophylaxis or adjunctive treatment of human cases of H5N1

  12. The role of filamentous hemagglutinin adhesin in adherence and biofilm formation in Acinetobacter baumannii ATCC19606(T).

    Science.gov (United States)

    Darvish Alipour Astaneh, Shakiba; Rasooli, Iraj; Mousavi Gargari, Seyed Latif

    2014-09-01

    Filamentous hemagglutinin adhesins (FHA) are key factors for bacterial attachment and subsequent cell accumulation on substrates. Here an FHA-like Outer membrane (OM) adhesin of Acinetobacter baumannii ATCC19606(T) was displayed on Escherichia coli. The candidate autotransporter (AT) genes were identified in A. baumannii ATCC19606(T) genome. The exoprotein (FhaB1) and transporter (FhaC1) were produced independently within the same cell (FhaB1C1). The fhaC1 was mutated. In vitro adherence to epithelial cells of the recombinant FhaB1C1 and the mutant strains were compared with A. baumanni ATCC19606(T). A bivalent chimeric protein (K) composed of immunologically important portions of fhaB1 (B) and fhaC1 (C) was constructed. The mice vaccinated with chimeric protein were challenged with A. baumannii ATCC19606(T) and FhaB1C1 producing recombinant E. coli. Mutations in the fhaC1 resulted in the absence of FhaB1 in the OM. Expression of FhaB1C1 enhanced the adherence of recombinant bacteria to A546 bronchial cell line. The results revealed association of FhaB1 with bacterial adhesion and biofilm formation. Immunization with a combination of recombinant B and K proteins proved protective against A. baumanni ATCC19606(T). The findings may be applied in active and passive immunization strategies against A. baumannii. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    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.

  15. 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

  16. First human case of avian influenza A (H5N6 in Yunnan province, China

    Directory of Open Access Journals (Sweden)

    Jibo He

    2015-08-01

    Full Text Available Objective: To report clinical, virological, and epidemiological features of the first death caused by a H5N6 avian influenza virus in Yunnan Province, China. Method: The case was described in clinical expression, chest radiography, blood test and treatment. Real-time RT-PCR was used to detect H5N6 virus RNA in clinical and environment samples. Epidemiological investigation was performed including case exposure history determinant, close contacts follow up, and environment sample collection. Results: The patient initially developed sore throat and coughs on 27 January 2015. The disease progressed to severe pneumonia, multiple organ dysfunction syndrome, and acute respiratory distress syndrome. And the patient died on 6 February. A highly pathogenic avian influenza A H5N6 virus was isolated from the tracheal aspirate specimen of the patient. The viral genome analyses revealed that the H5 hemmagglutinin gene belongs to 2.3.4.4 clade. Epidemiological investigation showed that the patient had exposure to wild bird. All close contacts of the patient did not present the same disease in seven consecutive days. A high H5 positive rate was detected in environmental samples from local live poultry markets. Conclusion: The findings suggest that studies on the source of the virus, transmission models, serologic investigations, vaccines, and enhancing surveillance in both humans and birds are necessary.

  17. Genome characterisation of the newly discovered avian influenza A H5N7 virus subtype combination

    DEFF Research Database (Denmark)

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

    2007-01-01

    In Denmark, in 2003, a previously unknown subtype combination of avian influenza A virus, H5N7 (A/Mallard/Denmark/64650/03), was isolated from a flock of 12,000 mallards. The H5N7 subtype combination might be a reassortant between recent European avian influenza A H5, H7, and a third subtype......) and the human-fatal A/Netherlands/219/03 (H7N7), respectively. The basic polymerase 1 and 2 genes were phylogenetically equidistant to both A/Duck/Denmark/65047/04 (H5N2) and A/Chicken/Netherlands/1/03 (H7N7). The nucleoprotein and matrix gene had highest nucleotide sequence similarity to the H6 subtypes A....../Duck/Hong Kong/3096/99 (H6N2) and A/WDk/ST/1737/2000 (H6N8), respectively. All genes of the H5N7 strain were of avian origin, and no further evidence of pathogenicity to humans has been found....

  18. Highly pathogenic avian influenza virus subtype H5N1 in Africa: a comprehensive phylogenetic analysis and molecular characterization of isolates.

    Directory of Open Access Journals (Sweden)

    Giovanni Cattoli

    Full Text Available Highly pathogenic avian influenza virus A/H5N1 was first officially reported in Africa in early 2006. Since the first outbreak in Nigeria, this virus spread rapidly to other African countries. From its emergence to early 2008, 11 African countries experienced A/H5N1 outbreaks in poultry and human cases were also reported in three of these countries. At present, little is known of the epidemiology and molecular evolution of A/H5N1 viruses in Africa. We have generated 494 full gene sequences from 67 African isolates and applied molecular analysis tools to a total of 1,152 A/H5N1 sequences obtained from viruses isolated in Africa, Europe and the Middle East between 2006 and early 2008. Detailed phylogenetic analyses of the 8 gene viral segments confirmed that 3 distinct sublineages were introduced, which have persisted and spread across the continent over this 2-year period. Additionally, our molecular epidemiological studies highlighted the association between genetic clustering and area of origin in a majority of cases. Molecular signatures unique to strains isolated in selected areas also gave us a clearer picture of the spread of A/H5N1 viruses across the continent. Mutations described as typical of human influenza viruses in the genes coding for internal proteins or associated with host adaptation and increased resistance to antiviral drugs have also been detected in the genes coding for transmembrane proteins. These findings raise concern for the possible human health risk presented by viruses with these genetic properties and highlight the need for increased efforts to monitor the evolution of A/H5N1 viruses across the African continent. They further stress how imperative it is to implement sustainable control strategies to improve animal and public health at a global level.

  19. H5N2 Highly Pathogenic Avian Influenza Viruses from the US 2014-2015 outbreak have an unusually long pre-clinical period in turkeys.

    Science.gov (United States)

    Spackman, Erica; Pantin-Jackwood, Mary J; Kapczynski, Darrell R; Swayne, David E; Suarez, David L

    2016-11-22

    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. Three isolates were selected for characterization in turkeys: the US index isolate from December 2014 (A/northern pintail/WA/40964/2014), and two poultry isolates from April 2015 (A/chicken/IA/13388/2015 and A/turkey/MN/12528/2015). Four week old broad-breasted white turkeys were inoculated with one of three doses (10 2 , 10 4 or 10 6 50% egg infectious doses [EID 50 ] per bird) of each of the isolates to evaluate infectious dose and pathogenesis. The mean bird infectious dose of A/northern pintail/WA/40964/2014 and A/turkey/MN/12528/2015 was 10 5 EID 50 per bird, but was 10 3 EID 50 per bird for A/chicken/IA/13388/2015, suggesting the latter had greater adaptation to gallinaceous birds. All three isolates had unusually long mean death time of 5.3-5.9 days post challenge, and the primary clinical signs were severe lethargy and neurological signs which started no more than 24 h before death (the average pre-clinical period was 4 days). Infected turkeys also shed high levels of virus by both the oropharyngeal and cloacal routes. The unusually long mean death times, high levels of virus in feces, and increased adaptation of the later viruses may have contributed to the rapid spread of the virus during the peak of the outbreak.

  20. 2.1 Natural History of Highly Pathogenic Avian Influenza H5N1

    Science.gov (United States)

    Sonnberg, Stephanie; Webby, Richard J.; Webster, Robert G.

    2013-01-01

    The ecology of highly pathogenic avian influenza (HPAI) H5N1 has significantly changed from sporadic outbreaks in terrestrial poultry to persistent circulation in terrestrial and aquatic poultry and potentially in wild waterfowl. A novel genotype of HPAI H5N1 arose in 1996 in southern China and through ongoing mutation, reassortment, and natural selection, has diverged into distinct lineages and expanded into multiple reservoir hosts. The evolution of Goose/Guangdong-lineage highly pathogenic H5N1 viruses is ongoing: while stable interactions exist with some reservoir hosts, these viruses are continuing to evolve and adapt to others, and pose an un-calculable risk to sporadic hosts, including humans. PMID:23735535

  1. Progress Towards the Rotational Spectrum of H_5^+ and its Isotopologues

    Science.gov (United States)

    McGuire, Brett A.; Wang, Yimin; Bowman, Joel M.; Weaver, Susanna L. Widicus

    2011-06-01

    The reaction of H_3^+ with H_2, arguably the most common bimolecular reaction in the universe, proceeds through the H_5^+ collisional complex. This reaction, and consequently H_5^+, greatly influence the chemical and physical processes in the interstellar medium, playing crucial roles in such varied processes as isotopic fractionation and the formation of complex organic molecules. A thorough understanding of the role of H_5^+ in interstellar chemistry is contingent upon its definitive astronomical detection, necessitating the acquisition of a laboratory rotational spectrum. Rotationally-resolved spectra of H_5^+ in the terahertz region have not yet been observed experimentally. The prediction of this spectrum based on a high-level theoretical study is therefore an important first step to guide experiment. The highly fluxional nature of H_5^+ presents major challenges for theory, especially for the pure rotational spectrum due to the difficulties in determining an accurate dipole moment from a correct description of the highly delocalized zero-point wavefunction. We have now completed this work using the most recent potential energy and dipole moment surfaces for H_5^+ and its isotopologues DH_4^+, D_2H_3^+, D_3H_2^+, D_4H^+, and D_5^+. Pure rotational spectra have been predicted for these species based on the optimized minimum-energy geometries and the zero-point averaged dipole moments calculated from our potential energy surface. We will discuss the implications of these results for the detection of each ion's rotational spectrum, show preliminary predictions of the rotational spectrum for those species possessing permanent dipole moments, and comment on the degree of expected spectral splitting arising from internal motion. Finally, we will report on progress toward the laboratory spectroscopic investigation of these species in the terahertz region.

  2. Progress Towards the Terahertz Rotational Spectrum of H_5^+ and its Isotopologues

    Science.gov (United States)

    McGuire, B. A.; Wang, Y.; Bowman, J.; Widicus Weaver, S. L.

    2011-05-01

    The H_5^+ collisional complex is readily formed from the reaction of H_3^+ with H_2, which is arguably the most common bimolecular reaction in the universe. This reaction, and consequently H_5^+, play critical roles in interstellar chemistry, influencing such varied processes as complex molecule formation and isotopic fractionation. A thorough understanding of the role of H_5^+ on these and other astrochemical processes is contingent upon its successful laboratory and interstellar detection. The experimental spectrum of H_5^+ in the terahertz region is currently not known and thus the theoretical/computational prediction of this spectrum is an important first step to guide experiment. The highly fluxional nature of H_5^+ presents major challenges for theory, especially for the pure rotational spectrum. This is because the dipole moment must be obtained from a correct description of the highly delocalized zero-point wavefunction. This has been done using the most recent potential energy and dipole moment surfaces for H_5^+ and its isotopologues DH_4^+, D_2H_3^+, D_3H_2^+, D_4H^+, and D_5^+. We will present calculated pure rotational spectra for these species using standard simulation codes but with zero-point averaged calculated dipole moments. We will discuss the implications of these results for the detection of the rotational spectrum for each ion, show preliminary predictions of the rotational spectrum for those species possessing permanent dipole moments, and comment on the degree of expected spectral splitting arising from internal motion. Finally, we will report on progress in the construction of a supersonic expansion discharge source coupled with a high-sensitivity cavity ringdown spectrometer to enable laboratory spectroscopic investigation of these species in the terahertz region.

  3. Seasonal patterns in human A (H5N1 virus infection: analysis of global cases.

    Directory of Open Access Journals (Sweden)

    Maya B Mathur

    Full Text Available Human cases of highly pathogenic avian influenza (HPAI A (H5N1 have high mortality. Despite abundant data on seasonal patterns in influenza epidemics, it is unknown whether similar patterns exist for human HPAI H5N1 cases worldwide. Such knowledge could help decrease avian-to-human transmission through increased prevention and control activities during peak periods.We performed a systematic search of published human HPAI H5N1 cases to date, collecting month, year, country, season, hemisphere, and climate data. We used negative binomial regression to predict changes in case incidence as a function of season. To investigate hemisphere as a potential moderator, we used AIC and the likelihood-ratio test to compare the season-only model to nested models including a main effect or interaction with hemisphere. Finally, we visually assessed replication of seasonal patterns across climate groups based on the Köppen-Geiger climate classification.We identified 617 human cases (611 with complete seasonal data occurring in 15 countries in Southeast Asia, Africa, and the Middle East. Case occurrence was much higher in winter (n = 285, p = 0.03 than summer (n = 64, and the winter peak occurred across diverse climate groups. There was no significant interaction between hemisphere and season.Across diverse climates, HPAI H5N1 virus infection in humans increases significantly in winter. This is consistent with increased poultry outbreaks and HPAI H5N1 virus transmission during cold and dry conditions. Prioritizing prevention and control activities among poultry and focusing public health messaging to reduce poultry exposures during winter months may help to reduce zoonotic transmission of HPAI H5N1 virus in resource-limited settings.

  4. Novel Polymerase Gene Mutations for Human Adaptation in Clinical Isolates of Avian H5N1 Influenza Viruses.

    Directory of Open Access Journals (Sweden)

    Yasuha Arai

    2016-04-01

    Full Text Available A major determinant in the change of the avian influenza virus host range to humans is the E627K substitution in the PB2 polymerase protein. However, the polymerase activity of avian influenza viruses with a single PB2-E627K mutation is still lower than that of seasonal human influenza viruses, implying that avian viruses require polymerase mutations in addition to PB2-627K for human adaptation. Here, we used a database search of H5N1 clade 2.2.1 virus sequences with the PB2-627K mutation to identify other polymerase adaptation mutations that have been selected in infected patients. Several of the mutations identified acted cooperatively with PB2-627K to increase viral growth in human airway epithelial cells and mouse lungs. These mutations were in multiple domains of the polymerase complex other than the PB2-627 domain, highlighting a complicated avian-to-human adaptation pathway of avian influenza viruses. Thus, H5N1 viruses could rapidly acquire multiple polymerase mutations that function cooperatively with PB2-627K in infected patients for optimal human adaptation.

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

    Directory of Open Access Journals (Sweden)

    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.

  6. 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.

  7. Contemporary Avian Influenza A Virus Subtype H1, H6, H7, H10, and H15 Hemagglutinin Genes Encode a Mammalian Virulence Factor Similar to the 1918 Pandemic Virus H1 Hemagglutinin

    OpenAIRE

    Qi, Li; Pujanauski, Lindsey M.; Davis, A. Sally; Schwartzman, Louis M.; Chertow, Daniel S.; Baxter, David; Scherler, Kelsey; Hartshorn, Kevan L.; Slemons, Richard D.; Walters, Kathie-Anne; Kash, John C.; Taubenberger, Jeffery K.

    2014-01-01

    ABSTRACT Zoonotic avian influenza virus infections may lead to epidemics or pandemics. The 1918 pandemic influenza virus has an avian influenza virus-like genome, and its H1 hemagglutinin was identified as a key mammalian virulence factor. A chimeric 1918 virus expressing a contemporary avian H1 hemagglutinin, however, displayed murine pathogenicity indistinguishable from that of the 1918 virus. Here, isogenic chimeric avian influenza viruses were constructed on an avian influenza virus backb...

  8. Serosurvey of antibody to highly pathogenic avian influenza (H5N1 ...

    African Journals Online (AJOL)

    Avian influenza is a disease of economic and public health importance that has been described in most domestic animals and humans. Highly pathogenic avian influenza H5N1 epidemic in Nigeria was observed in agro-ecological zones where pigs and chickens are raised in shared environment with chances of ...

  9. Probable Tiger-to-Tiger Transmission of Avian Influenza H5N1

    Science.gov (United States)

    Thanawongnuwech, Roongroje; Amonsin, Alongkorn; Tantilertcharoen, Rachod; Damrongwatanapokin, Sudarat; Theamboonlers, Apiradee; Payungporn, Sunchai; Nanthapornphiphat, Kamonchart; Ratanamungklanon, Somchuan; Tunak, Eakchai; Songserm, Thaweesak; Vivatthanavanich, Veravit; Lekdumrongsak, Thawat; Kesdangsakonwut, Sawang; Tunhikorn, Schwann

    2005-01-01

    During the second outbreak of avian influenza H5N1 in Thailand, probable horizontal transmission among tigers was demonstrated in the tiger zoo. Sequencing and phylogenetic analysis of those viruses showed no differences from the first isolate obtained in January 2004. This finding has implications for influenza virus epidemiology and pathogenicity in mammals. PMID:15890122

  10. Avian Influenza Virus A (H5N1), Detected through Routine Surveillance, in Child, Bangladesh

    Science.gov (United States)

    Alamgir, A.S.M.; Sultana, Rebecca; Islam, M. Saiful; Rahman, Mustafizur; Fry, Alicia M.; Shu, Bo; Lindstrom, Stephen; Nahar, Kamrun; Goswami, Doli; Haider, M. Sabbir; Nahar, Sharifun; Butler, Ebonee; Hancock, Kathy; Donis, Ruben O.; Davis, Charles T.; Zaman, Rashid Uz; Luby, Stephen P.; Uyeki, Timothy M.; Rahman, Mahmudur

    2009-01-01

    We identified avian influenza virus A (H5N1) infection in a child in Bangladesh in 2008 by routine influenza surveillance. The virus was of the same clade and phylogenetic subgroup as that circulating among poultry during the period. This case illustrates the value of routine surveillance for detection of novel influenza virus. PMID:19751601

  11. Novel Eurasian Highly Pathogenic Influenza A H5 Viruses in Wild Birds, Washington, USA, 2014

    Centers for Disease Control (CDC) Podcasts

    2015-03-24

    Sarah Gregory reads an abridged version of the article, Novel Eurasian Highly Pathogenic Influenza A H5 Viruses in Wild Birds, Washington, USA, 2014.  Created: 3/24/2015 by National Center for Emerging and Zoonotic Infectious Diseases (NCEZID).   Date Released: 4/13/2015.

  12. Molecular characteristic and pathogenicity of Indonesian H5N1 clade 2.3.2 viruses

    Directory of Open Access Journals (Sweden)

    Dharmayanti NLPI

    2013-06-01

    Full Text Available The outbreak of disease in late 2012 in Indonesia caused high duck mortality. The agent of the disease was identified as H5N1 clade 2.3.2. The disease caused economic loss to the Indonesian duck farmer. The clade 2.3.2 of H5N1 virus has not previously been identified, so this study was conducted to characterize 4 of H5N1 clade 2.3.2 viruses by DNA sequencing in eight genes segment virus namely HA, NA, NS, M, PB1, PB2, PA and NP. The pathogenicity test of clade 2.3.2 viruses in ducks was compared to clade 2.1.3 viruses which predominat circulating in Indonesia. Results of phylogenetic tree analysis showed that the four of clade 2.3.2 viruses isolated in 2012 was the new introduced virus from abroad. Further analysis showed eight genes were in one group with the clade 2.3.2 viruses, especially those from VietNam and did not belong to Indonesia viruses group. The pathogenicity test in ducks showed that virus H5N1 clade 2.3.2 and clade 2.1.3 have similar clinical symptoms and pathogenicity and cause death in 75% of ducks on days 3-6 after infection.

  13. validering af det noninvasive terumo digital termometer 0800H5- et case control studie

    DEFF Research Database (Denmark)

    Vamosi, Marianne

    2014-01-01

    , Parret T-test, P-værdi og BlandAltman. Statistik programmet R version. 3.0.0 er anvendt. Resultater I dette studie har analysen af Thermofocus 0800H5 og Terumo–digital c405 og vist standard deviasion (SD) på ± 0,46. ANOVA analysen viste en styrke (p værdi) p

  14. Highly pathogenic avian influenza H5N1 in Mainland China

    NARCIS (Netherlands)

    X.-L. Li (Xin-Lou); K. Liu (Kun); H.-W. Yao (Hong-Wu); Y. Sun (Ye); W.-J. Chen (Wan-Jun); R.-X. Sun (Ruo-Xi); S.J. de Vlas (Sake); L.Q. Fang (Lily); W.-C. Cao (Wu-Chun)

    2015-01-01

    textabstractHighly pathogenic avian influenza (HPAI) H5N1 has posed a significant threat to both humans and birds, and it has spanned large geographic areas and various ecological systems throughout Asia, Europe and Africa, but especially in mainland China. Great efforts in control and prevention of

  15. H5N1 avian influenza virus: human cases reported in southern China.

    NARCIS (Netherlands)

    Crofts, J.; Paget, J.; Karcher, F.

    2003-01-01

    Two cases of confirmed influenza due to the avian influenza A H5N1 virus were reported last week in Hong Kong (1). The cases occurred in a Hong Kong family who had recently visited Fujian province in southern China. The daughter, aged 8 years, died following a respiratory illness. The cause of her

  16. Avian influenza H5N1 viral and bird migration networks in Asia

    Science.gov (United States)

    Tian, Huaivu; Zhou, Sen; Dong, Lu; Van Boeckel, Thomas P.; Cui, Yujun; Newman, Scott H.; Takekawa, John Y.; Prosser, Diann J.; Xiao, Xiangming; Wu, Yarong; Cazelles, Bernard; Huang, Shanqian; Yang, Ruifu; Grenfell, Bryan T.; Xu, Bing

    2015-01-01

    The spatial spread of the highly pathogenic avian influenza virus H5N1 and its long-term persistence in Asia have resulted in avian influenza panzootics and enormous economic losses in the poultry sector. However, an understanding of the regional long-distance transmission and seasonal patterns of the virus is still lacking. In this study, we present a phylogeographic approach to reconstruct the viral migration network. We show that within each wild fowl migratory flyway, the timing of H5N1 outbreaks and viral migrations are closely associated, but little viral transmission was observed between the flyways. The bird migration network is shown to better reflect the observed viral gene sequence data than other networks and contributes to seasonal H5N1 epidemics in local regions and its large-scale transmission along flyways. These findings have potentially far-reaching consequences, improving our understanding of how bird migration drives the periodic reemergence of H5N1 in Asia.

  17. Hydrazinium(1+) hexafluorotitanate(IV), 2N2H5+.TiF62-

    International Nuclear Information System (INIS)

    Leban, I.

    1994-01-01

    The crystals exhibit racemic twinning. The structure consists of hydrazinium(1+), N 2 H 5 + , cations and usual octahedral hexafluorotitanate(IV) anions. They are linked together via hydrogen bonds of the types N-H..F and N-H..N. (orig.)

  18. Prevalence of Avian Origin H5 and H7 Influenza Virus Antibodies in ...

    African Journals Online (AJOL)

    As part of ongoing influenza surveillance efforts in livestock and companion animals in Nigeria, a study was conducted to investigate the prevalence of avian H5 and H7 influenza virus antibodies in exotic and Nigerian village dogs in Ibadan and Sagamu, two cities in Oyo and Ogun states respectively. One hundred and ...

  19. Influenza A H5N1 and HIV co-infection: case report

    Directory of Open Access Journals (Sweden)

    Simmons Cameron

    2010-06-01

    Full Text Available Abstract Background The role of adaptive immunity in severe influenza is poorly understood. The occurrence of influenza A/H5N1 in a patient with HIV provided a rare opportunity to investigate this. Case Presentation A 30-year-old male was admitted on day 4 of influenza-like-illness with tachycardia, tachypnea, hypoxemia and bilateral pulmonary infiltrates. Influenza A/H5N1 and HIV tests were positive and the patient was treated with Oseltamivir and broad-spectrum antibiotics. Initially his condition improved coinciding with virus clearance by day 6. He clinically deteriorated as of day 10 with fever recrudescence and increasing neutrophil counts and died on day 16. His admission CD4 count was 100/μl and decreased until virus was cleared. CD8 T cells shifted to a CD27+CD28- phenotype. Plasma chemokine and cytokine levels were similar to those found previously in fatal H5N1. Conclusions The course of H5N1 infection was not notably different from other cases. Virus was cleared despite profound CD4 T cell depletion and aberrant CD8 T cell activation but this may have increased susceptibility to a fatal secondary infection.

  20. Silent spread of highly pathogenic Avian Influenza H5N1 virus amongst vaccinated commercial layers

    NARCIS (Netherlands)

    Poetri, O.N.; Boven, M.; Claassen, I.J.T.M.; Koch, G.; Wibawan, I.W.; Stegeman, A.; Broek, van den J.; Bouma, A.

    2014-01-01

    The aim of this study was to determine whether a single vaccination of commercial layer type chickens with an inactivated vaccine containing highly pathogenic avian influenza virus strain H5N1 A/chicken/Legok/2003, carried out on the farm, was sufficient to protect against infection with the

  1. Non-Attenuation Of Highly Pathogenic Avian Influenza H5N1 By ...

    African Journals Online (AJOL)

    Avian influenza H5N1 represents one of the most researched viruses in laboratories world-wide in recent times with regards to its epidemiology, ecology, biology and geography. The virus has caused 409 human cases and 256 human fatalities to date. Some laboratory activities and other lab related works predispose ...

  2. Human influenza A (H5N1): a brief review and recommendations for travelers.

    Science.gov (United States)

    Hurtado, Timothy R

    2006-01-01

    Although avian influenza A (H5N1) is common in birds worldwide, it has only recently led to disease in humans. Humans who are infected with the disease (referred to as human influenza A [H5N1]) have a greater than 50% mortality rate. Currently there has not been documented sustained human-to-human transmission; however, should the virus mutate and make this possible, the world could experience an influenza pandemic. Probable risk factors for infection include slaughtering, defeathering, and butchering fowl; close contact with wild birds or caged poultry; ingestion of undercooked poultry products; direct contact with surfaces contaminated with poultry feces; and close contact with infected humans. Possible risk factors include swimming in or ingesting water contaminated with bird feces or dead birds and the use of unprocessed poultry feces as fertilizer. Clinically, early human influenza A (H5N1) resembles typical influenza illnesses, with fever and a preponderance of lower respiratory tract symptoms. Often, patients develop rapidly progressive respiratory failure and require ventilatory support. Treatment is primarily supportive care with the addition of antiviral medications. Currently, travelers to countries with both human and avian influenza A (H5N1) have a low risk of developing the disease. There are no current recommended travel restrictions. Travelers are advised to avoid contact with all birds, especially poultry; avoid surfaces contaminated with poultry feces; and avoid undercooked poultry products. The use of prophylactic antiviral medications is not recommended.

  3. Avian influenza virus (H5N1): a threat to human health

    NARCIS (Netherlands)

    Peiris, J. S. Malik; de Jong, Menno D.; Guan, Yi

    2007-01-01

    Pandemic influenza virus has its origins in avian influenza viruses. The highly pathogenic avian influenza virus subtype H5N1 is already panzootic in poultry, with attendant economic consequences. It continues to cross species barriers to infect humans and other mammals, often with fatal outcomes.

  4. Human infection with highly pathogenic H5N1 influenza virus

    NARCIS (Netherlands)

    Gambotto, Andrea; Barratt-Boyes, Simon M.; de Jong, Menno D.; Neumann, Gabriele; Kawaoka, Yoshihiro

    2008-01-01

    Highly pathogenic H5N1 influenza A viruses have spread relentlessly across the globe since 2003, and they are associated with widespread death in poultry, substantial economic loss to farmers, and reported infections of more than 300 people with a mortality rate of 60%. The high pathogenicity of

  5. Advances and Future Challenges in Recombinant Adenoviral Vectored H5N1 Influenza Vaccines

    Directory of Open Access Journals (Sweden)

    Jianfeng Zhang

    2012-11-01

    Full Text Available The emergence of a highly pathogenic avian influenza virus H5N1 has increased the potential for a new pandemic to occur. This event highlights the necessity for developing a new generation of influenza vaccines to counteract influenza disease. These vaccines must be manufactured for mass immunization of humans in a timely manner. Poultry should be included in this policy, since persistent infected flocks are the major source of avian influenza for human infections. Recombinant adenoviral vectored H5N1 vaccines are an attractive alternative to the currently licensed influenza vaccines. This class of vaccines induces a broadly protective immunity against antigenically distinct H5N1, can be manufactured rapidly, and may allow mass immunization of human and poultry. Recombinant adenoviral vectors derived from both human and non-human adenoviruses are currently being investigated and appear promising both in nonclinical and clinical studies. This review will highlight the current status of various adenoviral vectored H5N1 vaccines and will outline novel approaches for the future.

  6. H5N1 surveillance in migratory birds in Java, Indonesia.

    Science.gov (United States)

    Stoops, Arthur C; Barbara, Katie A; Indrawan, Mochamad; Ibrahim, Ima N; Petrus, Wicaksana B; Wijaya, Susan; Farzeli, Arik; Antonjaya, Ungke; Sin, Lim W; Hidayatullah, N; Kristanto, Ige; Tampubolon, A M; Purnama, S; Supriatna, Adam; Burgess, Timothy H; Williams, Maya; Putnam, Shannon D; Tobias, Steve; Blair, Patrick J

    2009-12-01

    We sought to elucidate the role of migratory birds in transmission of H5N1 in an enzoonotic area. Resident, captive, and migratory birds were sampled at five sites in Java, Indonesia. Mist nets were used to trap birds. Birds were identified to species. RNA was extracted from swabs and reverse transcriptase polymerase chain reaction (RT-PCR) conducted for the HA and M genes of H5N1. Antibodies were detected by enzyme-linked immunosorbent assay and hemagglutination inhibition test. Between October 2006 and September 2007, a total of 4,067 captive, resident, and migratory birds comprising 98 species in 23 genera were sampled. The most commonly collected birds were the common sandpiper (6% of total), striated heron (3%), and the domestic chicken (14%). The overall prevalence of H5N1 antibodies was 5.3%. A significantly higher percentage of captive birds (16.1%) showed antibody evidence of H5N1 exposure when compared to migratory or resident birds. The greatest number of seropositive birds in each category were Muschovy duck (captive), striated heron (resident), and the Pacific golden plover (migratory). Seven apparently well captive birds yielded molecular evidence of H5N1 infection. Following amplification, the HA, NA, and M genes were analyzed. Phylogenetic analysis of the HA gene showed that the isolates were 97% similar to EU124153.1 A/chicken/West Java/Garut May 2006, an isolate obtained in a similar region of West Java. While no known markers of neuraminidase inhibitor resistance were found within the NA gene, M segment analysis revealed the V27A mutation known to confer resistance to adamantanes. Our results demonstrate moderate serologic evidence of H5N1 infection in captive birds, sampled in five sites in Java, Indonesia, but only occasional infection in resident and migratory birds. These data imply that in an enzoonotic region of Indonesia the role of migratory birds in transmission of H5N1 is limited.

  7. Genetic drift evolution under vaccination pressure among H5N1 Egyptian isolates

    Directory of Open Access Journals (Sweden)

    Afifi Manal A

    2011-06-01

    Full Text Available Background The highly pathogenic H5N1 is a major avian pathogen that intensively affects the poultry industry in Egypt even in spite of the adoption of vaccination strategy. Antigenic drift is among the strategies the influenza virus uses to escape the immune system that might develop due to the pressure of extensive vaccination. H5N1 mutates in an intensified manner and is considered a potential candidate for the possible next pandemic with all the catastrophic consequences such an eventuality will entail. Methods H5N1 was isolated from the pooled organ samples of four different affected flocks in specific pathogen free embryonated chicken eggs (SPF-ECE. A reverse transcriptase polymerase chain reaction (RT-PCR was performed to the haemagglutingin and neuraminidase. Sequencing of the full length haemagglutingin was performed. Sequence analyses of the isolated strains were performed and compared to all available H5N1 from Egyptian human and avian strains in the flu database. Changes in the different amino acid that may be related to virus virulence, receptor affinity and epitope configuration were assigned and matched with all available Egyptian strains in the flu database. Results One out of the four strains was found to be related to the B2 Egyptian lineage, 2 were related to A1 lineage and the 4th was related to A2 lineage. Comparing data obtained from the current study by other available Egyptian H5N1 sequences remarkably demonstrates that amino acid changes in the immune escape variants are remarkably restricted to a limited number of locations on the HA molecule during antigenic drift. Molecular diversity in the HA gene, in relevance to different epitopes, were not found to follow a regular trend, suggesting abrupt cumulative sequence mutations. However a number of amino acids were found to be subjected to high mutation pressure. Conclusion The current data provides a comprehensive view of HA gene evolution among H5N1 subtype viruses in

  8. Highly pathogenic influenza A(H5N1 virus survival in complex artificial aquatic biotopes.

    Directory of Open Access Journals (Sweden)

    Viseth Srey Horm

    Full Text Available BACKGROUND: Very little is known regarding the persistence of Highly Pathogenic Avian Influenza (HPAI H5N1 viruses in aquatic environments in tropical countries, although environmental materials have been suggested to play a role as reservoirs and sources of transmission for H5N1 viruses. METHODOLOGY/PRINCIPAL FINDINGS: The survival of HPAI H5N1 viruses in experimental aquatic biotopes (water, mud, aquatic flora and fauna relevant to field conditions in Cambodia was investigated. Artificial aquatic biotopes, including simple ones containing only mud and water, and complex biotopes involving the presence of aquatic flora and fauna, were set up. They were experimentally contaminated with H5N1 virus. The persistence of HPAI H5N1 virus (local avian and human isolates was determined by virus isolation in embryonated chicken eggs and by real-time reverse-polymerase chain reaction. Persistence of infectious virus did not exceed 4 days, and was only identified in rain water. No infectious virus particles were detected in pond and lake water or mud even when high inoculum doses were used. However, viral RNA persisted up to 20 days in rain water and 7 days in pond or lake water. Viral RNA was also detected in mud samples, up to 14 days post-contamination in several cases. Infectious virus and viral RNA was detected in few cases in the aquatic fauna and flora, especially in bivalves and labyrinth fish, although these organisms seemed to be mostly passive carriers of the virus rather than host allowing virus replication. CONCLUSIONS/SIGNIFICANCE: Although several factors for the survival and persistence of HPAI viruses in the environment are still to be elucidated, and are particularly hard to control in laboratory conditions, our results, along with previous data, support the idea that environmental surveillance is of major relevance for avian influenza control programs.

  9. Avian influenza virus (H5N1; effects of physico-chemical factors on its survival

    Directory of Open Access Journals (Sweden)

    Hameed Sajid

    2009-03-01

    Full Text Available Abstract Present study was performed to determine the effects of physical and chemical agents on infective potential of highly pathogenic avian influenza (HPAI H5N1 (local strain virus recently isolated in Pakistan during 2006 outbreak. H5N1 virus having titer 108.3 ELD50/ml was mixed with sterilized peptone water to get final dilution of 4HA units and then exposed to physical (temperature, pH and ultraviolet light and chemical (formalin, phenol crystals, iodine crystals, CID 20, virkon®-S, zeptin 10%, KEPCIDE 300, KEPCIDE 400, lifebuoy, surf excel and caustic soda agents. Harvested amnio-allantoic fluid (AAF from embryonated chicken eggs inoculated with H5N1 treated virus (0.2 ml/egg was subjected to haemagglutination (HA and haemagglutination inhibition (HI tests. H5N1 virus lost infectivity after 30 min at 56°C, after 1 day at 28°C but remained viable for more than 100 days at 4°C. Acidic pH (1, 3 and basic pH (11, 13 were virucidal after 6 h contact time; however virus retained infectivity at pH 5 (18 h, 7 and 9 (more than 24 h. UV light was proved ineffectual in inactivating virus completely even after 60 min. Soap (lifebuoy®, detergent (surf excel® and alkali (caustic soda destroyed infectivity after 5 min at 0.1, 0.2 and 0.3% dilution. All commercially available disinfectants inactivated virus at recommended concentrations. Results of present study would be helpful in implementing bio-security measures at farms/hatcheries levels in the wake of avian influenza virus (AIV outbreak.

  10. Public health concerns of highly pathogenic avian influenza H5N1 endemicity in Africa

    Directory of Open Access Journals (Sweden)

    Olubunmi Gabriel Fasanmi

    2017-10-01

    Full Text Available Highly pathogenic avian influenza virus (HPAIV H5N1 was first officially reported in Africa in 2006; thereafter this virus has spread rapidly from Nigeria to 11 other African countries. This study was aimed at utilizing data from confirmed laboratory reports to carry out a qualitative evaluation of the factors responsible for HPAI H5N1 persistence in Africa and the public health implications; and to suggest appropriate control measures. Relevant publications were sought from data banks and repositories of FAO, OIE, WHO, and Google scholars. Substantiated data on HPAI H5N1 outbreaks in poultry in Africa and in humans across the world were mined. HPAI H5N1 affects poultry and human populations, with Egypt having highest human cases (346 globally. Nigeria had a reinfection from 2014 to 2015, with outbreaks in Cote d'Ivoire, Ghana, Niger, Nigeria, and Burkina Faso throughout 2016 unabated. The persistence of this virus in Africa is attributed to the survivability of HPAIV, ability to evolve other subtypes through genetic reassortment, poor biosecurity compliance at the live bird markets and poultry farms, husbandry methods and multispecies livestock farming, poultry vaccinations, and continuous shedding of HPAIV, transboundary transmission of HPAIV through poultry trades; and transcontinental migratory birds. There is, therefore, the need for African nations to realistically reassess their status, through regular surveillance and be transparent with HPAI H5N1 outbreak data. Also, it is important to have an understanding of HPAIV migration dynamics which will be helpful in epidemiological modeling, disease prevention, control and eradication measures.

  11. Features of pathology in mice experimentally infected with highly pathogenic H5N1 influenza virus

    International Nuclear Information System (INIS)

    Ryabchikova, E. I.; Taranov, O. S.; Malkova, E. M.; Gritsyk, O. B.; Demina, O. K.

    2009-01-01

    Avian influenza became a new threat and has set people thinking about possibility of new influenza pandemic which may be caused by highly pathogenic H5N1 influenza virus. The virus could acquire ability of fast spreading between the humans and new pandemics could kill millions. Influenza virus H5N1 exhibited its deadly essence by taking out many millions of birds in nature and aviculture; other millions of chicks and ducks were killed to prevent spread of the epizootic. The strains isolated in Russia belong to Qinghai group of H5N1 influenza virus, and were imported to Russia by migratory birds. We examined time-course changes in mice blood and lungs after intranasal infection with strains A /Chicken/ Kurgan/ 05/2005, A/ Duck/ Kurgan/08/ 2005 and A/ Chicken/ Suzdalka/ Nov-11/2005 differing in virulence for this animal species. Development of leucopenia and severe damage of hemopoiesis were found in mice infected with all H5N1 influenza virus strains. Pathological changes in mice lungs during the infection with above mentioned strains, and strain-specific features have been examined. Main characteristics of lung pathology in all mice were focal nature of the alterations, severe damage of bronchial epithelium and pronounced alteration of lung vasculature. Strain A/Chicken/Suzdalka/Nov-11/2005 induced massive apoptosis of infected bronchial cells which may be a part of mechanism responsible for avirulent properties of this strain. The most interesting finding was absence of serious direct virus damage of the lung evidencing for principal role of the host humoral mechanisms in pathogenesis of H5N1 influenza in mice.(author)

  12. Novel Highly Pathogenic Avian Influenza A(H5N6) Virus in the Netherlands, December 2017.

    Science.gov (United States)

    Beerens, Nancy; Koch, Guus; Heutink, Rene; Harders, Frank; Vries, D P Edwin; Ho, Cynthia; Bossers, Alex; Elbers, Armin

    2018-04-17

    A novel highly pathogenic avian influenza A(H5N6) virus affecting wild birds and commercial poultry was detected in the Netherlands in December 2017. Phylogenetic analysis demonstrated that the virus is a reassortant of H5N8 clade 2.3.4.4 viruses and not related to the Asian H5N6 viruses that caused human infections.

  13. 75 FR 69046 - Notice of Determination of the High Pathogenic Avian Influenza Subtype H5N1 Status of Czech...

    Science.gov (United States)

    2010-11-10

    ... Avian Influenza Subtype H5N1 Status of Czech Republic and Sweden AGENCY: Animal and Plant Health... the highly pathogenic avian influenza (HPAI) subtype H5N1 status of the Czech Republic and Sweden... status of the Czech Republic and Sweden relative to highly pathogenic avian influenza (HPAI) subtype H5N1...

  14. 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.

  15. The role of carbohydrate in determining the immunochemical properties of the hemagglutinin of influenza A virus

    International Nuclear Information System (INIS)

    Gitelman, A.K.; Berezin, V.A.; Kharitonenkov, I.G.

    1981-01-01

    Most of the carbohydrate was removed from influenza with MRC II (H3N2) and its purified hemagglutinin (HA) on treatment with glycosidases, including α-mannosidase, #betta#-N-acetylglucosaminidase, #betta#-galactosidase and α-fucosidase. The release of 50 per cent of the carbohydrate from intact virus particles significantly affected hemagglutinating activity. The ability of untreated and glycosidase-treated virus to inhibit the binding of antibodies directed against the hemagglutinin was almost indistinguishable by competitive radioimmunoassay (RIA). Up to 60 per cent of the carbohydrate from the purified HA of influenza virus could be removed. The antigenicity of glycosidase treated HA molecules decreased 8-fold compared to intact HAs as measured by competitive RIA. In addition, glycosidase digestion of 125 I-labeled HA resulted in a decrease in its reactivity in direct RIA. We conclude that the carbohydrate portion of the HA of influenza virus is not of major importance in defining the antigenicity of HA. (Author)

  16. Evolution of highly pathogenic avian H5N1 influenza viruses

    Energy Technology Data Exchange (ETDEWEB)

    Macken, Catherine A [Los Alamos National Laboratory; Green, Margaret A [Los Alamos National Laboratory

    2009-01-01

    Highly pathogenic avian H5N1 viruses have circulated in Southeast Asia for more than a decade, are now endemic in parts of this region, and have also spread to more than 60 countries on three continents. The evolution of these viruses is characterized by frequent reassortment events that have created a significant number of different genotypes, both transient and longer lasting. However, fundamental questions remain about the generation and perpetuation of this substantial genetic diversity. These gaps in understanding may, in part, be due to the difficulties of genotyping closely related viruses, and limitations in the size of the data sets used in analysis. Using our recently published novel genotyping procedure ('two-time test'), which is amenable to high throughput analysis and provides an increased level of resolution relative to previous analyses, we propose a detailed model for the evolution and diversification of avian H5N1 viruses. Our analysis suggests that (i) all current H5N1 genotypes are derived from a single, clearly defined sequence of initial reassortment events; (ii) reassortment of the polymerase and NP genes may have played an important role in avian H5N1 virus evolution; (iii) the current genotype Z viruses have diverged into three distinguishable sub-genotypes in the absence of reassortment; (iv) some potentially significant molecular changes appear to be correlated with particular genotypes (for example, reassortment of the internal genes is often paralleled by a change in the HA clade); and (v) as noted in earlier studies of avian influenza A virus evolution, novel segments are typically derived from different donors (i.e., there is no obvious pattern of gene linkage in reassortment). The model of avian H5N1 viral evolution by reassortment and mutation that emerges from our study provides a context within which significant amino acid changes may be revealed; it also may help in predicting the 'success' of newly emerging

  17. Ecological Determinants of Highly Pathogenic Avian Influenza (H5N1) Outbreaks in Bangladesh

    Science.gov (United States)

    Ahmed, Syed S. U.; Ersbøll, Annette K.; Biswas, Paritosh K.; Christensen, Jens P.; Hannan, Abu S. M. A.; Toft, Nils

    2012-01-01

    Background The agro-ecology and poultry husbandry of the south Asian and south-east Asian countries share common features, however, with noticeable differences. Hence, the ecological determinants associated with risk of highly pathogenic avian influenza (HPAI-H5N1) outbreaks are expected to differ between Bangladesh and e.g., Thailand and Vietnam. The primary aim of the current study was to establish ecological determinants associated with the risk of HPAI-H5N1 outbreaks at subdistrict level in Bangladesh. The secondary aim was to explore the performance of two different statistical modeling approaches for unmeasured spatially correlated variation. Methodology/Principal Findings An ecological study at subdistrict level in Bangladesh was performed with 138 subdistricts with HPAI-H5N1 outbreaks during 2007–2008, and 326 subdistricts with no outbreaks. The association between ecological determinants and HPAI-H5N1 outbreaks was examined using a generalized linear mixed model. Spatial clustering of the ecological data was modeled using 1) an intrinsic conditional autoregressive (ICAR) model at subdistrict level considering their first order neighbors, and 2) a multilevel (ML) model with subdistricts nested within districts. Ecological determinants significantly associated with risk of HPAI-H5N1 outbreaks at subdistrict level were migratory birds' staging areas, river network, household density, literacy rate, poultry density, live bird markets, and highway network. Predictive risk maps were derived based on the resulting models. The resulting models indicate that the ML model absorbed some of the covariate effect of the ICAR model because of the neighbor structure implied in the two different models. Conclusions/Significance The study identified a new set of ecological determinants related to river networks, migratory birds' staging areas and literacy rate in addition to already known risk factors, and clarified that the generalized concept of free grazing duck and

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

    Science.gov (United States)

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

    2017-06-15

    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 chimeric hemagglutinins, consisting of globular head domains from exotic influenza A viruses and stalk domains from influenza B viruses. Sequential vaccination with these constructs in mice leads to the induction of broadly reactive antibodies that bind to the conserved stalk domain of influenza B virus hemagglutinin. Vaccinated mice are protected from lethal challenge with diverse influenza B viruses. Results from serum transfer experiments and antibody-dependent cell-mediated cytotoxicity (ADCC) assays indicate that this protection is antibody mediated and based on Fc effector functions. The present data suggest that chimeric hemagglutinin-based vaccination is a viable strategy to broadly protect against influenza B virus infection. IMPORTANCE While current influenza virus vaccines are effective, they are affected by mismatches between vaccine strains and circulating strains. Furthermore, the antiviral drug oseltamivir is less effective for treating influenza B virus infections than for treating influenza A virus infections. A vaccine that induces broad and long-lasting protection against influenza B viruses is therefore urgently needed. Copyright © 2017 American Society for Microbiology.

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

    Science.gov (United States)

    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.

  20. Histatin 5 binds to Porphyromonas gingivalis hemagglutinin B (HagB) and alters HagB-induced chemokine responses

    Science.gov (United States)

    Borgwardt, Derek S.; Martin, Aaron D.; van Hemert, Jonathan R.; Yang, Jianyi; Fischer, Carol L.; Recker, Erica N.; Nair, Prashant R.; Vidva, Robinson; Chandrashekaraiah, Shwetha; Progulske-Fox, Ann; Drake, David; Cavanaugh, Joseph E.; Vali, Shireen; Zhang, Yang; Brogden, Kim A.

    2014-01-01

    Histatins are human salivary gland peptides with anti-microbial and anti-inflammatory activities. In this study, we hypothesized that histatin 5 binds to Porphyromonas gingivalis hemagglutinin B (HagB) and attenuates HagB-induced chemokine responses in human myeloid dendritic cells. Histatin 5 bound to immobilized HagB in a surface plasmon resonance (SPR) spectroscopy-based biosensor system. SPR spectroscopy kinetic and equilibrium analyses, protein microarray studies, and I-TASSER structural modeling studies all demonstrated two histatin 5 binding sites on HagB. One site had a stronger affinity with a KD1 of 1.9 μM and one site had a weaker affinity with a KD2 of 60.0 μM. Binding has biological implications and predictive modeling studies and exposure of dendritic cells both demonstrated that 20.0 μM histatin 5 attenuated (p < 0.05) 0.02 μM HagB-induced CCL3/MIP-1α, CCL4/MIP-1β, and TNFα responses. Thus histatin 5 is capable of attenuating chemokine responses, which may help control oral inflammation.

  1. 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.

  2. Fatal H5N6 Avian Influenza Virus Infection in a Domestic Cat and Wild Birds in China.

    Science.gov (United States)

    Yu, Zhijun; Gao, Xiaolong; Wang, Tiecheng; Li, Yanbing; Li, Yongcheng; Xu, Yu; Chu, Dong; Sun, Heting; Wu, Changjiang; Li, Shengnan; Wang, Haijun; Li, Yuanguo; Xia, Zhiping; Lin, Weishi; Qian, Jun; Chen, Hualan; Xia, Xianzhu; Gao, Yuwei

    2015-06-02

    H5N6 avian influenza viruses (AIVs) may pose a potential human risk as suggested by the first documented naturally-acquired human H5N6 virus infection in 2014. Here, we report the first cases of fatal H5N6 avian influenza virus (AIV) infection in a domestic cat and wild birds. These cases followed human H5N6 infections in China and preceded an H5N6 outbreak in chickens. The extensive migration routes of wild birds may contribute to the geographic spread of H5N6 AIVs and pose a risk to humans and susceptible domesticated animals, and the H5N6 AIVs may spread from southern China to northern China by wild birds. Additional surveillance is required to better understand the threat of zoonotic transmission of AIVs.

  3. [Influenza A/H5N1 virus outbreaks and prepardness to avert flu pandemic].

    Science.gov (United States)

    Haque, A; Lucas, B; Hober, D

    2007-01-01

    This review emphasizes the need to improve the knowledge of the biology of H5N1 virus, a candidate for causing the next influenza pandemic. In-depth knowledge of mode of infection, mechanisms of pathogenesis and immune response will help in devising an efficient and practical control strategy against this flu virus. We have discussed limitations of currently available vaccines and proposed novel approaches for making better vaccines against H5N1 influenza virus. They include cell-culture system, reverse genetics, adjuvant development. Our review has also underscored the concept of therapeutic vaccine (anti-disease vaccine), which is aimed at diminishing 'cytokine storm' seen in acute respiratory distress syndrome and/or hemophagocytosis.

  4. Characterization of (/sup 3/H)5-hydroxytryptamine uptake within rat cerebrovascular tree

    Energy Technology Data Exchange (ETDEWEB)

    Amenta, F.; Rossi, M. de; Mione, M.C.; Geppetti, P.

    1985-06-07

    The in vitro uptake of tritiated serotonin ((/sup 3/H)5HT) was studied in a preparation of rat extracerebral arteries. The uptake of (/sup 3/H)5HT was time- and temperature-dependent and of high affinity; linear regression analysis gave a Ksub(m) value of 6.48 X 10/sup 7/ M for the specific uptake. Histoautoradiographic studies showed the highest density of silver grains at the level of the adventitial-medial border of the basilar artery. Fluoxetine inhibited the accumulation of silver grains within the adventitial-medial border in the blood vessel studied. The present data further support the view that a neuronal serotonergic system may play a role in the control of blood flow in the cerebrovascular tree.

  5. Typing of Poultry Influenza Virus (H5 and H7 by Reverse Transcription- Polymerase Chain Reaction

    Directory of Open Access Journals (Sweden)

    Cesare Bonacina

    2010-01-01

    Full Text Available The ability of the influenza Orthomixovirus to undergo to continually antigenically changes that can affect its pathogenicity and its diffusion, explains the growing seriousness of this disease and the recent epizoozies in various parts of the world. There have been 15 HA and 9 NA type A sub-types of the influenza virus identified all of which are present in birds. Until now the very virulent avian influenza viruses identified were all included to the H5 and H7 sub-types. We here show that is possible to identify the H5 and H7 sub-types with reverse transcription-polymerase chain reaction (RT-PCR by using a set of specific primers for each HA sub-type. The RT-PCR is a quick and sensitive method of identifying the HA sub-types of the influenza virus directly from homogenised organs.

  6. Respon Imun Itik Bali terhadap Berbagai Dosis Vaksin Avian Influenza H5N1

    Directory of Open Access Journals (Sweden)

    Ida Bagus Kade Suardana

    2009-09-01

    Full Text Available A study was carried out to investigate the immune response of Bali ducks against various doses ofAvian Influenza H5N1 vaccine. The study was carried out using a complete Random-Split in Time researchdesign as many as 40 of Bali ducks of 3 months age were kept separately in 4 groups. The ducks werevaccinated twice in two week interval with AI H5N1 vaccine of 0 (as negative control, 1/2, 1, and 2 doses.Sera were collected one day before first vaccination, then every week until three weeks after the secondvaccination. All sera were tested by hemaglutination inhibition (HI test. The result shows that antibodylevel with double dose was significantly higher than single dose, half dose, and negative control (P<0.01.However antibody level in ducks vaccinated with single and half dose did not show any significant difference(P > 0.05.

  7. Ecological determinants of highly pathogenic avian influenza (H5N1) outbreaks in Bangladesh

    DEFF Research Database (Denmark)

    Ahmed, Syed Sayeem Uddin; Ersbøll, Annette Kjær; Biswas, Paritosh K.

    2012-01-01

    between Bangladesh and e. g., Thailand and Vietnam. The primary aim of the current study was to establish ecological determinants associated with the risk of HPAI-H5N1 outbreaks at subdistrict level in Bangladesh. The secondary aim was to explore the performance of two different statistical modeling...... approaches for unmeasured spatially correlated variation. Methodology/Principal Findings: An ecological study at subdistrict level in Bangladesh was performed with 138 subdistricts with HPAI-H5N1 outbreaks during 2007-2008, and 326 subdistricts with no outbreaks. The association between ecological...... derived based on the resulting models. The resulting models indicate that the ML model absorbed some of the covariate effect of the ICAR model because of the neighbor structure implied in the two different models. Conclusions/Significance: The study identified a new set of ecological determinants related...

  8. Neutron activation analysis of NBS oyster tissue (SRM 1566) and IAEA animal bone (H-5)

    International Nuclear Information System (INIS)

    Lepel, E.A.; Laul, J.C.

    1983-10-01

    Data have been presented for 35 elements determined by INAA for NBS oyster tissue (SRM 1566) and for 38 elements determined by INAA and RNAA for IAEA animal bone (H-5). The experimental data showed excellent agreement with published values wherever the comparison exists. Additional trace-element data in the ppb range have been presented for the elements Sc, Sb, Cs, La, Ce, Nd, Sm, Eu, Tb, Dy, Ho, Yb, Lu, Hf, Ta, W and Th in NBS oyster tissue. Also, additional trace-element data for IAEA animal bone (H-5) in the ppb range for the elements Al, Sc, Co, Rb, Cs, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Tm, Yb, lu, Hf, Ta and Th have been presented

  9. Quantitative transmission characteristics of different H5 low pathogenic avian influenza viruses in Muscovy ducks.

    Science.gov (United States)

    Niqueux, Éric; Picault, Jean-Paul; Amelot, Michel; Allée, Chantal; Lamandé, Josiane; Guillemoto, Carole; Pierre, Isabelle; Massin, Pascale; Blot, Guillaume; Briand, François-Xavier; Rose, Nicolas; Jestin, Véronique

    2014-01-10

    EU annual serosurveillance programs show that domestic duck flocks have the highest seroprevalence of H5 antibodies, demonstrating the circulation of notifiable avian influenza virus (AIV) according to OIE, likely low pathogenic (LP). Therefore, transmission characteristics of LPAIV within these flocks can help to understand virus circulation and possible risk of propagation. This study aimed at estimating transmission parameters of four H5 LPAIV (three field strains from French poultry and decoy ducks, and one clonal reverse-genetics strain derived from one of the former), using a SIR model to analyze data from experimental infections in SPF Muscovy ducks. The design was set up to accommodate rearing on wood shavings with a low density of 1.6 ducks/m(2): 10 inoculated ducks were housed together with 15 contact-exposed ducks. Infection was monitored by RNA detection on oropharyngeal and cloacal swabs using real-time RT-PCR with a cutoff corresponding to 2-7 EID50. Depending on the strain, the basic reproduction number (R0) varied from 5.5 to 42.7, confirming LPAIV could easily be transmitted to susceptible Muscovy ducks. The lowest R0 estimate was obtained for a H5N3 field strain, due to lower values of transmission rate and duration of infectious period, whereas reverse-genetics derived H5N1 strain had the highest R0. Frequency and intensity of clinical signs were also variable between strains, but apparently not associated with longer infectious periods. Further comparisons of quantitative transmission parameters may help to identify relevant viral genetic markers for early detection of potentially more virulent strains during surveillance of LPAIV. Copyright © 2013 Elsevier B.V. All rights reserved.

  10. Modelling H5N1 in Bangladesh across spatial scales: Model complexity and zoonotic transmission risk

    Directory of Open Access Journals (Sweden)

    Edward M. Hill

    2017-09-01

    Full Text Available Highly pathogenic avian influenza H5N1 remains a persistent public health threat, capable of causing infection in humans with a high mortality rate while simultaneously negatively impacting the livestock industry. A central question is to determine regions that are likely sources of newly emerging influenza strains with pandemic causing potential. A suitable candidate is Bangladesh, being one of the most densely populated countries in the world and having an intensifying farming system. It is therefore vital to establish the key factors, specific to Bangladesh, that enable both continued transmission within poultry and spillover across the human–animal interface. We apply a modelling framework to H5N1 epidemics in the Dhaka region of Bangladesh, occurring from 2007 onwards, that resulted in large outbreaks in the poultry sector and a limited number of confirmed human cases. This model consisted of separate poultry transmission and zoonotic transmission components. Utilising poultry farm spatial and population information a set of competing nested models of varying complexity were fitted to the observed case data, with parameter inference carried out using Bayesian methodology and goodness-of-fit verified by stochastic simulations. For the poultry transmission component, successfully identifying a model of minimal complexity, which enabled the accurate prediction of the size and spatial distribution of cases in H5N1 outbreaks, was found to be dependent on the administration level being analysed. A consistent outcome of non-optimal reporting of infected premises materialised in each poultry epidemic of interest, though across the outbreaks analysed there were substantial differences in the estimated transmission parameters. The zoonotic transmission component found the main contributor to spillover transmission of H5N1 in Bangladesh was found to differ from one poultry epidemic to another. We conclude by discussing possible explanations for

  11. Highly pathogenic avian influenza virus (H5N1) in experimentally infected adult mute swans.

    Science.gov (United States)

    Kalthoff, Donata; Breithaupt, Angele; Teifke, Jens P; Globig, Anja; Harder, Timm; Mettenleiter, Thomas C; Beer, Martin

    2008-08-01

    Adult, healthy mute swans were experimentally infected with highly pathogenic avian influenza virus A/Cygnus cygnus/Germany/R65/2006 subtype H5N1. Immunologically naive birds died, whereas animals with preexisting, naturally acquired avian influenza virus-specific antibodies became infected asymptomatically and shed virus. Adult mute swans are highly susceptible, excrete virus, and can be clinically protected by preexposure immunity.

  12. Influenza A aviária (H5N1: a gripe do frango Avian influenza A (H5N1: the bird flu

    Directory of Open Access Journals (Sweden)

    Cássio da Cunha Ibiapina

    2005-10-01

    Full Text Available Este estudo tem como objetivo rever a literatura sobre o vírus influenza A aviária (H5N1. O levantamento bibliográfico foi realizado nos bancos de dados eletrônicos Medline, MD Consult, HighWire, Medscape e Literatura Latinoamericana y del Caribe en Ciencias de la Salud (LILACS, Literatura Latinoamericana e do Caribe em Ciências da Saúde, e por pesquisa direta, referentes aos últimos dez anos. Foram selecionados 32 artigos originais abordando os surtos recentes de infecção por um subtipo de vírus influenza A aviária, o H5N1, em criações de aves domésticas na Ásia, que resultaram em importantes prejuízos econômicos e repercussões em saúde pública, além de casos de infecção humana de alta letalidade. A maioria dos casos está associada com a exposição direta a aves infectadas ou superfícies contaminadas com excrementos dessas aves, porém foi confirmada a transmissão entre humanos. O período de incubação foi de dois a quatro dias. As manifestações clínicas variaram de infecção assintomática e doença leve do trato respiratório superior a pneumonia grave e falência múltipla de órgãos. A radiografia de tórax pode apresentar infiltrado intersticial bilateral, colapso lobar, consolidação focal e broncograma aéreo sem derrame pleural. A presença de linfopenia indica pior prognóstico. O tratamento de suporte parece ser o único tratamento aceitável. Os fatores de risco para mau prognóstico incluem idade avançada, demora na hospitalização, envolvimento do trato respiratório inferior, baixa contagem de leucócitos totais e linfopenia à admissão. Controlar os surtos em aves domésticas e o contato entre seres humanos e tais aves deve ser a prioridade no manejo da doença em nível de saúde pública, e medidas e conhecimentos acerca da doença devem ser amplamente divulgados.The objective of this study was to review the literature related to avian influenza A (H5N1. The bibliographic research was

  13. Autoradiographic visualization on the role of central 3H-5-hydroxytryptamine in acupuncture analgesia

    International Nuclear Information System (INIS)

    Zhu Shoupeng; Shi Fuxi; Liu Zhonghao; Jiang Ji

    1990-10-01

    The role played by central 5-hydroxytryptamine (5-HT) in electroacupuncture analgesia has been studied in rats by means of autoradiography with isotopic tracers 3 H-5-HT. The purpose of the study is to determine the localization of 3 H-5-HT in the midbrain raphe nuclei and in the mesencephalon aquaeduct as well as periaquaeductal gray matter. Parallel experiments were studied by freezing microautoradiographic method and histo-fixative microautoradiographic method. The analgesic effect of acupuncture can be enhanced or lowered by the increment or the decrement of the 5-HT level in the midbrain raphe nuclei and in the mesencephalon aquaeduct as well as periaquaeductal gray matter. The results show that when the rats were subjected to electro-acupuncture analgesia, the microautoradiographic intensities of 3 H-5-HT both in the midbrain raphe nuclei and in the areas of mesencephalon aqueduct were significantly increased. It may be observed that the release of 5-HT in these regions of the brain is accelerated during acupuncture analgesia. From this it can be concluded that the midbrain raphe nuclei and the mesencephalon aquaeduct as well as the periaquaeductal gray matter are closely related to acupuncture analgesia. The results imply that 5-HT in these areas may be one of the most important neurochemical agents mediating acupuncture analgesia

  14. Risk factors and clusters of Highly Pathogenic Avian Influenza H5N1 outbreaks in Bangladesh

    Science.gov (United States)

    Loth, Leo; Gilbert, Marius; Osmani, Mozaffar G.; Kalam, Abul M.; Xiao, Xiangming

    2016-01-01

    Between March 2007 and July 2009, 325 Highly Pathogenic Avian Influenza (HPAI, subtype H5N1) outbreaks in poultry were reported in 154 out of a total of 486 sub-districts in Bangladesh. This study analyzed the temporal and spatial patterns of HPAI H5N1 outbreaks and quantified the relationship between several spatial risk factors and HPAI outbreaks in sub-districts in Bangladesh. We assessed spatial autocorrelation and spatial dependence, and identified clustering sub-districts with disease statistically similar to or dissimilar from their neighbors. Three significant risk factors associated to HPAI H5N1 virus outbreaks were identified; the quadratic log-transformation of human population density [humans per square kilometer, P = 0.01, OR 1.15 (95% CI: 1.03–1.28)], the log-transformation of the total commercial poultry population [number of commercial poultry per sub-district, P Bangladesh to target surveillance and to concentrate response efforts in areas where disease is likely to occur. Concentrating response efforts may help to combat HPAI more effectively, reducing the environmental viral load and so reducing the number of disease incidents. PMID:20554337

  15. Photodissociation of C3H5Br and C4H7Br at 234 nm

    International Nuclear Information System (INIS)

    Kim, Hyun Kook; Paul, Dababrata; Hong, Ki Ryong; Cho, Ha Na; Kim, Tae Kyu; Lee, Kyoung Seok

    2012-01-01

    The photodissociation dynamics of cyclopropyl bromide (C-3H 5 Br) and cyclobutyl bromide (C 4 H 7 Br) at 234 nm was investigated. A two-dimensional photofragment ion-imaging technique coupled with a [2+1] resonance enhanced multiphoton ionization scheme was utilized to obtain speed and angular distributions of the nascent Br( 2 P 3/2 ) and Br*( 2 P 1/2 ) atoms. The recoil anisotropies for the Br and Br* channels were measured to be βBr = 0.92 ± 0.03 and βBr* = 1.52 ± 0.04 for C 3 H 5 Br and βBr = 1.10 ± 0.03 and βBr* = 1.49 ± 0.05 for C 4 H 7 Br. The relative quantum yield for Br was found to be ΦBr = 0.13 ± 0.03 and for C 3 H 5 Br and C 4 H 7 Br, respectively. The soft radical limit of the impulsive model adequately modeled the related energy partitioning. The nonadiabatic transition probability from the 3A' and 4A' potential energy surfaces was estimated and discussed

  16. 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.

  17. The Rate Constant for the Reaction H + C2H5 at T = 295 - 150K

    Science.gov (United States)

    Pimentel, Andre S.; Payne, Walter A.; Nesbitt, Fred L.; Cody, Regina J.; Stief, Louis J.

    2004-01-01

    The reaction between the hydrogen atom and the ethyl (C2H3) radical is predicted by photochemical modeling to be the most important loss process for C2H5 radicals in the atmospheres of Jupiter and Saturn. This reaction is also one of the major sources for the methyl radicals in these atmospheres. These two simplest hydrocarbon radicals are the initial species for the synthesis of larger hydrocarbons. Previous measurements of the rate constant for the H + C2H5 reaction varied by a factor of five at room temperature, and some studies showed a dependence upon temperature while others showed no such dependence. In addition, the previous studies were at higher temperatures and generally higher pressures than that needed for use in planetary atmospheric models. The rate constant for the reaction H + C2H5 has been measured directly at T = 150, 202 and 295 K and at P = 1.0 Torr He for all temperatures and additionally at P = 0.5 and 2.0 Torr He at T = 202 K. The measurements were performed in a discharge - fast flow system. The decay of the C2H5 radical in the presence of excess hydrogen was monitored by low-energy electron impact mass spectrometry under pseudo-first order conditions. H atoms and C2H5 radicals were generated rapidly and simultaneously by the reaction of fluorine atoms with H2 and C2H6, respectively. The total rate constant was found to be temperature and pressure independent. The measured total rate constant at each temperature are: k(sub 1)(295K) = (1.02+/-0.24)x10(exp -10), k(sub 1)(202K) = (1.02+/-0.22)x10(exp -10) and k(sub 1)(150K) = (0.93+/-0.21)x10(exp -10), all in units of cu cm/molecule/s. The total rate constant derived from all the combined measurements is k(sub 1) = (l.03+/-0.17)x10(exp -10) cu cm/molecule/s. At room temperature our results are about a factor of two higher than the recommended rate constant and a factor of three lower than the most recently published study.

  18. Genetic characterization of highly pathogenic avian influenza A H5N8 viruses isolated from wild birds in Egypt.

    Science.gov (United States)

    Kandeil, Ahmed; Kayed, Ahmed; Moatasim, Yassmin; Webby, Richard J; McKenzie, Pamela P; Kayali, Ghazi; Ali, Mohamed A

    2017-07-01

    A newly emerged H5N8 influenza virus was isolated from green-winged teal in Egypt during December 2016. In this study, we provide a detailed characterization of full genomes of Egyptian H5N8 viruses and some virological features. Genetic analysis demonstrated that the Egyptian H5N8 viruses are highly pathogenic avian influenza viruses. Phylogenetic analysis revealed that the genome of the Egyptian H5N8 viruses was related to recently characterized reassortant H5N8 viruses of clade 2.3.4.4 isolated from different Eurasian countries. Multiple peculiar mutations were characterized in the Egyptian H5N8 viruses, which probably permits transmission and virulence of these viruses in mammals. The Egyptian H5N8 viruses preferentially bound to avian-like receptors rather than human-like receptors. Also, the Egyptian H5N8 viruses were fully sensitive to amantadine and neuraminidase inhibitors. Chicken sera raised against commercial inactivated avian influenza-H5 vaccines showed no or very low reactivity with the currently characterized H5N8 viruses in agreement with the genetic dissimilarity. Surveillance of avian influenza in waterfowl provides early warning of specific threats to poultry and human health and hence should be continued.

  19. Protection level of AI H5N1 vaccine clade 2.1.3 commercial against AI H5N1 clade 2.3.2 virus from Ducks to SPF chicken in laboratory conditions

    Directory of Open Access Journals (Sweden)

    Indriani R

    2015-03-01

    Full Text Available Highly Pathogenic Avian Influenza (HPAI subtype H5N1 clade 2.3.2 has infected chickens in farms, causing mortality and a decrease in egg production. Vaccination is one of the strategies to control disease of AI subtype H5N1. AI H5N1 clade 2.1.3 vaccine is available commercially. The effectiveness of two vaccines of AI H5N1 clade 2.1.3 (product A and B, and AI H5N1 clade 2.3.2 (Sukoharjo against AI H5N1 clade 2.3.2 (Sukoharjo virus SPF chickens was tested in laboratory. Four groups of SPF chickens were used in this study, there were (1 vaccinated with H5N1 clade 2.1.3 (product A, (2 vaccinated with H5N1 clade 2.1.3 (product B, (3 vaccinated with AI H5N1 clade 2.3.2 and (4 unvaccinated (as a control. Each vaccinated group consisted of 10 chicken except 8 chicken for control group. SPF chicken were vaccinated with 1 dose of vaccine at 3 weeks olds, and then after 3 weeks post vaccination (at 6 weeks olds. All group of chicken were challenged with 106 EID50 per 0.1 ml via intranasal. The results showed, chicken vaccinated with H5N1 clade 2.1.3 product A and B gave 100 and 80% protection respectively, but showed challenged virus shedding, whereas vaccine of H5N1 clade 2.3.2 gave 100% protection from mortality and without virus shedding. Vaccines of AI H5N1 clade 2.1.3 product A was better than vaccine product B, and when chicken vaccinated against H5N1 clade 2.3.2, H5N1 clade 2.3.2 vaccine was the best to be used. In order to protect chicken from AI subtype H5N1 clade 2.1.3 and 2.3.2 in the field, a bivalent vaccine of H5N1 clade 2.1.3 and 2.3.2 subtypes should be developed.

  20. Presence of serum antibodies to influenza A subtypes H5 and N1 in swans and ibises in French wetlands, irrespective of highly pathogenic H5N1 natural infection.

    Science.gov (United States)

    Niqueux, Eric; Guionie, Olivier; Schmitz, Audrey; Hars, Jean; Jestin, Véronique

    2010-03-01

    Highly pathogenic (HP) avian influenza A viruses (AIVs) subtype H5N1 (subclade 2.2) were detected in wild birds during outbreaks in France during winter 2006 and summer 2007 in la Dombes wetlands (eastern France) and in Moselle wetlands (northeastern France), respectively. Blood samples from apparently healthy wild birds were collected in 2006 and 2007 from the end of the outbreak to several weeks after the influenza A outbreak inside and outside the contaminated areas, and in 2008 outside the contaminated areas. The samples were tested for the presence and/or quantitation of serum antibodies to influenza A subtypes H5 and N1 using hemagglutination inhibition tests (HITs), a commercial N1-specific enzyme-linked immunosorbent assay kit, and virus neutralization assay. In the HIT, low pathogenicity (LP) and HP H5 AIVs (belonging to H5N1, H5N2, and H5N3 subtypes) were used as antigens. One hundred mute swans were bled in the la Dombes outbreak area in 2006. During 2007, 46 mallards, 69 common pochards, and 59 mute swans were sampled in the Moselle outbreak area. For comparison, blood samples were also collected in 2007 from 60 mute swans from the Marne department where no HP H5N1 influenza A cases have been reported, and in 2008 from 111 sacred ibises in western France where no HP H5N1 influenza A infections in wild birds have been reported either. Mute swans (irrespective of their origin and time of sampling) and sacred ibises (from an area with no known outbreaks) had the highest prevalence of positive sera in the H5 HIT (49-69% and 64%, respectively). The prevalence of anti-H5 antibodies in mallards and common pochards was lower (28% and 27%, respectively). Positive H5- and N1-antibody responses were also significantly associated in swans (irrespective of their origin and time of sampling) and in sacred ibises. However, in swans from the area without outbreaks, the HIT titer against an H5N1 LPAIV was significantly higher than against an H5N1 2.2.1 HPAIV, whereas no

  1. An enzyme-linked immunosorbent assay for detection of avian influenza virus subtypes H5 and H7 antibodies

    DEFF Research Database (Denmark)

    Jensen, Trine Hammer; Ajjouri, Gitte; Handberg, Kurt

    2013-01-01

    subtypes H5 and H7. The ELISAs were evaluated with polyclonal chicken anti-AIV antibodies against AIV subtypes: H1N2, H5N2, H5N7, H7N1, H7N7, H9N9, H10N4 and H16N3. RESULTS: Both the H5 and H7 ELISA proved to have a high sensitivity and specificity and the ELISAs detected H5 and H7 antibodies earlier......BACKGROUND: Avian influenza virus (AIV) subtypes H5 and H7 attracts particular attention because of the risk of their potential pathogenicity in poultry. The haemagglutination inhibition (HI) test is widely used as subtype specific test for serological diagnostics despite the laborious nature...

  2. Smartphone-Based Fluorescent Diagnostic System for Highly Pathogenic H5N1 Viruses.

    Science.gov (United States)

    Yeo, Seon-Ju; Choi, Kyunghan; Cuc, Bui Thi; Hong, Nguyen Ngoc; Bao, Duong Tuan; Ngoc, Nguyen Minh; Le, Mai Quynh; Hang, Nguyen Le Khanh; Thach, Nguyen Co; Mallik, Shyam Kumar; Kim, Hak Sung; Chong, Chom-Kyu; Choi, Hak Soo; Sung, Haan Woo; Yu, Kyoungsik; Park, Hyun

    2016-01-01

    Field diagnostic tools for avian influenza (AI) are indispensable for the prevention and controlled management of highly pathogenic AI-related diseases. More accurate, faster and networked on-site monitoring is demanded to detect such AI viruses with high sensitivity as well as to maintain up-to-date information about their geographical transmission. In this work, we assessed the clinical and field-level performance of a smartphone-based fluorescent diagnostic device with an efficient reflective light collection module using a coumarin-derived dendrimer-based fluorescent lateral flow immunoassay. By application of an optimized bioconjugate, a smartphone-based diagnostic device had a two-fold higher detectability as compared to that of the table-top fluorescence strip reader for three different AI subtypes (H5N3, H7N1, and H9N2). Additionally, in a clinical study of H5N1-confirmed patients, the smartphone-based diagnostic device showed a sensitivity of 96.55% (28/29) [95% confidence interval (CI): 82.24 to 99.91] and a specificity of 98.55% (68/69) (95% CI: 92.19 to 99.96). The measurement results from the distributed individual smartphones were wirelessly transmitted via short messaging service and collected by a centralized database system for further information processing and data mining. Smartphone-based diagnosis provided highly sensitive measurement results for H5N1 detection within 15 minutes. Because of its high sensitivity, portability and automatic reporting feature, the proposed device will enable agile identification of patients and efficient control of AI dissemination.

  3. Smartphone-Based Fluorescent Diagnostic System for Highly Pathogenic H5N1 Viruses

    Science.gov (United States)

    Yeo, Seon-Ju; Choi, Kyunghan; Cuc, Bui Thi; Hong, Nguyen Ngoc; Bao, Duong Tuan; Ngoc, Nguyen Minh; Le, Mai Quynh; Hang, Nguyen Le Khanh; Thach, Nguyen Co; Mallik, Shyam Kumar; Kim, Hak Sung; Chong, Chom-Kyu; Choi, Hak Soo; Sung, Haan Woo; Yu, Kyoungsik; Park, Hyun

    2016-01-01

    Field diagnostic tools for avian influenza (AI) are indispensable for the prevention and controlled management of highly pathogenic AI-related diseases. More accurate, faster and networked on-site monitoring is demanded to detect such AI viruses with high sensitivity as well as to maintain up-to-date information about their geographical transmission. In this work, we assessed the clinical and field-level performance of a smartphone-based fluorescent diagnostic device with an efficient reflective light collection module using a coumarin-derived dendrimer-based fluorescent lateral flow immunoassay. By application of an optimized bioconjugate, a smartphone-based diagnostic device had a two-fold higher detectability as compared to that of the table-top fluorescence strip reader for three different AI subtypes (H5N3, H7N1, and H9N2). Additionally, in a clinical study of H5N1-confirmed patients, the smartphone-based diagnostic device showed a sensitivity of 96.55% (28/29) [95% confidence interval (CI): 82.24 to 99.91] and a specificity of 98.55% (68/69) (95% CI: 92.19 to 99.96). The measurement results from the distributed individual smartphones were wirelessly transmitted via short messaging service and collected by a centralized database system for further information processing and data mining. Smartphone-based diagnosis provided highly sensitive measurement results for H5N1 detection within 15 minutes. Because of its high sensitivity, portability and automatic reporting feature, the proposed device will enable agile identification of patients and efficient control of AI dissemination. PMID:26877781

  4. EVALUATION OF OIL BASED AVIAN INFLUENZA VACCINE (H5NI PREPARED WITH DIFFERENT CONCENTRATIONS OF ADJUVANT

    Directory of Open Access Journals (Sweden)

    M. IQBAL, M. NISAR, ANWARUL-HAQ, S. NOOR AND Z. J. GILL

    2008-12-01

    Full Text Available Bird flu vaccine from H5N1 strain of avian influenza virus was prepared with two concentrations of adjuvant (Montanide ISA 70MVG. Two vaccines (I and II were prepared containing 50 and 60% Montanide, respectively. Immune response of both the vaccines as single, as well as booster, dose was evaluated in layer birds through haemagglutination inhibition test. Single dose of both vaccines showed poor immune response, while booster dose gave better response with both the vaccines. However, the vaccine prepared with 60% Montanide provided better immune response compared with the vaccine containing 50% montanide.

  5. Neutron activation analysis of NBS oyster tissue (SRM 1566) and IAEA animal bone (H-5)

    International Nuclear Information System (INIS)

    Lepel, E.A.; Laul, J.C.

    1984-03-01

    Instrumental and radiochemical neutron activation analysis (INAA and RNAA) were employed to measure about 37 major, minor, and trace elements in two standard reference materials: oyster tissue (SRM 1566) supplied by the National Bureau of Standards (NBS) and animal bone (H-5) supplied by the International Atomic Energy Agency (IAEA). Wherever the comparison exists, our data show excellent agreement with accepted values for each SRM. These SRM's are useful as reference standards for the analysis of biological materials. Additionally, the chondritic normalized rare earth element pattern of animal bone behaves as a smooth function of the ionic radii, as previously observed for biological materials

  6. Single phase cascaded H5 inverter with leakage current elimination for transformerless photovoltaic system

    DEFF Research Database (Denmark)

    Guo, Xiaoqiang; Jia, X.; Lu, Z.

    2016-01-01

    Leakage current reduction is one of the important issues for the transformelress PV systems. In this paper, the transformerless single-phase cascaded H-bridge PV inverter is investigated. The common mode model for the cascaded H4 inverter is analyzed. And the reason why the conventional cascade H4...... inverter fails to reduce the leakage current is clarified. In order to solve the problem, a new cascaded H5 inverter is proposed to solve the leakage current issue. Finally, the experimental results are presented to verify the effectiveness of the proposed topology with the leakage current reduction...... for the single-phase transformerless PV systems....

  7. Spatial and temporal patterns of global H5N1 outbreaks

    CSIR Research Space (South Africa)

    Si, YL

    2008-07-01

    Full Text Available -96. Walter, C., Mcbratney, A. B., Rossel, R. A. V. and Markus, J. A., 2005. Spatial point-process statistics: concepts and application to the analysis of lead contamination in urban soil. Environmetrics, 16, pp. 339-355. Ward, M. P., Maftei, D., Apostu... OF GLOBAL H5N1 OUTBREAKS Y.L. Si a,c, *, P. Debba b, A. K. Skidmore a, A. G. Toxopeus a, L. Li c a ITC, Department of Natural Resources, 7500AA Enschede, The Netherlands – (yali, skidmore, toxopeus)@itc.nl b Council for Scientific and Industrial...

  8. Spatio-temporal dynamics of global H5N1 outbreaks match bird migration patterns

    CSIR Research Space (South Africa)

    Si, Y

    2009-11-01

    Full Text Available , Chaitaweesub P, Songserm T, Chaisingh A, Hoonsuwan W, Buranathai C, Parakamawongsa T, Premashthira S, Amonsin A, Gilbert M, Nielen M, Stegeman A, 2005. Highly pathogenic avian influenza H5N1, Thailand, 2004. Emerg Infect Dis 11, 1664-1672. Ward MP, Maftei... (Fig. 4IIIa-b), eight clusters were located in western Russia and Kazakhstan. In southeast Asia, clusters were identified in Thailand between July and September (Fig. 4IIIa-c), reappearing in south Y. Si et al. - Geospatial Health 4(1), 2009, pp. 65...

  9. Widespread detection of highly pathogenic H5 influenza viruses in wild birds from the Pacific Flyway of the United States.

    Science.gov (United States)

    Bevins, S N; Dusek, R J; White, C L; Gidlewski, T; Bodenstein, B; Mansfield, K G; DeBruyn, P; Kraege, D; Rowan, E; Gillin, C; Thomas, B; Chandler, S; Baroch, J; Schmit, B; Grady, M J; Miller, R S; Drew, M L; Stopak, S; Zscheile, B; Bennett, J; Sengl, J; Brady, Caroline; Ip, H S; Spackman, E; Killian, M L; Torchetti, M K; Sleeman, J M; Deliberto, T J

    2016-07-06

    A novel highly pathogenic avian influenza virus belonging to the H5 clade 2.3.4.4 variant viruses was detected in North America in late 2014. Motivated by the identification of these viruses in domestic poultry in Canada, an intensive study was initiated to conduct highly pathogenic avian influenza surveillance in wild birds in the Pacific Flyway of the United States. A total of 4,729 hunter-harvested wild birds were sampled and highly pathogenic avian influenza virus was detected in 1.3% (n = 63). Three H5 clade 2.3.4.4 subtypes were isolated from wild birds, H5N2, H5N8, and H5N1, representing the wholly Eurasian lineage H5N8 and two novel reassortant viruses. Testing of 150 additional wild birds during avian morbidity and mortality investigations in Washington yielded 10 (6.7%) additional highly pathogenic avian influenza isolates (H5N8 = 3 and H5N2 = 7). The geographically widespread detection of these viruses in apparently healthy wild waterfowl suggest that the H5 clade 2.3.4.4 variant viruses may behave similarly in this taxonomic group whereby many waterfowl species are susceptible to infection but do not demonstrate obvious clinical disease. Despite these findings in wild waterfowl, mortality has been documented for some wild bird species and losses in US domestic poultry during the first half of 2015 were unprecedented.

  10. Influence of Novel Highly Pathogenic Avian Influenza A (H5N1 Virus Infection on Migrating Whooper Swans Fecal Microbiota

    Directory of Open Access Journals (Sweden)

    Na Zhao

    2018-02-01

    Full Text Available The migration of wild birds plays an important role in the transmission and spread of H5 highly pathogenic avian influenza (HPAI virus, posing a severe risk to animal and human health. Substantial evidence suggests that altered gut microbial community is implicated in the infection of respiratory influenza virus. However, the influence of H5N1 infection in gut microbiota of migratory birds remains unknown. In January 2015, a novel recombinant H5N1 virus emerged and killed about 100 migratory birds, mainly including whooper swans in Sanmenxia Reservoir Area of China. Here, we describe the first fecal microbiome diversity study of H5N1-infected migratory birds. By investigating the influence of H5N1 infection on fecal bacterial communities in infected and uninfected individuals, we found that H5N1 infection shaped the gut microbiota composition by a difference in the dominance of some genera, such as Aeromonas and Lactobacillus. We also found a decreased α diversity and increased β diversity in infectious individuals. Our results highlight that increases in changes in pathogen-containing gut communities occur when individuals become infected with H5N1. Our study may provide the first evidence that there are statistical association among H5N1 presence and fecal microbiota compositional shifts, and properties of the fecal microbiota may serve as the risk of gut-linked disease in migrates with H5N1 and further aggravate the disease transmission.

  11. Influence of Novel Highly Pathogenic Avian Influenza A (H5N1) Virus Infection on Migrating Whooper Swans Fecal Microbiota.

    Science.gov (United States)

    Zhao, Na; Wang, Supen; Li, Hongyi; Liu, Shelan; Li, Meng; Luo, Jing; Su, Wen; He, Hongxuan

    2018-01-01

    The migration of wild birds plays an important role in the transmission and spread of H5 highly pathogenic avian influenza (HPAI) virus, posing a severe risk to animal and human health. Substantial evidence suggests that altered gut microbial community is implicated in the infection of respiratory influenza virus. However, the influence of H5N1 infection in gut microbiota of migratory birds remains unknown. In January 2015, a novel recombinant H5N1 virus emerged and killed about 100 migratory birds, mainly including whooper swans in Sanmenxia Reservoir Area of China. Here, we describe the first fecal microbiome diversity study of H5N1-infected migratory birds. By investigating the influence of H5N1 infection on fecal bacterial communities in infected and uninfected individuals, we found that H5N1 infection shaped the gut microbiota composition by a difference in the dominance of some genera, such as Aeromonas and Lactobacillus . We also found a decreased α diversity and increased β diversity in infectious individuals. Our results highlight that increases in changes in pathogen-containing gut communities occur when individuals become infected with H5N1. Our study may provide the first evidence that there are statistical association among H5N1 presence and fecal microbiota compositional shifts, and properties of the fecal microbiota may serve as the risk of gut-linked disease in migrates with H5N1 and further aggravate the disease transmission.

  12. Fluoxetine-induced inhibition of synaptosomal [3H]5-HT release: Possible Ca2+-channel inhibition

    International Nuclear Information System (INIS)

    Stauderman, K.A.; Gandhi, V.C.; Jones, D.J.

    1992-01-01

    Fluoxetine, a selective 5-Ht uptake inhibitor, inhibited 15 mM K + -induced [ 3 H]5-HT release from rat spinal cord and cortical synaptosomes at concentrations > 0.5 uM. This effect reflected a property shared by another selective 5-HT uptake inhibitor paroxetine but not by less selective uptake inhibitors such as amitriptyline, desipramine, imipramine or nortriptyline. Inhibition of release by fluoxetine was inversely related to both the concentration of K + used to depolarize the synaptosomes and the concentration of external Ca 2+ . Experiments aimed at determining a mechanism of action revealed that fluoxetine did not inhibit voltage-independent release of [ 3 H]5-HT release induced by the Ca 2+ -ionophore A 23187 or Ca 2+ -independent release induced by fenfluramine. Moreover the 5-HT autoreceptor antagonist methiothepin did not reverse the inhibitory actions of fluoxetine on K + -induced release. Further studies examined the effects of fluoxetine on voltage-dependent Ca 2+ channels and Ca 2+ entry

  13. Geohydrologic data for test well USW H-5, Yucca Mountain area, Nye County, Nevada

    International Nuclear Information System (INIS)

    Bentley, C.B.; Robison, J.H.; Spengler, R.W.

    1983-01-01

    This report presents data on drilling operations, lithology, borehold geophysics, water-level monitoring, core analysis, ground-water chemistry, pumping tests, and packer-injection tests for test well USW H-5. The well is one of a series of test wells drilled in and near the southwestern part of the Nevada Test Site, Nye County, Nevada, in cooperation with the US Department of Energy. These test wells are part of the Nevada Nuclear Waste Storage Investigations to identify suitable sites for storage of high-level radioactive wastes. Test well USW H-5 was drilled to a total depth of 1219 meters through volcanic rocks consisting mostly of ash-flow tuff. Depth to water in the well ranged between 703.8 and 707.2 meters below land surface, at an approximate altitude of 704 meters above sea level. Drawdown in the well exceeded 6 meters after test pumping more than 3000 minutes at a rate of 10 liters per second. Borehole-flow surveys showed that about 90 percent of the water in the well is contributed by the zone between 707 and about 820 meters below land surface. Two composite water samples collected after well completion contained 206 and 220 milligrams per liter of dissolved solids. Sodium and bicarbonate were the predominant dissolved anion and cation. The concentration of dissolved silica was 48 milligrams per liter in both samples, which is a relatively large concentration for most natural waters. 6 references, 19 figures, 6 tables

  14. Avian Influenza H5N1 and the Wild Bird Trade in Hanoi, Vietnam

    Directory of Open Access Journals (Sweden)

    F. Brooks-Moizer

    2009-06-01

    Full Text Available Wildlife trade and emerging infectious diseases pose significant threats to human and animal health and global biodiversity. Legal and illegal trade in domestic and wild birds has played a significant role in the global spread of highly pathogenic avian influenza H5N1, which has killed more than 240 people, many millions of poultry, and an unknown number of wild birds and mammals, including endangered species, since 2003. This 2007 study provides evidence for a significant decline in the scale of the wild bird trade in Hanoi since previous surveys in 2000 (39.7% decline and 2003 (74.1% decline. We attribute this to the enforcement of Vietnam's Law 169/2005/QD UBND, introduced in 2005, which prohibits the movement and sale of wild and ornamental birds in cities. Nevertheless, 91.3% (21/23 of bird vendors perceived no risk of H5N1 infection from their birds, and the trade continues, albeit at reduced levels, in open market shops. These findings highlight the importance of continued law enforcement to maintain this trade reduction and the associated benefits to human and animal health and biodiversity conservation.

  15. Geohydrologic data for test well USW H-5, Yucca Mountain area, Nye County, Nevada

    Science.gov (United States)

    Bentley, C.B.; Robison, J.H.; Spengler, R.W.

    1983-01-01

    This report presents data on drilling operations, lithology, borehole geophysics, water-level monitoring, core analysis, ground-water chemistry, pumping tests, and packer-injection tests for test well USW H-5. The well is one of a series of test wells drilled in and near the southwestern part of the Nevada Test Site, Nye County, Nevada, in cooperation with the U.S. Department of Energy. These test wells are part of the Nevada Nuclear Waste Storage Investigations to identify suitable sites for storage of high-level radioactive wastes. Test well USW H-5 was drilled to a total depth of 1,219 meters through volcanic rocks consisting mostly of ash-flow tuff. Depth to water in the well ranged between 703.8 and 707.2 meters below land surface, at an approximate altitude of 704 meters above sea level. Drawdown in the well exceeded 6 meters after test pumping more than 3,000 minutes at a rate of 10 liters per second. Borehole-flow surveys showed that about 90 percent of the water in the well is contributed by the zone between 707 and about 820 meters below land surface. Two composite water samples collected after well completion contained 206 and 220 milligrams per liter of dissolved solids. Sodium and bicarbonate were the predominant dissolved anion and cation. The concentration of dissolved silica was 48 milligrams per liter in both samples, which is a relatively large concentration for most natural waters.

  16. Progress on H5Part: A Portable High Performance Parallel Data Interface for Electromagnetics Simulations

    International Nuclear Information System (INIS)

    Adelmann, Andreas; Gsell, Achim; Oswald, Benedikt; Schietinger, Thomas; Bethel, Wes; Shalf, John; Siegerist, Cristina; Stockinger, Kurt

    2007-01-01

    Significant problems facing all experimental and computational sciences arise from growing data size and complexity. Common to all these problems is the need to perform efficient data I/O on diverse computer architectures. In our scientific application, the largest parallel particle simulations generate vast quantities of six-dimensional data. Such a simulation run produces data for an aggregate data size up to several TB per run. Motivated by the need to address data I/O and access challenges, we have implemented H5Part, an open source data I/O API that simplifies the use of the Hierarchical Data Format v5 library (HDF5). HDF5 is an industry standard for high performance, cross-platform data storage and retrieval that runs on all contemporary architectures from large parallel supercomputers to laptops. H5Part, which is oriented to the needs of the particle physics and cosmology communities, provides support for parallel storage and retrieval of particles, structured and in the future unstructured meshes. In this paper, we describe recent work focusing on I/O support for particles and structured meshes and provide data showing performance on modern supercomputer architectures like the IBM POWER 5

  17. Crystallographic characterization of divalent organosamarium compound (C5H5)2Sm(THF)2

    International Nuclear Information System (INIS)

    Jagannatha Swamy, S.

    2002-01-01

    The single pot reaction between SmX 2 (X = Cl - , I - ) and BuLi in THF at -40 degC, followed by the addition of C 5 H 5 - Na + results in a dark red solution. Leaving the concentrated reaction mixture at -25 degC for two days in a deep freezer results in the formation of the crystals of the compound, (C 5 H 5 ) 2 ; Sm(THF) 2 . The compound is insoluble in any solvent and it has been characterized by conventional methods. The crystals are monoclinic with space group C2/c, and a = 13.416(1), b = 9.644(1), c = 14.129(2) pm, β109.873(9) 0 and z = 4 for ρcalcd = 1.64 g cm -3 . Least squares refinement on the basis of 1804 observed reflections has led to a final R value of 0.037 and R w = 0.054. (author)

  18. Preparation of quadri-subtype influenza virus-like particles using bovine immunodeficiency virus gag protein

    Energy Technology Data Exchange (ETDEWEB)

    Tretyakova, Irina; Hidajat, Rachmat; Hamilton, Garrett; Horn, Noah; Nickols, Brian; Prather, Raphael O. [Medigen, Inc., 8420 Gas House Pike, Suite S, Frederick, MD (United States); Tumpey, Terrence M. [Influenza Division, Centers for Disease Control and Prevention, 1600 Clifton Road N.E., Atlanta, GA (United States); Pushko, Peter, E-mail: ppushko@medigen-usa.com [Medigen, Inc., 8420 Gas House Pike, Suite S, Frederick, MD (United States)

    2016-01-15

    Influenza VLPs comprised of hemagglutinin (HA), neuraminidase (NA), and matrix (M1) proteins have been previously used for immunological and virological studies. Here we demonstrated that influenza VLPs can be made in Sf9 cells by using the bovine immunodeficiency virus gag (Bgag) protein in place of M1. We showed that Bgag can be used to prepare VLPs for several influenza subtypes including H1N1 and H10N8. Furthermore, by using Bgag, we prepared quadri-subtype VLPs, which co-expressed within the VLP the four HA subtypes derived from avian-origin H5N1, H7N9, H9N2 and H10N8 viruses. VLPs showed hemagglutination and neuraminidase activities and reacted with specific antisera. The content and co-localization of each HA subtype within the quadri-subtype VLP were evaluated. Electron microscopy showed that Bgag-based VLPs resembled influenza virions with the diameter of 150–200 nm. This is the first report of quadri-subtype design for influenza VLP and the use of Bgag for influenza VLP preparation. - Highlights: • BIV gag protein was configured as influenza VLP core component. • Recombinant influenza VLPs were prepared in Sf9 cells using baculovirus expression system. • Single- and quadri-subtype VLPs were prepared by using BIV gag as a VLP core. • Co-localization of H5, H7, H9, and H10 HA was confirmed within quadri-subtype VLP. • Content of HA subtypes within quadri-subtype VLP was determined. • Potential advantages of quadri-subtype VLPs as influenza vaccine are discussed.

  19. Preparation of quadri-subtype influenza virus-like particles using bovine immunodeficiency virus gag protein

    International Nuclear Information System (INIS)

    Tretyakova, Irina; Hidajat, Rachmat; Hamilton, Garrett; Horn, Noah; Nickols, Brian; Prather, Raphael O.; Tumpey, Terrence M.; Pushko, Peter

    2016-01-01

    Influenza VLPs comprised of hemagglutinin (HA), neuraminidase (NA), and matrix (M1) proteins have been previously used for immunological and virological studies. Here we demonstrated that influenza VLPs can be made in Sf9 cells by using the bovine immunodeficiency virus gag (Bgag) protein in place of M1. We showed that Bgag can be used to prepare VLPs for several influenza subtypes including H1N1 and H10N8. Furthermore, by using Bgag, we prepared quadri-subtype VLPs, which co-expressed within the VLP the four HA subtypes derived from avian-origin H5N1, H7N9, H9N2 and H10N8 viruses. VLPs showed hemagglutination and neuraminidase activities and reacted with specific antisera. The content and co-localization of each HA subtype within the quadri-subtype VLP were evaluated. Electron microscopy showed that Bgag-based VLPs resembled influenza virions with the diameter of 150–200 nm. This is the first report of quadri-subtype design for influenza VLP and the use of Bgag for influenza VLP preparation. - Highlights: • BIV gag protein was configured as influenza VLP core component. • Recombinant influenza VLPs were prepared in Sf9 cells using baculovirus expression system. • Single- and quadri-subtype VLPs were prepared by using BIV gag as a VLP core. • Co-localization of H5, H7, H9, and H10 HA was confirmed within quadri-subtype VLP. • Content of HA subtypes within quadri-subtype VLP was determined. • Potential advantages of quadri-subtype VLPs as influenza vaccine are discussed.

  20. Highly pathogenic avian influenza (H5N1: pathways of exposure at the animal-human interface, a systematic review.

    Directory of Open Access Journals (Sweden)

    Maria D Van Kerkhove

    Full Text Available BACKGROUND: The threat posed by highly pathogenic avian influenza A H5N1 viruses to humans remains significant, given the continued occurrence of sporadic human cases (499 human cases in 15 countries with a high case fatality rate (approximately 60%, the endemicity in poultry populations in several countries, and the potential for reassortment with the newly emerging 2009 H1N1 pandemic strain. Therefore, we review risk factors for H5N1 infection in humans. METHODS AND FINDINGS: Several epidemiologic studies have evaluated the risk factors associated with increased risk of H5N1 infection among humans who were exposed to H5N1 viruses. Our review shows that most H5N1 cases are attributed to exposure to sick poultry. Most cases are sporadic, while occasional limited human-to-human transmission occurs. The most commonly identified factors associated with H5N1 virus infection included exposure through contact with infected blood or bodily fluids of infected poultry via food preparation practices; touching and caring for infected poultry; [corrected] exposure to H5N1 via swimming or bathing in potentially virus laden ponds; and exposure to H5N1 at live bird markets. CONCLUSIONS: Research has demonstrated that despite frequent and widespread contact with poultry, transmission of the H5N1 virus from poultry to humans is rare. Available research has identified several risk factors that may be associated with infection including close direct contact with poultry and transmission via the environment. However, several important data gaps remain that limit our understanding of the epidemiology of H5N1 in humans. Although infection in humans with H5N1 remains rare, human cases continue to be reported and H5N1 is now considered endemic among poultry in parts of Asia and in Egypt, providing opportunities for additional human infections and for the acquisition of virus mutations that may lead to more efficient spread among humans and other mammalian species

  1. 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

  2. Immunity to current H5 highly pathogenic avian influenza viruses: From vaccines to adaptive immunity in wild birds

    Science.gov (United States)

    Following the 2014-2015 outbreaks of H5N2 and H5N8 highly pathogenic avian influenza (HPAI) in the U.S., studies were performed to assess the immunity required for protection against future outbreaks should they occur. We assessed the ability of vaccines to induce protection of chickens and turkeys...

  3. Experimental assessment of the pathogenicity of two avian influenza A H5 viruses in ostrich chicks (Struthio camelus) and chickens

    DEFF Research Database (Denmark)

    Manvell, R.J.; Jørgensen, Poul Henrik; Nielsen, O.L.

    1998-01-01

    Virus excretion, immune response, and, for chickens, deaths were recorded in 3-week-old ostriches and chickens inoculated by either the intramuscular or intranasal route with one of two influenza A viruses of subtype H5, One of the viruses, A/turkey/England/50-92/91 (H5N1) (50/92), was highly...

  4. Cross-protection against lethal H5N1 challenge ferrets with an adjuvanted pandemic influenza vaccine

    NARCIS (Netherlands)

    B. Baras (Benoît); K.J. Stittelaar (Koert); J.H. Simon (James); R.J.M.M. Thoolen (Robert); S.P. Mossman (Sally); F.H. Pistoor (Frank); G. van Amerongen (Geert); M.A. Wettendorff (Martine); E. Hanon (Emmanuel); A.D.M.E. Osterhaus (Albert)

    2008-01-01

    textabstractBackground. Unprecedented spread between birds and mammals of highly pathogenic avian influenza viruses (HPAI) of the H5N1 subtype has resulted in hundreds of human infections with a high fatality rate. This has highlighted the urgent need for the development of H5N1 vaccines that can be

  5. Spatio-temporal magnitude and direction of highly pathogenic avian influenza (H5N1) outbreaks in Bangladesh

    DEFF Research Database (Denmark)

    Ahmed, Syed Sayeem Uddin; Ersbøll, Annette Kjær; Biswas, Paritosh K.

    2011-01-01

    The number of outbreaks of HPAI-H5N1 reported by Bangladesh from 2007 through 2011 placed the country among the highest reported numbers worldwide. However, so far, the understanding of the epidemic progression, direction, intensity, persistence and risk variation of HPAI-H5N1 outbreaks over spac...

  6. Dinamika Seroprevalensi Virus Avian Influenza H5 pada Itik di Pasar Unggas Beringkit dan Galiran

    Directory of Open Access Journals (Sweden)

    I Gusti Ngurah Narendra Putra

    2013-11-01

    Full Text Available Normal 0 false false false EN-US X-NONE X-NONE Live Bird Market (LBM has a high potential for spreading Avian Influenza Virus (AIV between fowls or from fowl to human. Up to now, a dinamic of avian flue incidents at many LBMs in Bali has not been reported. This research aimed to reveal a dynamic of seroprevalences of avian influenza in ducks at Beringkit (Badung and Galiran (Kelungkung LBMs. A total of 35 duck blood samples was collected from each of LBMs. Sampling was conducted monthly from March to August, 2012 . AIV antibody of duck serum was measured using Rapid Hemagglutination Inhibition (Rapid HI test. Seroprevalence differences were analyzes with Chi-square (?2 Nonparametric statistical test. The results showed that seroprevalences of AIV H5 in ducks at Beringkit and Galiran LBMs were very high, ranged from 68.6% to 100% and 65.7% to 97.1% respectively. A Dynamic of AIV H5 seroprevalences in ducks at Beringkit and Galiran LBM had a similar pattern, except in July 2012. This indicates that VAI H5 has been circulating for a long time and has been to be an endemic virus infection in ducks at LBMs in Bali. It can be suggested that an Avian Influenza Virus monitoring should be done continuously over a long period. /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-qformat:yes; mso-style-parent:""; mso-padding-alt:0in 5.4pt 0in 5.4pt; mso-para-margin:0in; mso-para-margin-bottom:.0001pt; text-align:justify; line-height:150%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri","sans-serif"; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"Times New Roman"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin;}

  7. Inefficient transmission of H5N1 influenza viruses in a ferret contact model.

    Science.gov (United States)

    Yen, Hui-Ling; Lipatov, Aleksandr S; Ilyushina, Natalia A; Govorkova, Elena A; Franks, John; Yilmaz, Neziha; Douglas, Alan; Hay, Alan; Krauss, Scott; Rehg, Jerold E; Hoffmann, Erich; Webster, Robert G

    2007-07-01

    The abilities to infect and transmit efficiently among humans are essential for a novel influenza A virus to cause a pandemic. To evaluate the pandemic potential of widely disseminated H5N1 influenza viruses, a ferret contact model using experimental groups comprised of one inoculated ferret and two contact ferrets was used to study the transmissibility of four human H5N1 viruses isolated from 2003 to 2006. The effects of viral pathogenicity and receptor binding specificity (affinity to synthetic sialosaccharides with alpha2,3 or alpha2,6 linkages) on transmissibility were assessed. A/Vietnam/1203/04 and A/Vietnam/JP36-2/05 viruses, which possess "avian-like" alpha2,3-linked sialic acid (SA) receptor specificity, caused neurological symptoms and death in ferrets inoculated with 10(3) 50% tissue culture infectious doses. A/Hong Kong/213/03 and A/Turkey/65-596/06 viruses, which show binding affinity for "human-like" alpha2,6-linked SA receptors in addition to their affinity for alpha2,3-linked SA receptors, caused mild clinical symptoms and were not lethal to the ferrets. No transmission of A/Vietnam/1203/04 or A/Turkey/65-596/06 virus was detected. One contact ferret developed neutralizing antibodies to A/Hong Kong/213/03 but did not exhibit any clinical signs or detectable virus shedding. In two groups, one of two naïve contact ferrets had detectable virus after 6 to 8 days when housed together with the A/Vietnam/JP36-2/05 virus-inoculated ferrets. Infected contact ferrets showed severe clinical signs, although little or no virus was detected in nasal washes. This limited virus shedding explained the absence of secondary transmission from the infected contact ferret to the other naïve ferret that were housed together. Our results suggest that despite their receptor binding affinity, circulating H5N1 viruses retain molecular determinants that restrict their spread among mammalian species.

  8. Efficacy of a Recombinant Turkey Herpesvirus H5 Vaccine Against Challenge With H5N1 Clades 1.1.2 and 2.3.2.1 Highly Pathogenic Avian Influenza Viruses in Domestic Ducks (Anas platyrhynchos domesticus).

    Science.gov (United States)

    Pantin-Jackwood, Mary J; Kapczynski, Darrell R; DeJesus, Eric; Costa-Hurtado, Mar; Dauphin, Gwenaelle; Tripodi, Astrid; Dunn, John R; Swayne, David E

    2016-03-01

    Domestic ducks are the second most abundant poultry species in many Asian countries and have played a critical role in the epizootiology of H5N1 highly pathogenic avian influenza (HPAI).In this study, the protective efficacy of a live recombinant vector vaccine based on a turkey herpesvirus (HVT) expressing the H5 gene from a clade 2.2 H5N1 HPAI strain (A/Swan/Hungary/4999/ 2006) (rHVT-H5/2.2), given at 3 days of age, was examined in Pekin ducks (Anas platyrhynchos domesticus). The vaccine was given alone or in combination with an inactivated H5N1 clade 2.3.2.1 reverse genetic (rgGD/2.3.2.1) vaccine given at 16 days of age, either as a single vaccination or in a prime-boost regime. At 30 days of age, ducks were challenged with one of two H5N1 HPAI viruses: A/duck/Vietnam/NCVD-2721/2013 (clade 1.1.2) or A/duck/Vietnam/NCVD-1584/2012 (clade 2.3.2.1.C). These viruses produced 100% mortality in less than 5 days in nonvaccinated control ducks. Ducks vaccinated with the rgGD/2.3.2.1 vaccine, with or without the rHVT-H5/2.2 vaccine, were 90%-100% protected against mortality after challenge with either of the two H5N1 HPAI viruses. The rHVT-H5/2.2 vaccine alone, however, conferred only 30% protection against mortality after challenge with either H5N1 HPAI virus; the surviving ducks from these groups shed higher amount of virus and for longer than the single-vaccinated rgGD/2.3.2.1 group. Despite low protection, ducks vaccinated with the rHVT-H5/2.2 vaccine and challenged with the clade 1.1.2 Vietnam virus had a longer mean death time than nonvaccinated controls (P = 0.02). A booster effect was found on reduction of virus shedding when using both vaccines, with lower oropharyngeal viral titers at 4 days after challenge with either HPAI virus (P study demonstrates the suboptimal protection with the rHVT-H5/2.2 vaccine given alone in Pekin ducks against H5N1 HPAI viruses and only a minor additive effect on virus shedding reduction when used with an inactivated vaccine in a

  9. Insight into Alternative Approaches for Control of Avian Influenza in Poultry, with Emphasis on Highly Pathogenic H5N1

    Directory of Open Access Journals (Sweden)

    Hafez M. Hafez

    2012-11-01

    Full Text Available Highly pathogenic avian influenza virus (HPAIV of subtype H5N1 causes a devastating disease in poultry but when it accidentally infects humans it can cause death. Therefore, decrease the incidence of H5N1 in humans needs to focus on prevention and control of poultry infections. Conventional control strategies in poultry based on surveillance, stamping out, movement restriction and enforcement of biosecurity measures did not prevent the virus spreading, particularly in developing countries. Several challenges limit efficiency of the vaccines to prevent outbreaks of HPAIV H5N1 in endemic countries. Alternative and complementary approaches to reduce the current burden of H5N1 epidemics in poultry should be encouraged. The use of antiviral chemotherapy and natural compounds, avian-cytokines, RNA interference, genetic breeding and/or development of transgenic poultry warrant further evaluation as integrated intervention strategies for control of HPAIV H5N1 in poultry.

  10. Expression of Two N1 Clones with Single Amino Acid Dissimilarity of Avian Influenza H5N1 Virus

    Directory of Open Access Journals (Sweden)

    RISZA HARTAWAN

    2012-12-01

    Full Text Available Two clones of N1 gene derived from isolate A/Dk/Tangerang/Bbalitvet-ACIAR-TE11/2007 (H5N1 exhibit single mismatch of amino acid sequence at position 242 that is threonine and methionine for the clone #3 and #5, respectively. In order to evaluate the effect of the amino acid substitution, these clones were inserted into two different expression vectors that are pEGFP-C1 and pcDNA-3.3 TOPO® TA cloning. Subsequently, the respective recombinant clones were transfected into eukaryotic cells, including CEF, RK13 and VERO using Lipofectamine ‘plus’ reagent. As a result, the clone #3 retaining atypical sequence showed lower expression level rather than the clone #15 in both vectors and all type of cells. The 3D conformational modelling revealed that the mutation occurs in the inner part of glycoprotein embedded within envelope or matrix. Therefore, the missense mutation seems has no effect on the antigenic properties of neuraminidase but this substitution by any means causes lethal mutagenesis in the individual gene expression by reducing level of protein transcript.

  11. Neutron diffraction analysis of HRh[P(C6H5)3]4

    International Nuclear Information System (INIS)

    Bau, R.; Stevens, R.C.; McLean, M.; Koetzle, T.F.

    1987-01-01

    We have collected neutron diffraction data on a large single crystal of the title compound. The most surprising result is an extremely short Rh-H distance of 1.31(8) A, presumably caused by steric interactions involving the bulky triphenyl phosphine ligands. Crystallographic details: HRh[P(C 6 H 5 ) 3 ] 4 . 1 / 2 C 6 H 6 crystallizes in the space group Pa3, with a = b = c = 22.776(3) A, Z = 8. Data were collected at the Brookhaven High Flux Beam reactor at a temperature of -23 0 C, λ = 1.15882(7) A -1 . Least-squares refinement (in which the phenyl rings were treated as rigid groups) resulted in an R factor [based on data with f > 4σ(F)] of 0.12 for 914 reflections and 95 parameters. 10 refs

  12. Wild bird surveillance for highly pathogenic avian influenza H5 in North America

    Science.gov (United States)

    Flint, Paul L.; Pearce, John M.; Franson, J. Christian; Derksen, Dirk V.

    2015-01-01

    It is unknown how the current Asian origin highly pathogenic avian influenza H5 viruses arrived, but these viruses are now poised to become endemic in North America. Wild birds harbor these viruses and have dispersed them at regional scales. What is unclear is how the viruses may be moving from the wild bird reservoir into poultry holdings. Active surveillance of live wild birds is likely the best way to determine the true distribution of these viruses. We also suggest that sampling be focused on regions with the greatest risk for poultry losses and attempt to define the mechanisms of transfer to enhance biosecurity. Responding to the recent outbreaks of highly pathogenic avian influenza in North America requires an efficient plan with clear objectives and potential management outcomes.

  13. New avian influenza A virus subtype combination H5N7 identified in Danish mallard ducks

    DEFF Research Database (Denmark)

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

    2005-01-01

    sequence was most closely related to the HPAIV A/Chicken/Netheriancts/01/03 (H7N7) that infected chickens and humans in the Netherlands in 2003. Ten persons with direct or indirect contact with the Danish mallard ducks showed signs Of influenza-like illness 2-3 clays following the killing of the ducks......During the past years increasing incidences of influenza A zoonosis have made it of uppermost importance to possess methods for rapid and precise identification and characterisation of influenza A Viruses. We present here a convenient one-step RT-PCR method that will amplify full......-length haemagglutinin (HA) and neuraminidase (NA) directly from clinical samples and from all known subtypes of influenza A. We applied the method on samples collected in September 2003 from a Danish flock of mallards with general health problems and by this a previously undescribed influenza A subtype combination, H5N...

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

    Directory of Open Access Journals (Sweden)

    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.

  15. The special neuraminidase stalk-motif responsible for increased virulence and pathogenesis of H5N1 influenza A virus.

    Directory of Open Access Journals (Sweden)

    Hongbo Zhou

    Full Text Available The variation of highly pathogenic avian influenza H5N1 virus results in gradually increased virulence in poultry, and human cases continue to accumulate. The neuraminidase (NA stalk region of influenza virus varies considerably and may associate with its virulence. The NA stalk region of all N1 subtype influenza A viruses can be divided into six different stalk-motifs, H5N1/2004-like (NA-wt, WSN-like, H5N1/97-like, PR/8-like, H7N1/99-like and H5N1/96-like. The NA-wt is a special NA stalk-motif which was first observed in H5N1 influenza virus in 2000, with a 20-amino acid deletion in the 49(th to 68(th positions of the stalk region. Here we show that there is a gradual increase of the special NA stalk-motif in H5N1 isolates from 2000 to 2007, and notably, the special stalk-motif is observed in all 173 H5N1 human isolates from 2004 to 2007. The recombinant H5N1 virus with the special stalk-motif possesses the highest virulence and pathogenicity in chicken and mice, while the recombinant viruses with the other stalk-motifs display attenuated phenotype. This indicates that the special stalk-motif has contributed to the high virulence and pathogenicity of H5N1 isolates since 2000. The gradually increasing emergence of the special NA stalk-motif in H5N1 isolates, especially in human isolates, deserves attention by all.

  16. Characterization of Mason Gully (H5): The second recovered fall from the Desert Fireball Network

    Science.gov (United States)

    Dyl, Kathryn A.; Benedix, Gretchen K.; Bland, Phil A.; Friedrich, Jon M.; Spurný, Pavel; Towner, Martin C.; O'Keefe, Mary Claire; Howard, Kieren; Greenwood, Richard; Macke, Robert J.; Britt, Daniel T.; Halfpenny, Angela; Thostenson, James O.; Rudolph, Rebecca A.; Rivers, Mark L.; Bevan, Alex W. R.

    2016-03-01

    Mason Gully, the second meteorite recovered using the Desert Fireball Network (DFN), is characterized using petrography, mineralogy, oxygen isotopes, bulk chemistry, and physical properties. Geochemical data are consistent with its classification as an H5 ordinary chondrite. Several properties distinguish it from most other H chondrites. Its 10.7% porosity is predominantly macroscopic, present as intergranular void spaces rather than microscopic cracks. Modal mineralogy (determined via PS-XRD, element mapping via energy dispersive spectroscopy [EDS], and X-ray tomography [for sulfide, metal, and porosity volume fractions]) consistently gives an unusually low olivine/orthopyroxene ratio (0.67-0.76 for Mason Gully versus ~1.3 for typical H5 ordinary chondrites). Widespread "silicate darkening" is observed. In addition, it contains a bright green crystalline object at the surface of the recovered stone (diameter ≈ 1.5 mm), which has a tridymite core with minor α-quartz and a rim of both low- and high-Ca pyroxene. The mineralogy allows the calculation of the temperatures and ƒ(O2) characterizing thermal metamorphism on the parent body using both the two-pyroxene and the olivine-chromite geo-oxybarometers. These indicate that MG experienced a peak metamorphic temperature of ~900 °C and had a similar ƒ(O2) to Kernouvé (H6) that was buffered by the reaction between olivine, metal, and pyroxene. There is no evidence for shock, consistent with the observed porosity structure. Thus, while Mason Gully has some unique properties, its geochemistry indicates a similar thermal evolution to other H chondrites. The presence of tridymite, while rare, is seen in other OCs and likely exogenous; however, the green object itself may result from metamorphism.

  17. Evidence for common ancestry among viruses isolated from wild birds in Beringia and highly pathogenic intercontinental reassortant H5N1 and H5N2 influenza A viruses

    Science.gov (United States)

    Ramey, Andy M.; Reeves, Andrew; Teslaa, Joshua L.; Nashold, Sean W.; Donnelly, Tyrone F.; Bahl, Justin; Hall, Jeffrey S.

    2016-01-01

    Highly pathogenic clade 2.3.4.4 H5N8, H5N2, and H5N1 influenza A viruses were first detected in wild, captive, and domestic birds in North America in November–December 2014. In this study, we used wild waterbird samples collected in Alaska prior to the initial detection of clade 2.3.4.4 H5 influenza A viruses in North America to assess the evidence for: (1) dispersal of highly pathogenic influenza A viruses from East Asia to North America by migratory birds via Alaska and (2) ancestral origins of clade 2.3.4.4 H5 reassortant viruses in Beringia. Although we did not detect highly pathogenic influenza A viruses in our sample collection from western Alaska, we did identify viruses that contained gene segments sharing recent common ancestry with intercontinental reassortant H5N2 and H5N1 viruses. Results of phylogenetic analyses and estimates for times of most recent common ancestry support migratory birds sampled in Beringia as maintaining viral diversity closely related to novel highly pathogenic influenza A virus genotypes detected in North America. Although our results do not elucidate the route by which highly pathogenic influenza A viruses were introduced into North America, genetic evidence is consistent with the hypothesized trans-Beringian route of introduction via migratory birds.

  18. Recombinant Lactococcus lactis Expressing Haemagglutinin from a Polish Avian H5N1 Isolate and Its Immunological Effect in Preliminary Animal Trials

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    Agnieszka K. Szczepankowska

    2017-01-01

    Full Text Available Lactic acid bacteria (LAB are Gram-positive, nonpathogenic microorganisms that are gaining much interest as antigen producers for development of live vaccine vectors. Heterologous proteins of different origin have been successfully expressed in various LAB species, including Lactococcus lactis. Recombinant L. lactis strains have been shown to induce specific local and systemic immune responses against various antigens. Our study aimed at constructing a L. lactis strain expressing haemagglutinin of a Polish avian H5H1 influenza isolate and examining its effect on animals. Expression of the cloned H5 gene was achieved using the nisin-controlled gene expression system. Detection of the intracellular H5 antigen produced in L. lactis was performed by Western blot analysis and confirmed using mass spectrometry. The potential of L. lactis recombinant cells to induce an immune response was examined by setting up preliminary immunization trials on chickens and mice. Obtained sera were tested for specific antibodies by ELISA assays. The results of these studies are a promising step toward developing a vaccine against the bird flu using Lactococcus lactis cells as bioreactors for efficient antigen production and delivery to the mucosal surface.

  19. Recombinant Lactococcus lactis Expressing Haemagglutinin from a Polish Avian H5N1 Isolate and Its Immunological Effect in Preliminary Animal Trials.

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    Szczepankowska, Agnieszka K; Szatraj, Katarzyna; Sałański, Przemysław; Rózga, Agnieszka; Górecki, Roman K; Bardowski, Jacek K

    2017-01-01

    Lactic acid bacteria (LAB) are Gram-positive, nonpathogenic microorganisms that are gaining much interest as antigen producers for development of live vaccine vectors. Heterologous proteins of different origin have been successfully expressed in various LAB species, including Lactococcus lactis . Recombinant L. lactis strains have been shown to induce specific local and systemic immune responses against various antigens. Our study aimed at constructing a L. lactis strain expressing haemagglutinin of a Polish avian H5H1 influenza isolate and examining its effect on animals. Expression of the cloned H5 gene was achieved using the nisin-controlled gene expression system. Detection of the intracellular H5 antigen produced in L. lactis was performed by Western blot analysis and confirmed using mass spectrometry. The potential of L. lactis recombinant cells to induce an immune response was examined by setting up preliminary immunization trials on chickens and mice. Obtained sera were tested for specific antibodies by ELISA assays. The results of these studies are a promising step toward developing a vaccine against the bird flu using Lactococcus lactis cells as bioreactors for efficient antigen production and delivery to the mucosal surface.

  20. 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.

  1. 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.

  2. Influenza H5N1 virus infection of polarized human alveolar epithelial cells and lung microvascular endothelial cells

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    Yuen Kit M

    2009-10-01

    Full Text Available Abstract Background Highly pathogenic avian influenza (HPAI H5N1 virus is entrenched in poultry in Asia and Africa and continues to infect humans zoonotically causing acute respiratory disease syndrome and death. There is evidence that the virus may sometimes spread beyond respiratory tract to cause disseminated infection. The primary target cell for HPAI H5N1 virus in human lung is the alveolar epithelial cell. Alveolar epithelium and its adjacent lung microvascular endothelium form host barriers to the initiation of infection and dissemination of influenza H5N1 infection in humans. These are polarized cells and the polarity of influenza virus entry and egress as well as the secretion of cytokines and chemokines from the virus infected cells are likely to be central to the pathogenesis of human H5N1 disease. Aim To study influenza A (H5N1 virus replication and host innate immune responses in polarized primary human alveolar epithelial cells and lung microvascular endothelial cells and its relevance to the pathogenesis of human H5N1 disease. Methods We use an in vitro model of polarized primary human alveolar epithelial cells and lung microvascular endothelial cells grown in transwell culture inserts to compare infection with influenza A subtype H1N1 and H5N1 viruses via the apical or basolateral surfaces. Results We demonstrate that both influenza H1N1 and H5N1 viruses efficiently infect alveolar epithelial cells from both apical and basolateral surface of the epithelium but release of newly formed virus is mainly from the apical side of the epithelium. In contrast, influenza H5N1 virus, but not H1N1 virus, efficiently infected polarized microvascular endothelial cells from both apical and basolateral aspects. This provides a mechanistic explanation for how H5N1 virus may infect the lung from systemic circulation. Epidemiological evidence has implicated ingestion of virus-contaminated foods as the source of infection in some instances and our

  3. Spatial distribution and risk factors of highly pathogenic avian influenza (HPAI) H5N1 in China

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    Martin, Vincent; Pfeiffer, Dirk U.; Zhou, Xiaoyan; Xiao, Xiangming; Prosser, Diann J.; Guo, Fusheng; Gilbert, Marius

    2011-01-01

    Highly pathogenic avian influenza (HPAI) H5N1 was first encountered in 1996 in Guangdong province (China) and started spreading throughout Asia and the western Palearctic in 2004–2006. Compared to several other countries where the HPAI H5N1 distribution has been studied in some detail, little is known about the environmental correlates of the HPAI H5N1 distribution in China. HPAI H5N1 clinical disease outbreaks, and HPAI virus (HPAIV) H5N1 isolated from active risk-based surveillance sampling of domestic poultry (referred to as HPAIV H5N1 surveillance positives in this manuscript) were modeled separately using seven risk variables: chicken, domestic waterfowl population density, proportion of land covered by rice or surface water, cropping intensity, elevation, and human population density. We used bootstrapped logistic regression and boosted regression trees (BRT) with cross-validation to identify the weight of each variable, to assess the predictive power of the models, and to map the distribution of HPAI H5N1 risk. HPAI H5N1 clinical disease outbreak occurrence in domestic poultry was mainly associated with chicken density, human population density, and elevation. In contrast, HPAIV H5N1 infection identified by risk-based surveillance was associated with domestic waterfowl density, human population density, and the proportion of land covered by surface water. Both models had a high explanatory power (mean AUC ranging from 0.864 to 0.967). The map of HPAIV H5N1 risk distribution based on active surveillance data emphasized areas south of the Yangtze River, while the distribution of reported outbreak risk extended further North, where the density of poultry and humans is higher. We quantified the statistical association between HPAI H5N1 outbreak, HPAIV distribution and post-vaccination levels of seropositivity (percentage of effective post-vaccination seroconversion in vaccinated birds) and found that provinces with either outbreaks or HPAIV H5N1 surveillance

  4. Spatial distribution and risk factors of highly pathogenic avian influenza (HPAI H5N1 in China.

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    Vincent Martin

    2011-03-01

    Full Text Available Highly pathogenic avian influenza (HPAI H5N1 was first encountered in 1996 in Guangdong province (China and started spreading throughout Asia and the western Palearctic in 2004-2006. Compared to several other countries where the HPAI H5N1 distribution has been studied in some detail, little is known about the environmental correlates of the HPAI H5N1 distribution in China. HPAI H5N1 clinical disease outbreaks, and HPAI virus (HPAIV H5N1 isolated from active risk-based surveillance sampling of domestic poultry (referred to as HPAIV H5N1 surveillance positives in this manuscript were modeled separately using seven risk variables: chicken, domestic waterfowl population density, proportion of land covered by rice or surface water, cropping intensity, elevation, and human population density. We used bootstrapped logistic regression and boosted regression trees (BRT with cross-validation to identify the weight of each variable, to assess the predictive power of the models, and to map the distribution of HPAI H5N1 risk. HPAI H5N1 clinical disease outbreak occurrence in domestic poultry was mainly associated with chicken density, human population density, and elevation. In contrast, HPAIV H5N1 infection identified by risk-based surveillance was associated with domestic waterfowl density, human population density, and the proportion of land covered by surface water. Both models had a high explanatory power (mean AUC ranging from 0.864 to 0.967. The map of HPAIV H5N1 risk distribution based on active surveillance data emphasized areas south of the Yangtze River, while the distribution of reported outbreak risk extended further North, where the density of poultry and humans is higher. We quantified the statistical association between HPAI H5N1 outbreak, HPAIV distribution and post-vaccination levels of seropositivity (percentage of effective post-vaccination seroconversion in vaccinated birds and found that provinces with either outbreaks or HPAIV H5N1

  5. 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.

  6. 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.

  7. Memory T-cell immune response in healthy young adults vaccinated with live attenuated influenza A (H5N2) vaccine.

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    Chirkova, T V; Naykhin, A N; Petukhova, G D; Korenkov, D A; Donina, S A; Mironov, A N; Rudenko, L G

    2011-10-01

    Cellular immune responses of both CD4 and CD8 memory/effector T cells were evaluated in healthy young adults who received two doses of live attenuated influenza A (H5N2) vaccine. The vaccine was developed by reassortment of nonpathogenic avian A/Duck/Potsdam/1402-6/68 (H5N2) and cold-adapted A/Leningrad/134/17/57 (H2N2) viruses. T-cell responses were measured by standard methods of intracellular cytokine staining of gamma interferon (IFN-γ)-producing cells and a novel T-cell recognition of antigen-presenting cells by protein capture (TRAP) assay based on the trogocytosis phenomenon, namely, plasma membrane exchange between interacting immune cells. TRAP enables the detection of activated trogocytosis-positive T cells after virus stimulation. We showed that two doses of live attenuated influenza A (H5N2) vaccine promoted both CD4 and CD8 T-memory-cell responses in peripheral blood of healthy young subjects in the clinical study. Significant differences in geometric mean titers (GMTs) of influenza A (H5N2)-specific IFN-γ(+) cells were observed at day 42 following the second vaccination, while peak levels of trogocytosis(+) T cells were detected earlier, on the 21st day after the second vaccination. The inverse correlation of baseline levels compared to postvaccine fold changes in GMTs of influenza-specific CD4 and CD8 T cells demonstrated that baseline levels of these specific cells could be considered a predictive factor of vaccine immunogenicity.

  8. Understanding the cross-resistance of oseltamivir to H1N1 and H5N1 influenza A neuraminidase mutations using multidimensional computational analyses

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    Singh A

    2015-07-01

    Full Text Available Ashona Singh, Mahmoud E Soliman School of Health Sciences, University of KwaZulu-Natal, Westville, Durban, South Africa Abstract: This study embarks on a comprehensive description of the conformational contributions to resistance of neuraminidase (N1 in H1N1 and H5N1 to oseltamivir, using comparative multiple molecular dynamic simulations. The available data with regard to elucidation of the mechanism of resistance as a result of mutations in H1N1 and H5N1 neuraminidases is not well established. Enhanced post-dynamic analysis, such as principal component analysis, solvent accessible surface area, free binding energy calculations, and radius of gyration were performed to gain a precise insight into the binding mode and origin of resistance of oseltamivir in H1N1 and H5N1 mutants. Three significant features reflecting resistance in the presence of mutations H274Y and I222K, of the protein complexed with the inhibitor are: reduced flexibility of the a-carbon backbone; an improved ΔEele of ~15 (kcal/mol for H1N1 coupled with an increase in ΔGsol­ (~13 kcal/mol from wild-type to mutation; a low binding affinity in comparison with the wild-type of ~2 (kcal/mol and ~7 (kcal/mol with respect to each mutation for the H5N1 systems; and reduced hydrophobicity of the overall surface structure due to an impaired hydrogen bonding network. We believe the results of this study will ultimately provide a useful insight into the structural landscape of neuraminidase-associated binding of oseltamivir. Furthermore, the results can be used in the design and development of potent inhibitors of neuraminidases. Keywords: neuraminidase, molecular dynamics, resistance, mutation, binding free energy

  9. Seroprevalence of antibodies against highly pathogenic avian influenza A (H5N1 virus among poultry workers in Bangladesh, 2009.

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    Sharifa Nasreen

    Full Text Available We conducted a cross-sectional study in 2009 to determine the seroprevalence and risk factors for highly pathogenic avian influenza A (H5N1 [HPAI H5N1] virus antibodies among poultry workers at farms and live bird markets with confirmed/suspected poultry outbreaks during 2009 in Bangladesh. We tested sera by microneutralization assay using A/Bangladesh/207095/2008 (H5N1; clade 2.2.2 virus with confirmation by horse red blood cell hemagglutination inhibition and H5-specific Western blot assays. We enrolled 212 workers from 87 farms and 210 workers from three live bird markets. One hundred and two farm workers (48% culled poultry. One hundred and ninety-three farm workers (91% and 178 market workers (85% reported direct contact with poultry that died during a laboratory confirmed HPAI H5N1 poultry farm outbreak or market poultry die-offs from suspected HPAI H5N1. Despite exposure to sick poultry, no farm or market poultry workers were seropositive for HPAI H5N1 virus antibodies (95% confidence interval 0-1%.

  10. Novel Reassortant Influenza A(H5N8) Viruses among Inoculated Domestic and Wild Ducks, South Korea, 2014

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    Kang, Hyun-Mi; Lee, Eun-Kyoung; Song, Byung-Min; Jeong, Jipseol; Choi, Jun-Gu; Jeong, Joojin; Moon, Oun-Kyong; Yoon, Hachung; Cho, Youngmi; Kang, Young-Myong; Lee, Hee-Soo

    2015-01-01

    An outbreak of highly pathogenic avian influenza, caused by a novel reassortant influenza A (H5N8) virus, occurred among poultry and wild birds in South Korea in 2014. The aim of this study was to evaluate the pathogenesis in and mode of transmission of this virus among domestic and wild ducks. Three of the viruses had similar pathogenicity among infected domestic ducks: the H5N8 viruses were moderately pathogenic (0%–20% mortality rate); in wild mallard ducks, the H5N8 and H5N1 viruses did not cause severe illness or death; viral replication and shedding were greater in H5N8-infected mallards than in H5N1-infected mallards. Identification of H5N8 viruses in birds exposed to infected domestic ducks and mallards indicated that the viruses could spread by contact. We propose active surveillance to support prevention of the spread of this virus among wild birds and poultry, especially domestic ducks. PMID:25625281

  11. High antiviral effects of hibiscus tea extract on the H5 subtypes of low and highly pathogenic avian influenza viruses.

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    Baatartsogt, Tugsbaatar; Bui, Vuong N; Trinh, Dai Q; Yamaguchi, Emi; Gronsang, Dulyatad; Thampaisarn, Rapeewan; Ogawa, Haruko; Imai, Kunitoshi

    2016-10-01

    Viral neuraminidase inhibitors are widely used as synthetic anti-influenza drugs for the prevention and treatment of influenza. However, drug-resistant influenza A virus variants, including H5N1 highly pathogenic avian influenza viruses (HPAIVs), have been reported. Therefore, the discovery of novel and effective antiviral agents is warranted. We screened the antiviral effects of 11 herbal tea extracts (hibiscus, black tea, tencha, rosehip tea, burdock tea, green tea, jasmine tea, ginger tea, lavender tea, rose tea and oak tea) against the H5N1 HPAIV in vitro. Among the tested extracts, only the hibiscus extract and its fractionated extract (frHibis) highly and rapidly reduced the titers of all H5 HPAIVs and low pathogenic AIVs (LPAIVs) used in the pre-treatment tests of Madin-Darby canine kidney (MDCK) cells that were inoculated with a mixture of the virus and the extract. Immunogold electron microscopy showed that anti-H5 monoclonal antibodies could not bind to the deformed H5 virus particles pretreated with frHibis. In post-treatment tests of MDCK cells cultured in the presence of frHibis after infection with H5N1 HPAIV, the frHibis inhibited viral replication and the expression of viral antigens and genes. Among the plants tested, hibiscus showed the most prominent antiviral effects against both H5 HPAIV and LPAIV.

  12. Seroprevalence of avian influenza A (H5N1 virus among poultry workers in Jiangsu Province, China: an observational study

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    Huo Xiang

    2012-04-01

    Full Text Available Abstract Background Since 2003 to 06 Jan 2012, the number of laboratory confirmed human cases of infection with avian influenza in China was 41 and 27 were fatal. However, the official estimate of the H5N1 case-fatality rate has been described by some as an over estimation since there may be numerous undetected asymptomatic/mild cases of H5N1 infection. This study was conducted to better understand the real infection rate and evaluate the potential risk factors for the zoonotic spread of H5N1 viruses to humans. Methods A seroepidemiological survey was conducted in poultry workers, a group expected to have the highest level of exposure to H5N1-infected birds, from 3 counties with habitat lakes of wildfowl in Jiangsu province, China. Serum specimens were collected from 306 participants for H5N1 serological test. All participants were interviewed to collect information about poultry exposures. Results The overall seropositive rate was 2.61% for H5N1 antibodies. The poultry number was found associated with a 2.39-fold significantly increased subclinical infection risk after adjusted with age and gender. Conclusions Avian-to -human transmission of avian H5N1 virus remained low. Workers associated with raising larger poultry flocks have a higher risk on seroconversion.

  13. Genetic Characterization of Continually Evolving Highly Pathogenic H5N6 Influenza Viruses in China, 2012–2016

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    Li, Meng; Zhao, Na; Luo, Jing; Li, Yuan; Chen, Lin; Ma, Jiajun; Zhao, Lin; Yuan, Guohui; Wang, Chengmin; Wang, Yutian; Liu, Yanhua; He, Hongxuan

    2017-01-01

    H5N6 is a highly pathogenic avian influenza (HPAI) and a zoonotic disease that causes recurring endemics in East Asia. At least 155 H5N6 outbreaks, including 15 human infections, have been reported in China. These repeated outbreaks have increased concern that the H5N6 virus may cross over to humans and cause a pandemic. In February, 2016, peafowls in a breeding farm exhibited a highly contagious disease. Post-mortem examinations, including RT-PCR, and virus isolation, confirmed that the highly pathogenic H5N6 influenza virus was the causative agent, and the strain was named A/Pavo Cristatus/Jiangxi/JA1/2016. In animal experiments, it exhibited high pathogenicity in chickens and an estimated median lethal dose in mice of ~104.3 TCID50. A phylogenetic analysis showed that JA1/2016 was clustered in H5 clade 2.3.4.4. FG594-like H5N6 virus from Guangdong Province was the probable predecessor of JA1/2016, and the estimated divergence time was June 2014. Furthermore, we found that H5N6 influenza viruses can be classified into the two following groups: Group 1 and Group 2. Group 2 influenza viruses have not been detected since the end of 2014, whereas Group 1 influenza viruses have continually evolved and reassorted with the “gene pool” circulating in south China, resulting in the rise of novel subtypes of this influenza virus. An increase in the number of its identified hosts, the expanding range of its distribution, and the continual evolution of H5N6 AIVs enhance the risk that an H5N6 virus may spread to other continents and cause a pandemic. PMID:28293218

  14. Genetic Characterization of Continually Evolving Highly Pathogenic H5N6 Influenza Viruses in China, 2012-2016.

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    Li, Meng; Zhao, Na; Luo, Jing; Li, Yuan; Chen, Lin; Ma, Jiajun; Zhao, Lin; Yuan, Guohui; Wang, Chengmin; Wang, Yutian; Liu, Yanhua; He, Hongxuan

    2017-01-01

    H5N6 is a highly pathogenic avian influenza (HPAI) and a zoonotic disease that causes recurring endemics in East Asia. At least 155 H5N6 outbreaks, including 15 human infections, have been reported in China. These repeated outbreaks have increased concern that the H5N6 virus may cross over to humans and cause a pandemic. In February, 2016, peafowls in a breeding farm exhibited a highly contagious disease. Post-mortem examinations, including RT-PCR, and virus isolation, confirmed that the highly pathogenic H5N6 influenza virus was the causative agent, and the strain was named A/ Pavo Cristatus /Jiangxi/JA1/2016. In animal experiments, it exhibited high pathogenicity in chickens and an estimated median lethal dose in mice of ~10 4.3 TCID 50 . A phylogenetic analysis showed that JA1/2016 was clustered in H5 clade 2.3.4.4. FG594-like H5N6 virus from Guangdong Province was the probable predecessor of JA1/2016, and the estimated divergence time was June 2014. Furthermore, we found that H5N6 influenza viruses can be classified into the two following groups: Group 1 and Group 2. Group 2 influenza viruses have not been detected since the end of 2014, whereas Group 1 influenza viruses have continually evolved and reassorted with the "gene pool" circulating in south China, resulting in the rise of novel subtypes of this influenza virus. An increase in the number of its identified hosts, the expanding range of its distribution, and the continual evolution of H5N6 AIVs enhance the risk that an H5N6 virus may spread to other continents and cause a pandemic.

  15. Identification of Two novel reassortant avian influenza a (H5N6) viruses in whooper swans in Korea, 2016.

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    Jeong, Jipseol; Woo, Chanjin; Ip, Hon S; An, Injung; Kim, Youngsik; Lee, Kwanghee; Jo, Seong-Deok; Son, Kidong; Lee, Saemi; Oem, Jae-Ku; Wang, Seung-Jun; Kim, Yongkwan; Shin, Jeonghwa; Sleeman, Jonathan; Jheong, Weonhwa

    2017-03-21

    On November 20, 2016 two novel strains of H5N6 highly pathogenic avian influenza virus (HPAIVs) were isolated from three whooper swans (Cygnus cygnus) at Gangjin Bay in South Jeolla province, South Korea. Identification of HPAIVs in wild birds is significant as there is a potential risk of transmission of these viruses to poultry and humans. Phylogenetic analysis revealed that Gangjin H5N6 viruses classified into Asian H5 clade 2.3.4.4 lineage and were distinguishable from H5N8 and H5N1 HPAIVs previously isolated in Korea. With the exception of the polymerase acidic (PA) gene, the viruses were most closely related to A/duck/Guangdong/01.01SZSGXJK005-Y/2016 (H5N6) (98.90 ~ 99.74%). The PA genes of the two novel Gangjin H5N6 viruses were most closely related to AIV isolates previously characterized from Korea, A/hooded crane/Korea/1176/2016 (H1N1) (99.16%) and A/environment/Korea/W133/2006 (H7N7) (98.65%). The lack of more recent viruses to A/environment/Korea/W133/2006 (H7N7) indicates the need for analysis of recent wild bird AIVs isolated in Korea because they might provide further clues as to the origin of these novel reassortant H5N6 viruses. Although research on the origins and epidemiology of these infections is ongoing, the most likely route of infection for the whooper swans was through direct or indirect contact with reassortant viruses shed by migratory wild birds in Korea. As H5N6 HPAIVs can potentially be transmitted to poultry and humans, continuous monitoring of AIVs among wild birds will help to mitigate this risk.

  16. Seroprevalence survey of avian influenza A (H5) in wild migratory birds in Yunnan Province, Southwestern China.

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    Chang, Hua; Dai, Feiyan; Liu, Zili; Yuan, Feizhou; Zhao, Siyue; Xiang, Xun; Zou, Fengcai; Zeng, Bangquan; Fan, Yating; Duan, Gang

    2014-02-03

    Highly pathogenic avian influenza virus (HPAIV) is a highly contagious disease which is a zoonotic pathogen of significant economic and public health concern. The outbreaks caused by HPAIV H5N1 of Asian origin have caused animal and human disease and mortality in several countries of Southeast Asia, such as Bangladesh, Cambodia, China, India, Indonesia, Laos, Myanmar, Thailand and Viet Nam. For the first time since 1961, this HPAIV has also caused extensive mortality in wild birds and has sparked debate of the role wild birds have played in the spread of this virus. Other than confirmed mortality events, little is known of this virus in wild birds. There is no report on the seroprevalence of avian influenza H5 infection in wild migratory birds in Yunnan Province. In this study we examined live wild birds in Yunnan Province for H5 specific antibody to better understand the occurrence of this disease in free living birds. Sera from 440 wild birds were collected from in Kunming and Northern Ailaoshan of Yunnan Province, Southwestern China, and assayed for H5 antibodies using the hemagglutination inhibition (HI) assays. The investigation revealed that the seroprevalence of avian influenza H5 was as following: Ciconiiformes 2.6%, Strigiformes 13.04%, Passeriformes 20%, Cuculiformes 21.74%, Gruiformes 0%, Columbiformes 0%, Charadriiformes 0% and Coraciiformes 0%. Statistical analyses showed that there was a significant difference of prevalence between the orders (P avian influenza H5 antibodies were detected in 23 of 440 (5.23%) sera. Mean HI titer 23 positive sera against H5 were 5.4 log₂. The results of the present survey indicated that the proportion of wild birds had previously infected AIV H5 at other times of the year. To our knowledge, this is the first seroprevalence report of avian influenza H5 infection in wild migratory birds in China' s southwestern Yunnan Province. The results of the present survey have significant public health concerns.

  17. Psychosocial effects assocPsychosocial effects associated with highly pathogenic avian influenza (H5N1 in Nigeriaiated with highly pathogenic avian influenza (H5N1 in Nigeria

    Directory of Open Access Journals (Sweden)

    Chiara Rafanelli

    2010-12-01

    Full Text Available Highly pathogenic avian influenza H5N1 (HPAI H5N1 infected poultry in Nigeria in 2006. The outbreaks caused significant economic losses and had serious zoonotic repercussions. The outbreaks have also had psychosocial effects on Nigerian farmers. To date, empirical data on the effect of outbreaks on humans are scarce. In this study, field data on HPAI H5N1 in Nigeria were analysed. Although only one human case leading to death was reported in Nigeria, the fact that HPAI H5N1 caused a human death created a disruption in social order and in the well-being of farmers (stress, altered livelihood and trauma and affected the rural economy. The implication of the above on health communication, the importance of successful control measures in poultry and policy implementation are stressed. Further studies are encouraged.

  18. Spatiotemporal Structure of Molecular Evolution of H5N1 Highly Pathogenic Avian Influenza Viruses in Vietnam

    OpenAIRE

    Carrel, Margaret A.; Emch, Michael; Jobe, R. Todd; Moody, Aaron; Wan, Xiu-Feng

    2010-01-01

    Background Vietnam is one of the countries most affected by outbreaks of H5N1 highly pathogenic avian influenza viruses. First identified in Vietnam in poultry in 2001 and in humans in 2004, the virus has since caused 111 cases and 56 deaths in humans. In 2003/2004 H5N1 outbreaks, nearly the entire poultry population of Vietnam was culled. Our earlier study (Wan et al., 2008, PLoS ONE, 3(10): e3462) demonstrated that there have been at least six independent H5N1 introductions into Vietnam and...

  19. Retrospective space-time analysis of H5N1 Avian Influenza emergence in Thailand

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    Shanmugasundaram Jothiganesh

    2010-01-01

    Full Text Available Abstract Background The highly pathogenic avian influenza (HPAI H5N1 virus remains a worldwide threat to human and animal health, while the mechanisms explaining its epizootic emergence and re-emergence in poultry are largely unknown. Data from Thailand, a country that experienced significant epidemics in poultry and has recorded suspicious cases of HPAI on a daily basis since 2004, are used here to study the process of emergence. A spatial approach is employed to describe all HPAI H5N1 virus epizootics from 2004 to 2008 and to characterize the pattern of emergence: multiple independent introductions of the virus followed by moderate local spread vs. very rare emergences followed by strong local spread and rare long range diffusion jumps. Sites where epizootics originate (by foreign introduction, local persistence, or long range jump were selected from those to which the disease subsequently spreads using a filter based on relative date and position. The spatial distribution of these selected foci was statistically analyzed, and to differentiate environmental factors from long range diffusion, we investigate the relationship of these foci with environmental exposure factors and with rearing characteristics. Results During each wave of epizootics, the temporal occurrence of cases did not show a temporal interruption of more than a week. All foci were globally clustered; i.e., more than 90% of cases had a previous case within a 10 km range and a 21 day period of time, showing a strong local spread. We were able to estimate 60 km as the maximum distance for the local farm to farm dissemination process. The remaining "emergent" cases have occurred randomly over Thailand and did not show specific location, clusters, or trends. We found that these foci are not statistically related to specific environmental conditions or land cover characteristics, and most of them may be interpreted as long range diffusion jumps due to commercial practices

  20. Effects of plant density on recombinant hemagglutinin yields in an Agrobacterium-mediated transient gene expression system using Nicotiana benthamiana plants.

    Science.gov (United States)

    Fujiuchi, Naomichi; Matsuda, Ryo; Matoba, Nobuyuki; Fujiwara, Kazuhiro

    2017-08-01

    Agrobacterium-mediated transient expression systems enable plants to rapidly produce a wide range of recombinant proteins. To achieve economically feasible upstream production and downstream processing, it is beneficial to obtain high levels of two yield-related quantities of upstream production: recombinant protein content per fresh mass of harvested biomass (g gFM -1 ) and recombinant protein productivity per unit area-time (g m -2 /month). Here, we report that the density of Nicotiana benthamiana plants during upstream production had significant impacts on the yield-related quantities of recombinant hemagglutinin (HA). The two quantities were smaller at a high plant density of 400 plants m -2 than at a low plant density of 100 plants m -2 . The smaller quantities at the high plant density were attributed to: (i) a lower HA content in young leaves, which usually have high HA accumulation potentials; (ii) a lower biomass allocation to the young leaves; and (iii) a high area-time requirement for plants. Thus, plant density is a key factor for improving upstream production in Agrobacterium-mediated transient expression systems. Biotechnol. Bioeng. 2017;114: 1762-1770. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  1. Experimental infection of clade 1.1.2 (H5N1), clade 2.3.2.1c (H5N1) and clade 2.3.4.4 (H5N6) highly pathogenic avian influenza viruses in dogs.

    Science.gov (United States)

    Lyoo, K S; Na, W; Phan, L V; Yoon, S W; Yeom, M; Song, D; Jeong, D G

    2017-12-01

    Since the emergence of highly pathogenic avian influenza (HPAI) H5N1 in Asia, the haemagglutinin (HA) gene of this virus lineage has continued to evolve in avian populations, and H5N1 lineage viruses now circulate concurrently worldwide. Dogs may act as an intermediate host, increasing the potential for zoonotic transmission of influenza viruses. Virus transmission and pathologic changes in HPAI clade 1.1.2 (H5N1)-, 2.3.2.1c (H5N1)- and 2.3.4.4 (H5N6)-infected dogs were investigated. Mild respiratory signs and antibody response were shown in dogs intranasally infected with the viruses. Lung histopathology showed lesions that were associated with moderate interstitial pneumonia in the infected dogs. In this study, HPAI H5N6 virus replication in dogs was demonstrated for the first time. Dogs have been suspected as a "mixing vessel" for reassortments between avian and human influenza viruses to occur. The replication of these three subtypes of the H5 lineage of HPAI viruses in dogs suggests that dogs could serve as intermediate hosts for avian-human influenza virus reassortment if they are also co-infected with human influenza viruses. © 2017 Blackwell Verlag GmbH.

  2. 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...

  3. Characterization of a novel influenza A virus hemagglutinin subtype (H16) obtained from black-headed gulls.

    NARCIS (Netherlands)

    R.A.M. Fouchier (Ron); V.J. Munster (Vincent); A. Wallensten (Anders); T.M. Bestebroer (Theo); S. Herfst (Sander); D.J. Smith (Derek James); G.F. Rimmelzwaan (Guus); B. Olsen (Björn); A.D.M.E. Osterhaus (Albert)

    2005-01-01

    textabstractIn wild aquatic birds and poultry around the world, influenza A viruses carrying 15 antigenic subtypes of hemagglutinin (HA) and 9 antigenic subtypes of neuraminidase (NA) have been described. Here we describe a previously unidentified antigenic subtype of HA (H16), detected in viruses

  4. Radiological and clinical course of pneumonia in patients with avian influenza H5N1

    International Nuclear Information System (INIS)

    Bay, Ali; Etlik, Omer; Oner, A. Faik; Unal, Ozkan; Arslan, Halil; Bora, Aydin; Davran, Ramazan; Yuca, Sevil Ari; Dogan, Murat

    2007-01-01

    Introduction: We evaluated chest X-ray and clinical findings of patients with lower respiratory tract infection due to influenza H5N1 and presented the radiological findings and clinical course of the infection. Materials and methods: Between December 2005 and February 2006, eight hospitalized patients (median age 10, 5-15 years) with avian-flu were evaluated in this study. All patients were evaluated with chest X-ray and four of them with CT scan. Post mortem pathological characterization were also available for three of the patients. Results: A rapidly progressive pneumonia with high mortality rate was observed especially for cases with late admission. The major radiologic abnormalities were extensive pneumonic infiltration with segmental and multifocal distribution, mostly located in lower zones of the lung. No pleural effusion and hilar lymphadenopathy was noted. Conclusion: Avian flu may be presented as rapidly progressive pneumonia. The chest radiography has an important role in diagnosis and should be obtained daily because of rapid change of the findings that may necessitate prompt action

  5. Identifying risk factors of highly pathogenic avian influenza (H5N1 subtype) in Indonesia.

    Science.gov (United States)

    Loth, Leo; Gilbert, Marius; Wu, Jianmei; Czarnecki, Christina; Hidayat, Muhammad; Xiao, Xiangming

    2011-10-01

    Highly pathogenic avian influenza (HPAI), subtype H5N1, was first officially reported in Indonesia in 2004. Since then the disease has spread and is now endemic in large parts of the country. This study investigated the statistical relationship between a set of risk factors and the presence or absence of HPAI in Indonesia during 2006 and 2007. HPAI was evaluated through participatory disease surveillance (PDS) in backyard village chickens (the study population), and risk factors included descriptors of people and poultry distribution (separating chickens, ducks and production sectors), poultry movement patterns and agro-ecological conditions. The study showed that the risk factors "elevation", "human population density" and "rice cropping" were significant in accounting for the spatial variation of the PDS-defined HPAI cases. These findings were consistent with earlier studies in Thailand and Vietnam. In addition "commercial poultry population", and two indicators of market locations and transport; "human settlements" and "road length", were identified as significant risk factors in the models. In contrast to several previous studies carried out in Southeast Asia, domestic backyard ducks were not found to be a significant risk factor in Indonesia. The study used surrogate estimates of market locations and marketing chains and further work should focus on the actual location of the live bird markets, and on the flow of live poultry and poultry products between them, so that patterns of possible transmission, and regions of particular risk could be better inferred. Copyright © 2011 Elsevier B.V. All rights reserved.

  6. Electrical energy conversion system design with single-phase inverter and H5 transformerless topology

    Directory of Open Access Journals (Sweden)

    Luis Gerardo González-Morales

    2014-01-01

    Full Text Available Este artículo presenta el diseño de un sistema de conversión de energía eléctrica (SCEE para el aprovechamiento de energía solar y luego acondicionamiento e inyección de energía a la red eléctrica comercial. Se utiliza un inversor puente completo monofásico con la topología sin transformador H5 acoplado a un filtro LCL. El sistema es identificado mediante el modelo de estado promediado y de pequeña señal, el esquema de control presenta un control en cascada sintonizado mediante la asignación de polos, además de un algoritmo de seguimiento de máxima potencia de tipo perturbar y observar que permite aumentar la eficiencia del sistema, operando en el punto óptimo de funcionamiento del panel solar. En el diseño se describen aspectos técnicos fundamentales en el dimensionamiento y selección de componentes para su desarrollo experimental, así como el diseño de la estructura de control para un buen desempeño ante perturbaciones del sistema.

  7. Migratory birds, the H5N1 influenza virus and the scientific method

    Directory of Open Access Journals (Sweden)

    Stilianakis Nikolaos I

    2008-05-01

    Full Text Available Abstract Background The role of migratory birds and of poultry trade in the dispersal of highly pathogenic H5N1 is still the topic of intense and controversial debate. In a recent contribution to this journal, Flint argues that the strict application of the scientific method can help to resolve this issue. Discussion We argue that Flint's identification of the scientific method with null hypothesis testing is misleading and counterproductive. There is far more to science than the testing of hypotheses; not only the justification, bur also the discovery of hypotheses belong to science. We also show why null hypothesis testing is weak and that Bayesian methods are a preferable approach to statistical inference. Furthermore, we criticize the analogy put forward by Flint between involuntary transport of poultry and long-distance migration. Summary To expect ultimate answers and unequivocal policy guidance from null hypothesis testing puts unrealistic expectations on a flawed approach to statistical inference and on science in general.

  8. Pandemic influenza – including a risk assessment of H5N1

    Science.gov (United States)

    Taubenberger, J.K.; Morens, D.M.

    2009-01-01

    Summary Influenza pandemics and epidemics have apparently occurred since at least the Middle Ages. When pandemics appear, 50% or more of an affected population can be infected in a single year, and the number of deaths caused by influenza can dramatically exceed what is normally expected. Since 1500, there appear to have been 13 or more influenza pandemics. In the past 120 years there were undoubted pandemics in 1889, 1918, 1957, 1968, and 1977. Although most experts believe we will face another influenza pandemic, it is impossible to predict when it will appear, where it will originate, or how severe it will be. Nor is there agreement about the subtype of influenza virus most likely to cause the next pandemic. The continuing spread of H5N1 highly pathogenic avian influenza viruses has heightened interest in pandemic prediction. Despite uncertainties in the historical record of the pre-virology era, study of previous pandemics may help guide future pandemic planning and lead to a better understanding of the complex ecobiology underlying the formation of pandemic strains of influenza A viruses. PMID:19618626

  9. Pandemic influenza--including a risk assessment of H5N1.

    Science.gov (United States)

    Taubenberger, J K; Morens, D M

    2009-04-01

    Influenza pandemics and epidemics have apparently occurred since at least the Middle Ages. When pandemics appear, 50% or more of an affected population can be infected in a single year, and the number of deaths caused by influenza can dramatically exceed what is normally expected. Since 1500, there appear to have been 13 or more influenza pandemics. In the past 120 years there were undoubted pandemics in 1889, 1918, 1957, 1968, and 1977. Although most experts believe we will face another influenza pandemic, it is impossible to predict when it will appear, where it will originate, or how severe it will be. Nor is there agreement about the subtype of influenza virus most likely to cause the next pandemic. The continuing spread of H5N1 highly pathogenic avian influenza viruses has heightened interest in pandemic prediction. Despite uncertainties in the historical record of the pre-virology era, study of previous pandemics may help guide future pandemic planning and lead to a better understanding of the complex ecobiology underlying the formation of pandemic strains of influenza A viruses.

  10. Radiological and clinical course of pneumonia in patients with avian influenza H5N1

    Energy Technology Data Exchange (ETDEWEB)

    Bay, Ali [Yuzuncu Yil University, Faculty of Medicine, Department of Pediatrics, Van (Turkey)]. E-mail: bayalibay@yahoo.com; Etlik, Omer [Yuzuncu Yil University, Faculty of Medicine, Department of Radiology, Van (Turkey); Oner, A. Faik [Yuzuncu Yil University, Faculty of Medicine, Department of Pediatrics, Van (Turkey); Unal, Ozkan [Yuzuncu Yil University, Faculty of Medicine, Department of Radiology, Van (Turkey); Arslan, Halil [Yuzuncu Yil University, Faculty of Medicine, Department of Radiology, Van (Turkey); Bora, Aydin [Yuzuncu Yil University, Faculty of Medicine, Department of Radiology, Van (Turkey); Davran, Ramazan [Yuzuncu Yil University, Faculty of Medicine, Department of Radiology, Van (Turkey); Yuca, Sevil Ari [Yuzuncu Yil University, Faculty of Medicine, Department of Pediatrics, Van (Turkey); Dogan, Murat [Yuzuncu Yil University, Faculty of Medicine, Department of Pediatrics, Van (Turkey)

    2007-02-15

    Introduction: We evaluated chest X-ray and clinical findings of patients with lower respiratory tract infection due to influenza H5N1 and presented the radiological findings and clinical course of the infection. Materials and methods: Between December 2005 and February 2006, eight hospitalized patients (median age 10, 5-15 years) with avian-flu were evaluated in this study. All patients were evaluated with chest X-ray and four of them with CT scan. Post mortem pathological characterization were also available for three of the patients. Results: A rapidly progressive pneumonia with high mortality rate was observed especially for cases with late admission. The major radiologic abnormalities were extensive pneumonic infiltration with segmental and multifocal distribution, mostly located in lower zones of the lung. No pleural effusion and hilar lymphadenopathy was noted. Conclusion: Avian flu may be presented as rapidly progressive pneumonia. The chest radiography has an important role in diagnosis and should be obtained daily because of rapid change of the findings that may necessitate prompt action.

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

    Directory of Open Access Journals (Sweden)

    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.

  12. Identifikasi Flu Burung H5N1 pada Unggas di Sekitar Kasus Flu Burung pada Manusia Tahun 2011 di Bekasi (AVIAN INFLUENZA H5N1 IDENTIFICATION IN AVIAN SPECIES SURROUNDING AVIAN INFLUENZA H5N1 HUMAN CASES IN BEKASI, WEST JAVA, 2011

    Directory of Open Access Journals (Sweden)

    Dyah Ayu Hewajuli

    2014-05-01

    Full Text Available H5N1 subtype Avian Influenza (AI virus is the causal agent  of AI disease in humans. In Indonesia,the first human AI occurred in Tangerang 2005.  Human AI in Indonesia has now spread into 12 provinces,including West Java, Jakarta, Banten, North Sumatra, East Java, Central Java, Lampung, South Sulawesi,West Sumatra, South Sumatra, Riau, and Bali. Until 2011, the total human AI cases were 182 cases  with150 deaths. This study was conducted to identify of H5N1 AI virus in birds in area surrounding a humanAI human case  in Bekasi city  in March 2011 and to investigate its role in the spread of AI to humans usingmethods of Hemaglutination Inhibition (HI , and Reverse Transcriptase-Polymerase Chain Reaction(RT-PCR. The result showed that 80% of birds in the area surrounding AI  surrounding H5N1 AI humancase in Bekasi 2011 were antibody negative  against  H5N1-AI virus. Antibody against H5N1-AI viruswith the titer less than 4 log 2 was detected in 4.4%  of birds and  with antibody titer 04 4-7 log 2 in 15%of birds. By RT-PCR, H5N1 AI virus was not detected in 47.6% of bird samples. H5 positive and N1negative  AI virus was detected in  30.2% samples.  Only 11.2% samples showed positive for H5N1 AI virus.The results suggest that H5N1-AI virus affecting birds may have a positive role in transmitting to thevirus to human in Bekasi 2011.

  13. Experimental infection of swans and geese with highly pathogenic avian influenza virus (H5N1) of Asian lineage.

    Science.gov (United States)

    Brown, Justin D; Stallknecht, David E; Swayne, David E

    2008-01-01

    The role of wild birds in the epidemiology of the Asian lineage highly pathogenic avian influenza (HPAI) virus subtype H5N1 epizootic and their contribution to the spread of the responsible viruses in Eurasia and Africa are unclear. To better understand the potential role of swans and geese in the epidemiology of this virus, we infected 4 species of swans and 2 species of geese with an HPAI virus of Asian lineage recovered from a whooper swan in Mongolia in 2005, A/whooper swan/Mongolia/244/2005 (H5N1). The highest mortality rates were observed in swans, and species-related differences in clinical illness and viral shedding were evident. These results suggest that the potential for HPAI (H5N1) viral shedding and the movement of infected birds may be species-dependent and can help explain observed deaths associated with HPAI (H5N1) infection in anseriforms in Eurasia.

  14. Fluorescence biosensor based on CdTe quantum dots for specific detection of H5N1 avian influenza virus

    Science.gov (United States)

    Hoa Nguyen, Thi; Dieu Thuy Ung, Thi; Hien Vu, Thi; Tran, Thi Kim Chi; Quyen Dong, Van; Khang Dinh, Duy; Liem Nguyen, Quang

    2012-09-01

    This report highlights the fabrication of fluorescence biosensors based on CdTe quantum dots (QDs) for specific detection of H5N1 avian influenza virus. The core biosensor was composed of (i) the highly luminescent CdTe/CdS QDs, (ii) chromatophores extracted from bacteria Rhodospirillum rubrum, and (iii) the antibody of β-subunit. This core part was linked to the peripheral part of the biosensor via a biotin-streptavidin-biotin bridge and finally connected to the H5N1 antibody to make it ready for detecting H5N1 avian influenza virus. Detailed studies of each constituent were performed showing the image of QDs-labeled chromatophores under optical microscope, proper photoluminescence (PL) spectra of CdTe/CdS QDs, chromatophores and the H5N1 avian influenza viruses.

  15. Antibodies to H5 subtype avian influenza virus and Japanese encephalitis virus in northern pintails (Anas acuta) sampled in Japan

    Science.gov (United States)

    Ramey, Andy M.; Spackman, Erica; Yeh, Jung-Yong; Fujita, Go; Konishi, Kan; Reed, John A.; Wilcox, Benjamin R.; Brown, Justin D.; Stallknecht, David E.

    2013-01-01

    Blood samples from 105 northern pintails (Anas acuta) captured on Hokkaido, Japan were tested for antibodies to avian influenza virus (AIV), Japanese encephalitis virus (JEV), and West Nile virus (WNV) to assess possible involvement of this species in the spread of economically important and potentially zoonotic pathogens. Antibodies to AIV were detected in 64 of 105 samples (61%). Of the 64 positives, 95% and 81% inhibited agglutination of two different H5 AIV antigens (H5N1 and H5N9), respectively. Antibodies to JEV and WNV were detected in five (5%) and none of the samples, respectively. Results provide evidence for prior exposure of migrating northern pintails to H5 AIV which couldhave implications for viral shedding and disease occurrence. Results also provide evidence for limited involvement of this species in the transmission and spread of flaviviruses during spring migration.

  16. Migration of whooper swans and outbreaks of highly pathogenic avian influenza H5N1 virus in eastern Asia.

    Directory of Open Access Journals (Sweden)

    Scott H Newman

    Full Text Available Evaluating the potential involvement of wild avifauna in the emergence of highly pathogenic avian influenza H5N1 (hereafter H5N1 requires detailed analyses of temporal and spatial relationships between wild bird movements and disease emergence. The death of wild swans (Cygnus spp. has been the first indicator of the presence of H5N1 in various Asian and European countries; however their role in the geographic spread of the disease remains poorly understood. We marked 10 whooper swans (Cygnus cygnus with GPS transmitters in northeastern Mongolia during autumn 2006 and tracked their migratory movements in relation to H5N1 outbreaks. The prevalence of H5N1 outbreaks among poultry in eastern Asia during 2003-2007 peaked during winter, concurrent with whooper swan movements into regions of high poultry density. However outbreaks involving poultry were detected year round, indicating disease perpetuation independent of migratory waterbird presence. In contrast, H5N1 outbreaks involving whooper swans, as well as other migratory waterbirds that succumbed to the disease in eastern Asia, tended to occur during seasons (late spring and summer and in habitats (areas of natural vegetation where their potential for contact with poultry is very low to nonexistent. Given what is known about the susceptibility of swans to H5N1, and on the basis of the chronology and rates of whooper swan migration movements, we conclude that although there is broad spatial overlap between whooper swan distributions and H5N1 outbreak locations in eastern Asia, the likelihood of direct transmission between these groups is extremely low. Thus, our data support the hypothesis that swans are best viewed as sentinel species, and moreover, that in eastern Asia, it is most likely that their infections occurred through contact with asymptomatic migratory hosts (e.g., wild ducks at or near their breeding grounds.

  17. Migration of whooper swans and outbreaks of highly pathogenic avian influenza H5N1 virus in eastern Asia.

    Science.gov (United States)

    Newman, Scott H; Iverson, Samuel A; Takekawa, John Y; Gilbert, Martin; Prosser, Diann J; Batbayar, Nyambyar; Natsagdorj, Tseveenmyadag; Douglas, David C

    2009-05-28

    Evaluating the potential involvement of wild avifauna in the emergence of highly pathogenic avian influenza H5N1 (hereafter H5N1) requires detailed analyses of temporal and spatial relationships between wild bird movements and disease emergence. The death of wild swans (Cygnus spp.) has been the first indicator of the presence of H5N1 in various Asian and European countries; however their role in the geographic spread of the disease remains poorly understood. We marked 10 whooper swans (Cygnus cygnus) with GPS transmitters in northeastern Mongolia during autumn 2006 and tracked their migratory movements in relation to H5N1 outbreaks. The prevalence of H5N1 outbreaks among poultry in eastern Asia during 2003-2007 peaked during winter, concurrent with whooper swan movements into regions of high poultry density. However outbreaks involving poultry were detected year round, indicating disease perpetuation independent of migratory waterbird presence. In contrast, H5N1 outbreaks involving whooper swans, as well as other migratory waterbirds that succumbed to the disease in eastern Asia, tended to occur during seasons (late spring and summer) and in habitats (areas of natural vegetation) where their potential for contact with poultry is very low to nonexistent. Given what is known about the susceptibility of swans to H5N1, and on the basis of the chronology and rates of whooper swan migration movements, we conclude that although there is broad spatial overlap between whooper swan distributions and H5N1 outbreak locations in eastern Asia, the likelihood of direct transmission between these groups is extremely low. Thus, our data support the hypothesis that swans are best viewed as sentinel species, and moreover, that in eastern Asia, it is most likely that their infections occurred through contact with asymptomatic migratory hosts (e.g., wild ducks) at or near their breeding grounds.

  18. Experimental infection of highly pathogenic avian influenza virus H5N1 in black-headed gulls (Chroicocephalus ridibundus)

    OpenAIRE

    Ramis , Antonio; van Amerongen , Geert; van de Bildt , Marco; Leijten , Loneke; Vanderstichel , Raphael; Osterhaus , Albert; Kuiken , Thijs

    2014-01-01

    Historically, highly pathogenic avian influenza viruses (HPAIV) rarely resulted in infection or clinical disease in wild birds. However, since 2002, disease and mortality from natural HPAIV H5N1 infection have been observed in wild birds including gulls. We performed an experimental HPAIV H5N1 infection of black-headed gulls (Chroicocephalus ridibundus) to determine their susceptibility to infection and disease from this virus, pattern of viral shedding, clinical signs, pathological changes a...

  19. H5N1 Influenza A Virus PB1-F2 Relieves HAX-1-Mediated Restriction of Avian Virus Polymerase PA in Human Lung Cells.

    Science.gov (United States)

    Mazel-Sanchez, B; Boal-Carvalho, I; Silva, F; Dijkman, R; Schmolke, M

    2018-06-01

    Highly pathogenic influenza A viruses (IAV) from avian hosts were first reported to directly infect humans 20 years ago. However, such infections are rare events, and our understanding of factors promoting or restricting zoonotic transmission is still limited. One accessory protein of IAV, PB1-F2, was associated with pathogenicity of pandemic and zoonotic IAV. This short (90-amino-acid) peptide does not harbor an enzymatic function. We thus identified host factors interacting with H5N1 PB1-F2, which could explain its importance for virulence. PB1-F2 binds to HCLS1-associated protein X1 (HAX-1), a recently identified host restriction factor of the PA subunit of IAV polymerase complexes. We demonstrate that the PA of a mammal-adapted H1N1 IAV is resistant to HAX-1 imposed restriction, while the PA of an avian-origin H5N1 IAV remains sensitive. We also showed HAX-1 sensitivity for PAs of A/Brevig Mission/1/1918 (H1N1) and A/Shanghai/1/2013 (H7N9), two avian-origin zoonotic IAV. Inhibition of H5N1 polymerase by HAX-1 can be alleviated by its PB1-F2 through direct competition. Accordingly, replication of PB1-F2-deficient H5N1 IAV is attenuated in the presence of large amounts of HAX-1. Mammal-adapted H1N1 and H3N2 viruses do not display this dependence on PB1-F2 for efficient replication in the presence of HAX-1. We propose that PB1-F2 plays a key role in zoonotic transmission of avian H5N1 IAV into humans. IMPORTANCE Aquatic and shore birds are the natural reservoir of influenza A viruses from which the virus can jump into a variety of bird and mammal host species, including humans. H5N1 influenza viruses are a good model for this process. They pose an ongoing threat to human and animal health due to their high mortality rates. However, it is currently unclear what restricts these interspecies jumps on the host side or what promotes them on the virus side. Here we show that a short viral peptide, PB1-F2, helps H5N1 bird influenza viruses to overcome a human restriction

  20. Lethal infection by a novel reassortant H5N1 avian influenza A virus in a zoo-housed tiger.

    Science.gov (United States)

    He, Shang; Shi, Jianzhong; Qi, Xian; Huang, Guoqing; Chen, Hualan; Lu, Chengping

    2015-01-01

    In early 2013, a Bengal tiger (Panthera tigris) in a zoo died of respiratory distress. All specimens from the tiger were positive for HPAI H5N1, which were detected by real-time PCR, including nose swab, throat swab, tracheal swab, heart, liver, spleen, lung, kidney, aquae pericardii and cerebrospinal fluid. One stain of virus, A/Tiger/JS/1/2013, was isolated from the lung sample. Pathogenicity experiments showed that the isolate was able to replicate and cause death in mice. Phylogenetic analysis indicated that HA and NA of A/Tiger/JS/1/2013 clustered with A/duck/Vietnam/OIE-2202/2012 (H5N1), which belongs to clade 2.3.2.1. Interestingly, the gene segment PB2 shared 98% homology with A/wild duck/Korea/CSM-28/20/2010 (H4N6), which suggested that A/Tiger/JS/1/2013 is a novel reassortant H5N1 subtype virus. Immunohistochemical analysis also confirmed that the tiger was infected by this new reassortant HPAI H5N1 virus. Overall, our results showed that this Bengal tiger was infected by a novel reassortant H5N1, suggesting that the H5N1 virus can successfully cross species barriers from avian to mammal through reassortment. Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.