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Sample records for ai h5n1 virus

  1. Characterization of low-pathogenicity H5N1 avian influenza viruses from North America

    USGS Publications Warehouse

    Spackman, Erica; Swayne, D.E.; Suarez, D.L.; Senne, D.A.; Pedersen, J.C.; Killian, M.L.; Pasick, J.; Handel, K.; Pillai, S.P.S.; Lee, C.-W.; Stallknecht, D.; Slemons, R.; Ip, H.S.; Deliberto, T.

    2007-01-01

    Wild-bird surveillance in North America for avian influenza (AI) viruses with a goal of early identification of the Asian H5N1 highly pathogenic AI virus has identified at least six low-pathogenicity H5N1 AI viruses between 2004 and 2006. The hemagglutinin (HA) and neuraminidase (NA) genes from all 6 H5N1 viruses and an additional 38 North American wild-bird-origin H5 subtype and 28 N1 subtype viruses were sequenced and compared with sequences available in GenBank by phylogenetic analysis. Both HA and NA were phylogenetically distinct from those for viruses from outside of North America and from those for viruses recovered from mammals. Four of the H5N1 AI viruses were characterized as low pathogenicity by standard in vivo pathotyping tests. One of the H5N1 viruses, A/MuteSwan/MI/451072-2/06, was shown to replicate to low titers in chickens, turkeys, and ducks. However, transmission of A/MuteSwan/MI/451072-2/06 was more efficient among ducks than among chickens or turkeys based on virus shed. The 50% chicken infectious dose for A/MuteSwan/MI/451072-2/06 and three other wild-waterfowl-origin H5 viruses were also determined and were between 10 5.3 and 107.5 50% egg infective doses. Finally, seven H5 viruses representing different phylogenetic clades were evaluated for their antigenic relatedness by hemagglutination inhibition assay, showing that the antigenic relatedness was largely associated with geographic origin. Overall, the data support the conclusion that North American H5 wild-bird-origin AI viruses are low-pathogenicity wild-bird-adapted viruses and are antigenically and genetically distinct from the highly pathogenic Asian H5N1 virus lineage. Copyright ?? 2007, American Society for Microbiology. All Rights Reserved.

  2. Characterization of low-pathogenicity H5N1 avian influenza viruses from North America

    USGS Publications Warehouse

    Spackman, Erica; Swayne, David E.; Suarez, David L.; Senne, Dennis A.; Pedersen, Janice C.; Killian, Mary Lea; Pasick, John; Handel, Katherine; Somanathan Pillai, Smitha; Lee, Chang-Won; Stallknecht, David; Slemons, Richard; Ip, Hon S.; Deliberto, Tom

    2007-01-01

    Wild-bird surveillance in North America for avian influenza (AI) viruses with a goal of early identification of the Asian H5N1 highly pathogenic AI virus has identified at least six low-pathogenicity H5N1 AI viruses between 2004 and 2006. The hemagglutinin (HA) and neuraminidase (NA) genes from all 6 H5N1 viruses and an additional 38 North American wild-bird-origin H5 subtype and 28 N1 subtype viruses were sequenced and compared with sequences available in GenBank by phylogenetic analysis. Both HA and NA were phylogenetically distinct from those for viruses from outside of North America and from those for viruses recovered from mammals. Four of the H5N1 AI viruses were characterized as low pathogenicity by standard in vivo pathotyping tests. One of the H5N1 viruses, A/MuteSwan/MI/451072-2/06, was shown to replicate to low titers in chickens, turkeys, and ducks. However, transmission of A/MuteSwan/MI/451072-2/06 was more efficient among ducks than among chickens or turkeys based on virus shed. The 50% chicken infectious dose for A/MuteSwan/MI/451072-2/06 and three other wild-waterfowl-origin H5 viruses were also determined and were between 105.3 and 107.5 50% egg infective doses. Finally, seven H5 viruses representing different phylogenetic clades were evaluated for their antigenic relatedness by hemagglutination inhibition assay, showing that the antigenic relatedness was largely associated with geographic origin. Overall, the data support the conclusion that North American H5 wild-bird-origin AI viruses are low-pathogenicity wild-bird-adapted viruses and are antigenically and genetically distinct from the highly pathogenic Asian H5N1 virus lineage.

  3. H5N1 influenza viruses: facts, not fear.

    PubMed

    Palese, Peter; Wang, Taia T

    2012-02-14

    The ongoing controversy over publication of two studies involving the transmission in ferrets of H5N1 (H5) subtype influenza viruses and the recommendations of the National Science Advisory Board for Biosecurity to redact key details in the manuscripts call for an examination of relevant scientific facts. In addition, there are calls in the media to destroy the viruses, curtail future research in this area, and protect the public from such "frightening" research efforts. Fear needs to be put to rest with solid science and not speculation.

  4. Recombinant parainfluenza virus 5 expressing hemagglutinin of influenza A virus H5N1 protected mice against lethal highly pathogenic avian influenza virus H5N1 challenge.

    PubMed

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

    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.

  5. Characterization of H5N1 Influenza Virus Variants with Hemagglutinin Mutations Isolated from Patients

    PubMed Central

    Arai, Yasuha; Daidoji, Tomo; Kawashita, Norihito; Ibrahim, Madiha S.; El-Gendy, Emad El-Din M.; Hiramatsu, Hiroaki; Kubota-Koketsu, Ritsuko; Takagi, Tatsuya; Murata, Takeomi; Takahashi, Kazuo; Okuno, Yoshinobu; Nakaya, Takaaki; Suzuki, Yasuo; Ikuta, Kazuyoshi

    2015-01-01

    ABSTRACT A change in viral hemagglutinin (HA) receptor binding specificity from α2,3- to α2,6-linked sialic acid is necessary for highly pathogenic avian influenza (AI) virus subtype H5N1 to become pandemic. However, details of the human-adaptive change in the H5N1 virus remain unknown. Our database search of H5N1 clade 2.2.1 viruses circulating in Egypt identified multiple HA mutations that had been selected in infected patients. Using reverse genetics, we found that increases in both human receptor specificity and the HA pH threshold for membrane fusion were necessary to facilitate replication of the virus variants in human airway epithelia. Furthermore, variants with enhanced replication in human cells had decreased HA stability, apparently to compensate for the changes in viral receptor specificity and membrane fusion activity. Our findings showed that H5N1 viruses could rapidly adapt to growth in the human airway microenvironment by altering their HA properties in infected patients and provided new insights into the human-adaptive mechanisms of AI viruses. PMID:25852160

  6. Genotypic diversity of H5N1 highly pathogenic avian influenza viruses.

    PubMed

    Zhao, Zi-Ming; Shortridge, Kennedy F; Garcia, Maricarmen; Guan, Yi; Wan, Xiu-Feng

    2008-09-01

    Besides enormous economic losses to the poultry industry, recent H5N1 highly pathogenic avian influenza viruses (HPAIVs) originating in eastern Asia have posed serious threats to public health. Up to April 17, 2008, 381 human cases had been confirmed with a mortality of more than 60 %. Here, we attempt to identify potential progenitor genes for H5N1 HPAIVs since their first recognition in 1996; most were detected in the Eurasian landmass before 1996. Combinations among these progenitor genes generated at least 21 reassortants (named H5N1 progenitor reassortant, H5N1-PR1-21). H5N1-PR1 includes A/Goose/Guangdong/1/1996(H5N1). Only reassortants H5N1-PR2 and H5N1-PR7 were associated with confirmed human cases: H5N1-PR2 in the Hong Kong H5N1 outbreak in 1997 and H5N1-PR7 in laboratory confirmed human cases since 2003. H5N1-PR7 also contains a majority of the H5N1 viruses causing avian influenza outbreaks in birds, including the first wave of genotype Z, Qinghai-like and Fujian-like virus lineages. Among the 21 reassortants identified, 13 are first reported here. This study illustrates evolutionary patterns of H5N1 HPAIVs, which may be useful toward pandemic preparedness as well as avian influenza prevention and control.

  7. The spread of avian influenza H5N1 virus; a pandemic threat to mankind.

    PubMed

    Chutinimitkul, Salin; Payungporn, Sunchai; Chieochansin, Thaweesak; Suwannakarn, Kamol; Theamboonlers, Apiradee; Poovorawan, Yong

    2006-09-01

    Influenza A H5N1 virus infection presents a major public health problem in Asian and Eurasian countries. The World Health organization has voiced their concerns about a potential pandemic with the imminent threat to humankind. In 1997, an outbreak of highly pathogenic H5N1 virus emerged and caused severe systemic disease among poultry and humans in Hong Kong. This article reviews the magnitude of the 2004-2006 outbreaks in various countries and highlights the highly pathogenic avian influenza (HPAI) subtype H5N1 virus as the cause of a major epidemic with potentially vast repercussions on economics, public health and society at large. Not only has this avian influenza (AI) virus infected poultry but has also proven highly pathogenic and fatal to mammalian species including humans and felines. The present review draws a comprehensive picture encompassing epidemiology, inter-species transmission and genetic characterization of this highly virulent virus. Moreover, laboratory diagnostic techniques, vaccination strategies and antiviral therapies aimed at outbreak control and management are also discussed.

  8. Multiple introductions of highly pathogenic avian influenza H5N1 viruses into Bangladesh

    PubMed Central

    Marinova-Petkova, Atanaska; Feeroz, Mohammed M; Rabiul Alam, SM; Kamrul Hasan, M; Akhtar, Sharmin; Jones-Engel, Lisa; Walker, David; McClenaghan, Laura; Rubrum, Adam; Franks, John; Seiler, Patrick; Jeevan, Trushar; McKenzie, Pamela; Krauss, Scott; Webby, Richard J; Webster, Robert G

    2014-01-01

    Highly pathogenic H5N1 and low pathogenic H9N2 influenza viruses are endemic to poultry markets in Bangladesh and have cocirculated since 2008. H9N2 influenza viruses circulated constantly in the poultry markets, whereas highly pathogenic H5N1 viruses occurred sporadically, with peaks of activity in cooler months. Thirty highly pathogenic H5N1 influenza viruses isolated from poultry were characterized by antigenic, molecular, and phylogenetic analyses. Highly pathogenic H5N1 influenza viruses from clades 2.2.2 and 2.3.2.1 were isolated from live bird markets only. Phylogenetic analysis of the 30 H5N1 isolates revealed multiple introductions of H5N1 influenza viruses in Bangladesh. There was no reassortment between the local H9N2 influenza viruses and H5N1 genotype, despite their prolonged cocirculation. However, we detected two reassortant H5N1 viruses, carrying the M gene from the Chinese H9N2 lineage, which briefly circulated in the Bangladesh poultry markets and then disappeared. On the other hand, interclade reassortment occurred within H5N1 lineages and played a role in the genesis of the currently dominant H5N1 viruses in Bangladesh. Few ‘human-like' mutations in H5N1 may account for the limited number of human cases. Antigenically, clade 2.3.2.1 H5N1 viruses in Bangladesh have evolved since their introduction and are currently mainly homogenous, and show evidence of recent antigenic drift. Although reassortants containing H9N2 genes were detected in live poultry markets in Bangladesh, these reassortants failed to supplant the dominant H5N1 lineage. PMID:26038508

  9. T-705 (favipiravir) activity against lethal H5N1 influenza A viruses.

    PubMed

    Kiso, Maki; Takahashi, Kazumi; Sakai-Tagawa, Yuko; Shinya, Kyoko; Sakabe, Saori; Le, Quynh Mai; Ozawa, Makoto; Furuta, Yousuke; Kawaoka, Yoshihiro

    2010-01-12

    The neuraminidase inhibitors oseltamivir and zanamivi are used to treat H5N1 influenza. However, oseltamivir-resistant H5N1 viruses have been isolated from oseltamivir-treated patients. Moreover, reassortment between H5N1 viruses and oseltamvir-resistant human H1N1 viruses currently circulating could create oseltamivir-resistant H5N1 viruses, rendering the oseltamivir stockpile obsolete. Therefore, there is a need for unique and effective antivirals to combat H5N1 influenza viruses. The investigational drug T-705 (favipiravir; 6-fluoro-3-hydroxy-2-pyrazinecarboxamide) has antiviral activity against seasonal influenza viruses and a mouse-adapted H5N1 influenza virus derived from a benign duck virus. However, its efficacy against highly pathogenic H5N1 viruses, which are substantially more virulent, remains unclear. Here, we demonstrate that T-705 effectively protects mice from lethal infection with oseltamivir-sensitive or -resistant highly pathogenic H5N1 viruses. Furthermore, our biochemical analysis suggests that T-705 ribofuranosyl triphosphate, an active form of T-705, acts like purines or purine nucleosides in human cells and does not inhibit human DNA synthesis. We conclude that T-705 shows promise as a therapeutic agent for the treatment of highly pathogenic H5N1 influenza patients.

  10. Determinants of pathogenicity of H5N1 highly pathogenic avian influenza viruses in ducks

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ducks have been implicated in the dissemination and evolution of the H5N1 highly pathogenic avian influenza (HPAI) viruses. The pathogenicity of H5N1 HPAI viruses in domestic ducks has increased over time with some viruses producing 100% mortality in very short time. The determinants of pathogenic...

  11. Pathogenicity of two Egyptian H5N1 highly pathogenic avian influenza viruses in domestic ducks

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Domestic ducks have been implicated in the dissemination and evolution of H5N1 highly pathogenic avian influenza (HPAI) viruses. Interestingly, the pathogenicity of H5N1 HPAI viruses in domestic ducks has increased over time with some viruses producing 100% mortality in ducks. These changes in vir...

  12. Protective Efficacy of the Inactivated H5N1 Influenza Vaccine Re-6 Against Different Clades of H5N1 Viruses Isolated in China and the Democratic People's Republic of Korea.

    PubMed

    Zeng, Xianying; Deng, Guohua; Liu, Liling; Li, Yanbing; Shi, Jianzhong; Chen, Pucheng; Feng, Huapeng; Liu, Jingli; Guo, Xingfu; Mao, Shenggang; Yang, Fan; Chen, Zhiyu; Tian, Guobin; Chen, Hualan

    2016-05-01

    An inactivated H5N1 avian influenza (AI) vaccine (Re-6) that bears the HA and NA genes from a clade 2.3.2.1 H5N1 virus, A/duck/Guangdong/S1322/10 (DK/GD/S1322/10), has been used in domestic poultry in China and other Southeast Asian countries to control clade 2.3.2.1 H5N1viruses since 2012. The efficacy of this vaccine against H5N1 viruses isolated in recent years has not been reported. In this study, we evaluated the protection efficacy of the Re-6 vaccine in chickens against challenge with four clade 2.3.2.1 H5N1 viruses, one clade 2.3.4.4 H5N1 virus, and one clade 7.2 H5N1 virus; these viruses were isolated in mainland China, Hong Kong, and the Democratic People's Republic of Korea between 2011 and 2015. The vaccinated chickens were completely protected (no disease signs, virus shedding, or death) from the challenge with the four clade 2.3.2.1 H5N1 viruses. In the clade 7.2 virus-challenged group, all of the vaccinated chickens remained healthy and survived for the entire 2-wk observation period; virus shedding was only detected from 1 of 10 chickens on day 3 postchallenge. In the clade 2.3.4.4 virus-challenged group, 8 of the 10 vaccinated chickens remained healthy and survived the 2-wk observation period; however, virus shedding was detected from 8 of 10 chickens on day 5 postchallenge. These results indicate that the Re-6 vaccine provides solid protection against clade 2.3.2.1, good protection against clade 7.2, and poor protection against clade 2.3.4.4. PMID:27309061

  13. Genetic characterization of HPAI (H5N1) viruses from poultry and wild vultures, Burkina Faso.

    PubMed

    Ducatez, Mariette F; Tarnagda, Zekiba; Tahita, Marc C; Sow, Adama; de Landtsheer, Sebastien; Londt, Brandon Z; Brown, Ian H; Osterhaus, D M E; Fouchier, Ron A M; Ouedraogo, Jean-Bosco B; Muller, Claude P

    2007-04-01

    Genetic analysis of highly pathogenic avian influenza (H5N1) viruses from poultry and hooded vultures in Burkina Faso shows that these viruses belong to 1 of 3 sublineages initially found in Nigeria and later in other African countries. Hooded vultures could potentially be vectors or sentinels of influenza subtype H5N1, as are cats and swans elsewhere. PMID:17553279

  14. Increased virulence in ducks of H5N1 highly pathogenic avian influenza viruses from Egypt

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The pathogenicity of H5N1 highly pathogenic avian influenza (HPAI) viruses in domestic ducks has increased over time. These changes in virulence have been reported with viruses from countries with high population of domestic ducks. Since 2006, H5N1 HPAI outbreaks in Egypt have been occurring in po...

  15. Genetic Characterization of HPAI (H5N1) Viruses from Poultry and Wild Vultures, Burkina Faso

    PubMed Central

    Ducatez, Mariette F.; Tarnagda, Zekiba; Tahita, Marc C.; Sow, Adama; de Landtsheer, Sebastien; Londt, Brandon Z.; Brown, Ian H.; Osterhaus, Albert D.M.E.; Fouchier, Ron A.M.; Ouedraogo, Jean-Bosco B.

    2007-01-01

    Genetic analysis of highly pathogenic avian influenza (H5N1) viruses from poultry and hooded vultures in Burkina Faso shows that these viruses belong to 1 of 3 sublineages initially found in Nigeria and later in other African countries. Hooded vultures could potentially be vectors or sentinels of influenza subtype H5N1, as are cats and swans elsewhere. PMID:17553279

  16. Airborne transmission of H5N1 high pathogenicity avian influenza viruses during simulated home slaughter

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Most H5N1 human infections have occurred following exposure to H5N1 high pathogenicity avian influenza (HPAI) virus-infected poultry, especially when poultry are home slaughtered or slaughtered in live poultry markets. Previous studies have demonstrated that slaughter of clade 1 isolate A/Vietnam/1...

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

    PubMed

    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

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

    PubMed Central

    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

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

    PubMed

    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.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Highly pathogenic (HP) H5N1 avian influenza (AI) viruses continue to circulate in Asia and other regions of the world. Vaccination is used as part of H5N1 HPAI control programs in many countries; however, eradication of the disease has not been possible due to the emergence and spread of new viruses...

  1. Detection prevalence of H5N1 avian influenza virus among stray cats in eastern China.

    PubMed

    Zhao, Fu-Rong; Zhou, Dong-Hui; Zhang, Yong-Guang; Shao, Jun-Jun; Lin, Tong; Li, Yang-Fan; Wei, Ping; Chang, Hui-Yun

    2015-08-01

    Since 1997, more and more cases of the infectious H5N1 avian influenza virus (AIV) in humans have been reported all over the world but the transmission of H5N1 avian influenza virus to stray cats has been little demonstrated. The objective of this pilot investigation was to determine the prevalence of H5N1 AIV antibodies in stray cats in eastern China where is the dominant enzootic H5N1 highly pathogenic avian influenza virus (HP AIV). A total of 1,020 nasal swab and 1,020 serum samples were collected and tested. Evidence of HPAI H5N1 virus antibodies was present in two of the 1,020 serum samples that were positive by HI assay and NT assay, respectively. The results imply little transmission and that the Clade 2.3.2 HPAIV H5N1 infections in poultry did not significantly affect the rural animal shelters or suburban environment in eastern China. In future studies, these results can be used as baseline seroepidemiological levels for H5N1 AIV among cats in China.

  2. Detection prevalence of H5N1 avian influenza virus among stray cats in eastern China.

    PubMed

    Zhao, Fu-Rong; Zhou, Dong-Hui; Zhang, Yong-Guang; Shao, Jun-Jun; Lin, Tong; Li, Yang-Fan; Wei, Ping; Chang, Hui-Yun

    2015-08-01

    Since 1997, more and more cases of the infectious H5N1 avian influenza virus (AIV) in humans have been reported all over the world but the transmission of H5N1 avian influenza virus to stray cats has been little demonstrated. The objective of this pilot investigation was to determine the prevalence of H5N1 AIV antibodies in stray cats in eastern China where is the dominant enzootic H5N1 highly pathogenic avian influenza virus (HP AIV). A total of 1,020 nasal swab and 1,020 serum samples were collected and tested. Evidence of HPAI H5N1 virus antibodies was present in two of the 1,020 serum samples that were positive by HI assay and NT assay, respectively. The results imply little transmission and that the Clade 2.3.2 HPAIV H5N1 infections in poultry did not significantly affect the rural animal shelters or suburban environment in eastern China. In future studies, these results can be used as baseline seroepidemiological levels for H5N1 AIV among cats in China. PMID:25952001

  3. The TLR4-TRIF Pathway Protects against H5N1 Influenza Virus Infection

    PubMed Central

    Shinya, Kyoko; Ito, Mutsumi; Makino, Akiko; Tanaka, Motoko; Miyake, Kensuke; Eisfeld, Amie J.

    2012-01-01

    Prestimulation of the TLR4 pathway with lipopolysaccharide (LPS) protects mice from lethal infection with H5N1 influenza virus. Here, we reveal that the TLR4-TRIF pathway is required for this protective effect by using mice whose TLR4-related molecules were knocked out. Microarray analysis of primary mouse lung culture cells that were LPS pretreated and infected with an H5N1 virus indicated that TLR3 mRNA was upregulated. Primary lung culture cells of TLR3 knockout mice showed no response to LPS pretreatment against H5N1 virus infection, suggesting that TLR3 is also involved in the preventive effect of LPS. Our data suggest that the TLR4-TRIF axis has an important role in stimulating protective innate immunity against H5N1 influenza A virus infection and that TLR3 signaling is involved in this pathway. PMID:22031950

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

    PubMed Central

    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, and the next pandemic may well arise from a low-pathogenicity virus. The rationale for particular concern about an H5N1 pandemic is not its inevitability but its potential severity. An H5N1 pandemic is an event of low probability but one of high human health impact and poses a predicament for public health. Here, we review the ecology and evolution of highly pathogenic avian influenza H5N1 viruses, assess the pandemic risk, and address aspects of human H5N1 disease in relation to its epidemiology, clinical presentation, pathogenesis, diagnosis, and management. PMID:17428885

  5. Influenza viruses and the evolution of avian influenza virus H5N1.

    PubMed

    Skeik, Nedaa; Jabr, Fadi I

    2008-05-01

    Although small in size and simple in structure, influenza viruses are sophisticated organisms with highly mutagenic genomes and wide antigenic diversity. They are species-specific organisms. Mutation and reassortment have resulted in newer viruses such as H5N1, with new resistance against anti-viral medications, and this might lead to the emergence of a fully transmissible strain, as occurred in the 1957 and 1968 pandemics. Influenza viruses are no longer just a cause of self-limited upper respiratory tract infections; the H5N1 avian influenza virus can cause severe human infection with a mortality rate exceeding 50%. The case death rate of H5N1 avian influenza infection is 20 times higher than that of the 1918 infection (50% versus 2.5%), which killed 675000 people in the USA and almost 40 million people worldwide. While the clock is still ticking towards what seems to be inevitable pandemic influenza, on April 17, 2007 the U.S. Food and Drug Administration (FDA) approved the first vaccine against the avian influenza virus H5N1 for humans at high risk. However, more research is needed to develop a more effective and affordable vaccine that can be given at lower doses.

  6. Lemna (duckweed) expressed hemagglutinin from avian influenza H5N1 protects chickens against H5N1 high pathogenicity avian influenza virus challenge

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In the last two decades, transgenic plants have been explored as safe and cost effective alternative expression platforms for producing recombinant proteins. In this study, a synthetic hemagglutinin (HA) gene from the high pathogenicity avian influenza (HPAI) virus A/chicken/Indonesia/7/2003 (H5N1)...

  7. Airborne Transmission of Influenza A/H5N1 Virus Between Ferrets

    PubMed Central

    Herfst, Sander; Schrauwen, Eefje J. A.; Linster, Martin; Chutinimitkul, Salin; de Wit, Emmie; Munster, Vincent J.; Sorrell, Erin M.; Bestebroer, Theo M.; Burke, David F.; Smith, Derek J.; Rimmelzwaan, Guus F.; Osterhaus, Albert D. M. E.; Fouchier, Ron A. M.

    2016-01-01

    Highly pathogenic avian influenza A/H5N1 virus can cause morbidity and mortality in humans but thus far has not acquired the ability to be transmitted by aerosol or respiratory droplet (“airborne transmission”) between humans. To address the concern that the virus could acquire this ability under natural conditions, we genetically modified A/H5N1 virus by site-directed mutagenesis and subsequent serial passage in ferrets. The genetically modified A/H5N1 virus acquired mutations during passage in ferrets, ultimately becoming airborne transmissible in ferrets. None of the recipient ferrets died after airborne infection with the mutant A/H5N1 viruses. Four amino acid substitutions in the host receptor-binding protein hemagglutinin, and one in the polymerase complex protein basic polymerase 2, were consistently present in airborne-transmitted viruses. The transmissible viruses were sensitive to the antiviral drug oseltamivir and reacted well with antisera raised against H5 influenza vaccine strains. Thus, avian A/H5N1 influenza viruses can acquire the capacity for airborne transmission between mammals without recombination in an intermediate host and therefore constitute a risk for human pandemic influenza. PMID:22723413

  8. Characterization of a highly pathogenic avian influenza H5N1 virus isolated from an ostrich.

    PubMed

    Yang, Penghui; Dongmei; Wang, Cheng; Tang, Chong; Xing, Li; Luo, Deyan; Zhan, Zhongpeng; Duan, Yueqiang; Jia, Weihong; Peng, Daxin; Liu, Xiufan; Wang, Xiliang

    2010-06-11

    The continued spread of a highly pathogenic avian influenza (HPAI) H5N1 virus among poultry and wild birds has posed a potential threat to human public health. An influenza pandemic happens, when a new subtype that has not previously circulated in humans emerges. Almost all of the influenza pandemics in history have originated from avian influenza viruses (AIV). Birds are significant reservoirs of influenza viruses. In the present study, we performed a survey of avian influenza virus in ostriches and H5N1 virus (A/Ostrich/SuZhou/097/03, China097) was isolated. This H5N1 virus is highly pathogenic to both chickens and mice. It is also able to replicate in the lungs of, and to cause death in, BALB/c mice following intranasal administration. It forms plaques in chicken embryo fibroblast (CEF) cells in the absence of trypsin. The hemagglutinin (HA) gene of the virus is genetically similar to A/Goose/Guangdong/1/96(H5N1) and belongs to clade 0. The HA sequence contains multiple basic amino acids adjacent to the cleavage site, a motif associated with HPAI viruses. More importantly, the existence of H5N1 isolates in ostriches highlights the potential threat of wild bird infections to veterinary and public health. PMID:20497905

  9. Greater virulence of highly pathogenic H5N1 influenza virus in cats than in dogs.

    PubMed

    Kim, Heui Man; Park, Eun Hye; Yum, Jung; Kim, Hyun Soo; Seo, Sang Heui

    2015-01-01

    Highly pathogenic H5N1 influenza virus continues to infect animals and humans. We compared the infectivity and pathogenesis of H5N1 virus in domestic cats and dogs to find out which animal is more susceptible to H5N1 influenza virus. When cats and dogs were infected with the H5N1 virus, cats suffered from severe outcomes including death, whereas dogs did not show any mortality. Viruses were shed in the nose and rectum of cats and in the nose of dogs. Viruses were detected in brain, lung, kidney, intestine, liver, and serum in the infected cats, but only in the lung in the infected dogs. Genes encoding inflammatory cytokines and chemokines, Toll-like receptors, and apoptotic factors were more highly expressed in the lungs of cats than in those of dogs. Our results suggest that the intensive monitoring of dogs is necessary to prevent human infection by H5N1 influenza virus, since infected dogs may not show clear clinical signs, in contrast to infected cats.

  10. The evolution of H5N1 influenza viruses in ducks in southern China.

    PubMed

    Chen, H; Deng, G; Li, Z; Tian, G; Li, Y; Jiao, P; Zhang, L; Liu, Z; Webster, R G; Yu, K

    2004-07-13

    The pathogenicity of avian H5N1 influenza viruses to mammals has been evolving since the mid-1980s. Here, we demonstrate that H5N1 influenza viruses, isolated from apparently healthy domestic ducks in mainland China from 1999 through 2002, were becoming progressively more pathogenic for mammals, and we present a hypothesis explaining the mechanism of this evolutionary direction. Twenty-one viruses isolated from apparently healthy ducks in southern China from 1999 through 2002 were confirmed to be H5N1 subtype influenza A viruses. These isolates are antigenically similar to A/Goose/Guangdong/1/96 (H5N1) virus, which was the source of the 1997 Hong Kong "bird flu" hemagglutinin gene, and all are highly pathogenic in chickens. The viruses form four pathotypes on the basis of their replication and lethality in mice. There is a clear temporal pattern in the progressively increasing pathogenicity of these isolates in the mammalian model. Five of six H5N1 isolates tested replicated in inoculated ducks and were shed from trachea or cloaca, but none caused disease signs or death. Phylogenetic analysis of the full genome indicated that most of the viruses are reassortants containing the A/Goose/Guangdong/1/96-like hemagglutinin gene and the other genes from unknown Eurasian avian influenza viruses. This study is a characterization of the H5N1 avian influenza viruses recently circulating in ducks in mainland China. Our findings suggest that immediate action is needed to prevent the transmission of highly pathogenic avian influenza viruses from the apparently healthy ducks into chickens or mammalian hosts.

  11. Evolution of highly pathogenic avian H5N1 influenza viruses

    SciTech Connect

    Macken, Catherine A; Green, Margaret A

    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 avian H5N1 viruses.

  12. Influenza (H5N1) viruses in poultry, Russian Federation, 2005-2006.

    PubMed

    Lipatov, Aleksandr S; Evseenko, Vasily A; Yen, Hui-Ling; Zaykovskaya, Anna V; Durimanov, Alexander G; Zolotykh, Sergey I; Netesov, Sergey V; Drozdov, Ilya G; Onishchenko, Gennadiy G; Webster, Robert G; Shestopalov, Alexander M

    2007-04-01

    We studied 7 influenza (H5N1) viruses isolated from poultry in western Siberia and the European part of the Russian Federation during July 2005-February 2006. Full genome sequences showed high homology to Qinghai-like influenza (H5N1) viruses. Phylogenetic analysis not only showed a close genetic relationship between the H5N1 strains isolated from poultry and wild migratory waterfowls but also suggested genetic reassortment among the analyzed isolates. Analysis of deduced amino acid sequences of the M2 and neuraminidase proteins showed that all isolates are potentially sensitive to currently available antiviral drugs. Pathogenicity testing showed that all studied viruses were highly pathogenic in chickens; for 3 isolates tested in mice and 2 tested in ferrets, pathogenicity was heterogeneous. Pathogenicity in mammalian models was generally correlated with Lys at residue 627 of polymerase basic protein 2.

  13. Susceptibility of Canada Geese (Branta canadensis) to highly pathogenic avian influenza virus (H5N1).

    PubMed

    Pasick, John; Berhane, Yohannes; Embury-Hyatt, Carissa; Copps, John; Kehler, Helen; Handel, Katherine; Babiuk, Shawn; Hooper-McGrevy, Kathleen; Li, Yan; Mai Le, Quynh; Lien Phuong, Song

    2007-12-01

    Migratory birds have been implicated in the long-range spread of highly pathogenic avian influenza (HPAI) A virus (H5N1) from Asia to Europe and Africa. Although sampling of healthy wild birds representing a large number of species has not identified possible carriers of influenza virus (H5N1) into Europe, surveillance of dead and sick birds has demonstrated mute (Cygnus olor) and whooper (C. cygnus) swans as potential sentinels. Because of concerns that migratory birds could spread H5N1 subtype to the Western Hemisphere and lead to its establishment within free-living avian populations, experimental studies have addressed the susceptibility of several indigenous North American duck and gull species. We examined the susceptibility of Canada geese (Branta canadensis) to HPAI virus (H5N1). Large populations of this species can be found in periagricultural and periurban settings and thus may be of potential epidemiologic importance if H5N1 subtype were to establish itself in North American wild bird populations.

  14. Pathogenicity of reassortant H5N1 highly pathogenic avian influenza viruses in domestic ducks

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The pathogenicity of H5N1 highly pathogenic avian influenza (HPAI) viruses in domestic ducks has increased over time. These changes in virulence have been reported with viruses from countries with high population of domestic ducks, including Egypt. In order to understand which viral genes are contri...

  15. Highly Pathogenic Influenza A(H5N1) Virus Survival in Complex Artificial Aquatic Biotopes

    PubMed Central

    Horm, Viseth Srey; Gutiérrez, Ramona A.; Nicholls, John M.; Buchy, Philippe

    2012-01-01

    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. PMID:22514622

  16. Pathogenicity, Transmission and Antigenic Variation of H5N1 Highly Pathogenic Avian Influenza Viruses

    PubMed Central

    Jiao, Peirong; Song, Hui; Liu, Xiaoke; Song, Yafen; Cui, Jin; Wu, Siyu; Ye, Jiaqi; Qu, Nanan; Zhang, Tiemin; Liao, Ming

    2016-01-01

    H5N1 highly pathogenic avian influenza (HPAI) was one of the most important avian diseases in poultry production of China, especially in Guangdong province. In recent years, new H5N1 highly pathogenic avian influenza viruses (HPAIV) still emerged constantly, although all poultry in China were immunized with H5N1 vaccinations compulsorily. To better understand the pathogenicity and transmission of dominant clades of the H5N1 HPAIVs in chicken from Guangdong in 2012, we chose a clade 7.2 avian influenza virus named A/Chicken/China/G2/2012(H5N1) (G2) and a clade 2.3.2.1 avian influenza virus named A/Duck/China/G3/2012(H5N1) (G3) in our study. Our results showed that the chickens inoculated with 103 EID50 of G2 or G3 viruses all died, and the titers of virus replication detected in several visceral organs were high but different. In the naive contact groups, virus shedding was not detected in G2 group and all chickens survived, but virus shedding was detected in G3 group and all chickens died. These results showed that the two clades of H5N1 HPAIVs had high pathogenicity in chickens and the contact transmission of them was different in chickens. The results of cross reactive HI assay showed that antigens of G2 and G3 were very different from those of current commercial vaccines isolates (Re-4, Re-6, and D7). And to evaluate the protective efficacy of three vaccines against most isolates form Guangdong belonging to clade 2.3.2.1 in 2012, G3 was chosen to challenge the three vaccines such as Re-4, Re-6, and D7. First, chickens were immunized with 0.3 ml Re-4, Re-6, and D7 inactivated vaccines by intramuscular injection, respectively, and then challenged with 106 EID50 of G3 on day 28 post-vaccination. The D7 vaccine had 100% protection against G3 for chickens, the Re-6 vaccine had 88.9%, and the Re-4 vaccine only had 66.7%. Our results suggested that the D7 vaccine could prevent and control H5N1 virus outbreaks more effectively in Guangdong. From the above, it was

  17. Extended parallel process model and H5N1 influenza virus.

    PubMed

    Siu, Wanda

    2008-04-01

    This study integrated the Extended Parallel Process Model and forewarning cues to assess the promotion of preventive measures against the H5N1 influenza virus, a significant health threat that affects Asia, Europe, and the USA. There are two types of forewarning, (1) telling the audience that they will hear messages intended to persuade them and (2) telling the audience the topic and stance of the impending persuasive message. Analysis of ratings by 265 undergraduates indicated that forewarnings of the topic and stance of a promotional message on the H5N1 virus facilitated elaboration of coping-related thoughts which enhance perceived self-efficacy and a stronger behavioral intention to combat H5N1. Conversely, the elaboration of danger-related thoughts evoked some fear but enhanced source perception.

  18. Avian Influenza A(H5N1) Virus in Egypt

    PubMed Central

    Kandeil, Ahmed; El-Shesheny, Rabeh; Kayed, Ahmed S.; Maatouq, Asmaa M.; Cai, Zhipeng; McKenzie, Pamela P.; Webby, Richard J.; El Refaey, Samir; Kandeel, Amr; Ali, Mohamed A.

    2016-01-01

    In Egypt, avian influenza A subtype H5N1 and H9N2 viruses are enzootic in poultry. The control plan devised by veterinary authorities in Egypt to prevent infections in poultry focused mainly on vaccination and ultimately failed. Recently, widespread H5N1 infections in poultry and a substantial increase in the number of human cases of H5N1 infection were observed. We summarize surveillance data from 2009 through 2014 and show that avian influenza viruses are established in poultry in Egypt and are continuously evolving genetically and antigenically. We also discuss the epidemiology of human infection with avian influenza in Egypt and describe how the true burden of disease is underestimated. We discuss the failures of relying on vaccinating poultry as the sole intervention tool. We conclude by highlighting the key components that need to be included in a new strategy to control avian influenza infections in poultry and humans in Egypt. PMID:26886164

  19. Avian Influenza A(H5N1) Virus in Egypt.

    PubMed

    Kayali, Ghazi; Kandeil, Ahmed; El-Shesheny, Rabeh; Kayed, Ahmed S; Maatouq, Asmaa M; Cai, Zhipeng; McKenzie, Pamela P; Webby, Richard J; El Refaey, Samir; Kandeel, Amr; Ali, Mohamed A

    2016-03-01

    In Egypt, avian influenza A subtype H5N1 and H9N2 viruses are enzootic in poultry. The control plan devised by veterinary authorities in Egypt to prevent infections in poultry focused mainly on vaccination and ultimately failed. Recently, widespread H5N1 infections in poultry and a substantial increase in the number of human cases of H5N1 infection were observed. We summarize surveillance data from 2009 through 2014 and show that avian influenza viruses are established in poultry in Egypt and are continuously evolving genetically and antigenically. We also discuss the epidemiology of human infection with avian influenza in Egypt and describe how the true burden of disease is underestimated. We discuss the failures of relying on vaccinating poultry as the sole intervention tool. We conclude by highlighting the key components that need to be included in a new strategy to control avian influenza infections in poultry and humans in Egypt.

  20. Experimental infection and pathology of clade 2.2 H5N1 virus in gulls

    PubMed Central

    Gulyaeva, Marina A.; Zaykovskaia, Anna V.; Shestopalova, Lidia V.; Shestopalov, Aleksander M.

    2016-01-01

    During 2006, H5N1 HPAI caused an epizootic in wild birds, resulting in a die-off of Laridae in the Novosibirsk region at Chany Lake. In the present study, we infected common gulls (Larus canus) with a high dose of the H5N1 HPAI virus isolated from a common gull to determine if severe disease could be induced over the 28 day experimental period. Moderate clinical signs including diarrhea, conjunctivitis, respiratory distress and neurological signs were observed in virus-inoculated birds, and 50% died. The most common microscopic lesions observed were necrosis of the pancreas, mild encephalitis, mild myocarditis, liver parenchymal hemorrhages, lymphocytic hepatitis, parabronchi lumen hemorrhages and interstitial pneumonia. High viral titers were shed from the oropharyngeal route and virus was still detected in one bird at 25 days after infection. In the cloaca, the virus was detected sporadically in lower titers. The virus was transmitted to direct contact gulls. Thus, infected gulls can pose a significant risk of H5N1 HPAIV transmission to other wild migratory waterfowl and pose a risk to more susceptible poultry species. These findings have important implications regarding the mode of transmission and potential risks of H5N1 HPAI spread by gulls. PMID:26243601

  1. Single-Domain Antibodies Targeting Neuraminidase Protect against an H5N1 Influenza Virus Challenge

    PubMed Central

    Cardoso, Francisco Miguel; Ibañez, Lorena Itatí; Van den Hoecke, Silvie; De Baets, Sarah; Smet, Anouk; Roose, Kenny; Schepens, Bert; Descamps, Francis J.; Fiers, Walter; Muyldermans, Serge

    2014-01-01

    ABSTRACT Influenza virus neuraminidase (NA) is an interesting target of small-molecule antiviral drugs. We isolated a set of H5N1 NA-specific single-domain antibodies (N1-VHHm) and evaluated their in vitro and in vivo antiviral potential. Two of them inhibited the NA activity and in vitro replication of clade 1 and 2 H5N1 viruses. We then generated bivalent derivatives of N1-VHHm by two methods. First, we made N1-VHHb by genetically joining two N1-VHHm moieties with a flexible linker. Second, bivalent N1-VHH-Fc proteins were obtained by genetic fusion of the N1-VHHm moiety with the crystallizable region of mouse IgG2a (Fc). The in vitro antiviral potency against H5N1 of both bivalent N1-VHHb formats was 30- to 240-fold higher than that of their monovalent counterparts, with 50% inhibitory concentrations in the low nanomolar range. Moreover, single-dose prophylactic treatment with bivalent N1-VHHb or N1-VHH-Fc protected BALB/c mice against a lethal challenge with H5N1 virus, including an oseltamivir-resistant H5N1 variant. Surprisingly, an N1-VHH-Fc fusion without in vitro NA-inhibitory or antiviral activity also protected mice against an H5N1 challenge. Virus escape selection experiments indicated that one amino acid residue close to the catalytic site is required for N1-VHHm binding. We conclude that single-domain antibodies directed against influenza virus NA protect against H5N1 virus infection, and when engineered with a conventional Fc domain, they can do so in the absence of detectable NA-inhibitory activity. IMPORTANCE Highly pathogenic H5N1 viruses are a zoonotic threat. Outbreaks of avian influenza caused by these viruses occur in many parts of the world and are associated with tremendous economic loss, and these viruses can cause very severe disease in humans. In such cases, small-molecule inhibitors of the viral NA are among the few treatment options for patients. However, treatment with such drugs often results in the emergence of resistant viruses

  2. Highly Pathogenic Avian Influenza Virus (H5N1) Isolated from Whooper Swans, Japan

    PubMed Central

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

    2008-01-01

    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. PMID:18760011

  3. Chlorine inactivation of H5N1 highly pathogenic avian influenza virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two Asian strains of H5N1 highly pathogenic avian influenza virus were studied to determine their resistance to chlorination. Experiments were conducted at two pH levels (pH 7 and 8) at 5 C. CT (chlorine concentration x exposure time) values were calculated for different levels of inactivation. R...

  4. Evolution and adaptation of hemagglutinin gene of human H5N1 influenza virus.

    PubMed

    Wei, Kaifa; Chen, Yanfeng; Chen, Juan; Wu, Lingjuan; Xie, Daoxin

    2012-06-01

    The H5N1 HPAI virus has brought heavy loss to poultry industry. Although, there exists limited human-to-human transmission, it poses potential serious risks to public health. HA is responsible for receptor-binding and membrane-fusion and contains the host receptor-binding sites and major epitopes for neutralizing antibodies. To investigate molecular adaption of HPAI H5N1 viruses, we performed a phylogenetic analysis of HA sequences with 240 HPAI virus strains isolated from human. The topology of the tree reveals overall clustering of strains in four major clusters based on geographic location, and shows antigenic diversity of HA of human H5N1 isolates co-circulating in Asia, Africa, and Europe. The four clusters possess distinct features within the cleavage site and glycosylation sites, respectively. We identified six sites apparently evolving under positive selection, five of which persist in the population. Three positively selected sites are found to be located either within or flanking the receptor-binding sites, suggesting that selection at these sites may increase the affinity to human-type receptor. Furthermore, some sites are also associated with glycosylation and antigenic changes. In addition, two sites are found to be selected differentially in the two clusters. The analyses provide us deep insight into the adaptive evolution of human H5N1 viruses, show us several candidate mutations that could cause a pandemic, and suggest that efficiency measures should be taken to deal with potential risks.

  5. Intersubtype Reassortments of H5N1 Highly Pathogenic Avian Influenza Viruses Isolated from Quail.

    PubMed

    Nguyen, Tinh Huu; Than, Van Thai; Thanh, Hien Dang; Hung, Vu-Khac; Nguyen, Duc Tan; Kim, Wonyong

    2016-01-01

    H5N1 highly pathogenic avian influenza (HPAI) viruses are considered a threat to national animal industries, causing production losses and high mortality in domestic poultry. In recent years, quail has become a popular terrestrial poultry species raised for production of meat and eggs in Asia. In this study, to better understand the roles of quail in H5N1 viral evolution, two H5N1-positive samples, designated A/quail/Vietnam/CVVI-49/2010 (CVVI-49/2010) and A/quail/Vietnam/CVVI-50/2014 (CVVI-50/2014), were isolated from quail during H5N1 outbreaks in Vietnam, and their whole genome were analyzed. The phylogenetic analysis reveals new evolutionary variation in the worldwide H5N1 viruses. The quail HA genes were clustered into clades 1.1.1 (CVVI-49/2010) and clade 2.3.2.1c (CVVI-50/2014), which may have evolved from viruses circulating from chickens and/or ducks in Cambodia, mainland of China, Taiwan, Indonesia, and South Korea in recent years. Interestingly, the M2 gene of the CVVI-49/2010 strain contained amino acid substitutions at position 26L-I and 31S-N that are related to amantadine-resistance. In particular, the CVVI-50/2014 strain revealed evidence of multiple intersubtype reassortment events between virus clades 2.3.2.1c, 2.3.2.1b, and 2.3.2.1a. Data from this study supports the possible role of quail as an important intermediate host in avian influenza virus evolution. Therefore, additional surveillance is needed to monitor these HPAI viruses both serologically and virologically in quail. PMID:26900963

  6. Intersubtype Reassortments of H5N1 Highly Pathogenic Avian Influenza Viruses Isolated from Quail.

    PubMed

    Nguyen, Tinh Huu; Than, Van Thai; Thanh, Hien Dang; Hung, Vu-Khac; Nguyen, Duc Tan; Kim, Wonyong

    2016-01-01

    H5N1 highly pathogenic avian influenza (HPAI) viruses are considered a threat to national animal industries, causing production losses and high mortality in domestic poultry. In recent years, quail has become a popular terrestrial poultry species raised for production of meat and eggs in Asia. In this study, to better understand the roles of quail in H5N1 viral evolution, two H5N1-positive samples, designated A/quail/Vietnam/CVVI-49/2010 (CVVI-49/2010) and A/quail/Vietnam/CVVI-50/2014 (CVVI-50/2014), were isolated from quail during H5N1 outbreaks in Vietnam, and their whole genome were analyzed. The phylogenetic analysis reveals new evolutionary variation in the worldwide H5N1 viruses. The quail HA genes were clustered into clades 1.1.1 (CVVI-49/2010) and clade 2.3.2.1c (CVVI-50/2014), which may have evolved from viruses circulating from chickens and/or ducks in Cambodia, mainland of China, Taiwan, Indonesia, and South Korea in recent years. Interestingly, the M2 gene of the CVVI-49/2010 strain contained amino acid substitutions at position 26L-I and 31S-N that are related to amantadine-resistance. In particular, the CVVI-50/2014 strain revealed evidence of multiple intersubtype reassortment events between virus clades 2.3.2.1c, 2.3.2.1b, and 2.3.2.1a. Data from this study supports the possible role of quail as an important intermediate host in avian influenza virus evolution. Therefore, additional surveillance is needed to monitor these HPAI viruses both serologically and virologically in quail.

  7. Intersubtype Reassortments of H5N1 Highly Pathogenic Avian Influenza Viruses Isolated from Quail

    PubMed Central

    Nguyen, Tinh Huu; Than, Van Thai; Thanh, Hien Dang; Hung, Vu-Khac; Nguyen, Duc Tan; Kim, Wonyong

    2016-01-01

    H5N1 highly pathogenic avian influenza (HPAI) viruses are considered a threat to national animal industries, causing production losses and high mortality in domestic poultry. In recent years, quail has become a popular terrestrial poultry species raised for production of meat and eggs in Asia. In this study, to better understand the roles of quail in H5N1 viral evolution, two H5N1-positive samples, designated A/quail/Vietnam/CVVI-49/2010 (CVVI-49/2010) and A/quail/Vietnam/CVVI-50/2014 (CVVI-50/2014), were isolated from quail during H5N1 outbreaks in Vietnam, and their whole genome were analyzed. The phylogenetic analysis reveals new evolutionary variation in the worldwide H5N1 viruses. The quail HA genes were clustered into clades 1.1.1 (CVVI-49/2010) and clade 2.3.2.1c (CVVI-50/2014), which may have evolved from viruses circulating from chickens and/or ducks in Cambodia, mainland of China, Taiwan, Indonesia, and South Korea in recent years. Interestingly, the M2 gene of the CVVI-49/2010 strain contained amino acid substitutions at position 26L-I and 31S-N that are related to amantadine-resistance. In particular, the CVVI-50/2014 strain revealed evidence of multiple intersubtype reassortment events between virus clades 2.3.2.1c, 2.3.2.1b, and 2.3.2.1a. Data from this study supports the possible role of quail as an important intermediate host in avian influenza virus evolution. Therefore, additional surveillance is needed to monitor these HPAI viruses both serologically and virologically in quail. PMID:26900963

  8. Temperature Drops and the Onset of Severe Avian Influenza A H5N1 Virus Outbreaks

    PubMed Central

    Liu, Chung-Ming; Lin, Shu-Hua; Chen, Ying-Chen; Lin, Katherine Chun-Min; Wu, Tsung-Shu Joseph; King, Chwan-Chuen

    2007-01-01

    Global influenza surveillance is one of the most effective strategies for containing outbreaks and preparing for a possible pandemic influenza. Since the end of 2003, highly pathogenic avian influenza viruses (HPAI) H5N1 have caused many outbreaks in poultries and wild birds from East Asia and have spread to at least 48 countries. For such a fast and wide-spreading virulent pathogen, prediction based on changes of micro- and macro-environment has rarely been evaluated. In this study, we are developing a new climatic approach by investigating the conditions that occurred before the H5N1 avian influenza outbreaks for early predicting future HPAI outbreaks and preventing pandemic disasters. The results show a temperature drop shortly before these outbreaks in birds in each of the Eurasian regions stricken in 2005 and 2006. Dust storms, like those that struck near China's Lake Qinghai around May 4, 2005, exacerbated the spread of this HPAI H5N1 virus, causing the deaths of a record number of wild birds and triggering the subsequent spread of H5N1. Weather monitoring could play an important role in the early warning of outbreaks of this potentially dangerous virus. PMID:17297505

  9. Pathogenicity of two Egyptian H5N1 highly pathogenic avian influenza viruses in domestic ducks.

    PubMed

    Wasilenko, J L; Arafa, A M; Selim, A A; Hassan, M K; Aly, M M; Ali, A; Nassif, S; Elebiary, E; Balish, A; Klimov, A; Suarez, D L; Swayne, D E; Pantin-Jackwood, M J

    2011-01-01

    Domestic ducks have been implicated in the dissemination and evolution of H5N1 highly pathogenic avian influenza (HPAI) viruses. In this study, two H5N1 HPAI viruses belonging to clade 2.2.1 isolated in Egypt in 2007 and 2008 were analyzed for their pathogenicity in domestic Pekin ducks. Both viruses produced clinical signs and mortality, but the 2008 virus was more virulent, inducing early onset of neurological signs and killing all ducks with a mean death time (MDT) of 4.1 days. The 2007 virus killed 3/8 ducks with a MDT of 7 days. Full-genome sequencing and phylogenetic analysis were used to examine differences in the virus genes that might explain the differences observed in pathogenicity. The genomes differed in 49 amino acids, with most of the differences found in the hemagglutinin protein. This increase in pathogenicity in ducks observed with certain H5N1 HPAI viruses has implications for the control of the disease, since vaccinated ducks infected with highly virulent strains shed viruses for longer periods of time, perpetuating the virus in the environment and increasing the possibility of transmission to susceptible birds.

  10. Highly pathogenic avian influenza H5N1 virus delays apoptotic responses via activation of STAT3

    PubMed Central

    Hui, Kenrie P. Y.; Li, Hung Sing; Cheung, Man Chun; Chan, Renee W. Y.; Yuen, Kit M.; Mok, Chris K. P.; Nicholls, John M.; Peiris, J. S. Malik; Chan, Michael C. W.

    2016-01-01

    Highly pathogenic avian influenza (HPAI) H5N1 virus continues to pose pandemic threat, but there is a lack of understanding of its pathogenesis. We compared the apoptotic responses triggered by HPAI H5N1 and low pathogenic H1N1 viruses using physiologically relevant respiratory epithelial cells. We demonstrated that H5N1 viruses delayed apoptosis in primary human bronchial and alveolar epithelial cells (AECs) compared to H1N1 virus. Both caspase-8 and -9 were activated by H5N1 and H1N1 viruses in AECs, while H5N1 differentially up-regulated TRAIL. H5N1-induced apoptosis was reduced by TRAIL receptor silencing. More importantly, STAT3 knock-down increased apoptosis by H5N1 infection suggesting that H5N1 virus delays apoptosis through activation of STAT3. Taken together, we demonstrate that STAT3 is involved in H5N1-delayed apoptosis compared to H1N1. Since delay in apoptosis prolongs the duration of virus replication and production of pro-inflammatory cytokines and TRAIL from H5N1-infected cells, which contribute to orchestrate cytokine storm and tissue damage, our results suggest that STAT3 may play a previously unsuspected role in H5N1 pathogenesis. PMID:27344974

  11. Production of antibody labeled gold nanoparticles for influenza virus H5N1 diagnosis kit development

    NASA Astrophysics Data System (ADS)

    Pham, Van Dong; Hoang, Ha; Hoang Phan, Trong; Conrad, Udo; Chu, Hoang Ha

    2012-12-01

    Preparation of colloidal gold conjugated antibodies specific for influenza A/H5N1 and its use in developing a virus A/H5N1 rapid diagnostic kit is presented. Colloidal gold nanoparticles (AuNPs) were prepared through citrate reduction. Single chain antibodies specific to H5N1 (scFv7 and scFv24) were produced using pTI2 + vector and E. coli strain HB2151. These antibodies were purified by affinity chromatography technique employing HiTrap Chelating HP columns pre-charged with Ni2 + . The method for preparation of antibody-colloidal gold conjugate was based on electrostatic force binding antibody with colloidal gold. The effect of factors such as pH and concentration of antibody has been quantitatively analyzed using spectroscopic methods after adding 1 wt% NaCl which induced AuNP aggregation. The morphological study by scanning electron microscopy (SEM) showed that the average size of the spherical AuNPs was 23 nm with uniform sizes. The spectroscopic properties of colloidal AuNPs showed the typical surface plasmon resonance band at 523 nm in UV-visible spectrum. The optimal pH of conjugated colloidal gold was found between 8.0 and 10.0. The activity of synthesized antibody labeled AuNPs for detection of H5N1 flu virus was checked by dot blot immunological method. The results confirmed the ability in detection of the A/H5N1 virus of the prepared antibody labeled gold particles and opened up the possibility of using them in manufacturing rapid detection kit for this virus.

  12. Production of antibody labeled gold nanoparticles for influenza virus H5N1 diagnosis kit development

    NASA Astrophysics Data System (ADS)

    Pham, Van Dong; Hoang, Ha; Hoang Phan, Trong; Conrad, Udo; Chu, Hoang Ha

    2012-12-01

    Preparation of colloidal gold conjugated antibodies specific for influenza A/H5N1 and its use in developing a virus A/H5N1 rapid diagnostic kit is presented. Colloidal gold nanoparticles (AuNPs) were prepared through citrate reduction. Single chain antibodies specific to H5N1 (scFv7 and scFv24) were produced using pTI2 + vector and E. coli strain HB2151. These antibodies were purified by affinity chromatography technique employing HiTrap Chelating HP columns pre-charged with Ni2 + . The method for preparation of antibody–colloidal gold conjugate was based on electrostatic force binding antibody with colloidal gold. The effect of factors such as pH and concentration of antibody has been quantitatively analyzed using spectroscopic methods after adding 1 wt% NaCl which induced AuNP aggregation. The morphological study by scanning electron microscopy (SEM) showed that the average size of the spherical AuNPs was 23 nm with uniform sizes. The spectroscopic properties of colloidal AuNPs showed the typical surface plasmon resonance band at 523 nm in UV-visible spectrum. The optimal pH of conjugated colloidal gold was found between 8.0 and 10.0. The activity of synthesized antibody labeled AuNPs for detection of H5N1 flu virus was checked by dot blot immunological method. The results confirmed the ability in detection of the A/H5N1 virus of the prepared antibody labeled gold particles and opened up the possibility of using them in manufacturing rapid detection kit for this virus.

  13. A Novel Humanized Antibody Neutralizes H5N1 Influenza Virus via Two Different Mechanisms

    PubMed Central

    Tan, Yunrui; Ng, Qingyong; Jia, Qiang

    2015-01-01

    ABSTRACT Highly pathogenic avian influenza virus subtype H5N1 continues to be a severe threat to public health, as well as the poultry industry, because of its high lethality and antigenic drift rate. Neutralizing monoclonal antibodies (MAbs) can serve as a useful tool for preventing, treating, and detecting H5N1. In the present study, humanized H5 antibody 8A8 was developed from a murine H5 MAb. Both the humanized and mouse MAbs presented positive activity in hemagglutination inhibition (HI), virus neutralization, and immunofluorescence assays against a wide range of H5N1 strains. Interestingly, both human and murine 8A8 antibodies were able to detect H5 in Western blot assays under reducing conditions. Further, by sequencing of escape mutants, the conformational epitope of 8A8 was found to be located within the receptor binding domain (RBD) of H5. The linear epitope of 8A8 was identified by Western blotting of overlapping fragments and substitution mutant forms of HA1. Reverse genetic H5N1 strains with individual mutations in either the conformational or the linear epitope were generated and characterized in a series of assays, including HI, postattachment, and cell-cell fusion inhibition assays. The results indicate that for 8A8, virus neutralization mediated by RBD blocking relies on the conformational epitope while binding to the linear epitope contributes to the neutralization by inhibiting membrane fusion. Taken together, the results of this study show that a novel humanized H5 MAb binds to two types of epitopes on HA, leading to virus neutralization via two mechanisms. IMPORTANCE Recurrence of the highly pathogenic avian influenza virus subtype H5N1 in humans and poultry continues to be a serious public health concern. Preventive and therapeutic measures against influenza A viruses have received much interest in the context of global efforts to combat the current and future pandemics. Passive immune therapy is considered to be the most effective and

  14. Molecular epidemiology of influenza A (H5N1) viruses, Bangladesh, 2007-2011.

    PubMed

    Hoque, Md Ahasanul; Tun, Hein Min; Hassan, Mohammad Mahmudul; Khan, Shahneaz Ali; Islam, Skm Azizul; Islam, Md Nurul; Giasuddin, Md; Osmani, Tabm Muzaffar Goni; Islam, Ariful; Thornton, Ronald Norman; Burgess, Graham William; Skerratt, Lee Francis; Selleck, Paul; Brun, Edgar; Debnath, Nitish Chandra; Leung, Frederick Chi-Ching

    2013-09-01

    To investigate the origins, evolution and patterns of spread of HPAI H5N1 outbreaks in Bangladesh, we performed a phylogenetic reconstruction analysis using Bayesian methods. The analysis was conducted using 81 hemagglutinin (HA) gene sequences from the H5N1 viruses isolated in Bangladesh from 2007 to 2011, together with 264 publicly available HA sequences of clade 2.2, 2.3.2 and 2.3.4 retrieved from GenBank. Our study provides evidence that clade 2.2.2 viruses that caused outbreaks in Bangladesh were lineages independent from the viruses introduced earlier into India. Furthermore, the Bangladesh clade 2.2.2 descendents subsequently spread to India and Bhutan. This has implications for avian influenza control in southern Asia suggesting multiple routes of entry of the virus including one pathway that spread to neighboring countries via Bangladesh.

  15. Subclinical Infection with Avian Influenza A H5N1 Virus in Cats

    PubMed Central

    Weikel, Joachim; Möstl, Karin; Revilla-Fernández, Sandra; Wodak, Eveline; Bagó, Zoltan; Vanek, Elisabeth; Benetka, Viviane; Hess, Michael; Thalhammer, Johann G.

    2007-01-01

    Avian influenza A virus subtype H5N1 was transmitted to domestic cats by close contact with infected birds. Virus-specific nucleic acids were detected in pharyngeal swabs from 3 of 40 randomly sampled cats from a group of 194 animals (day 8 after contact with an infected swan). All cats were transferred to a quarantine station and monitored for clinical signs, virus shedding, and antibody production until day 50. Despite unfamiliar handling, social distress and the presence of other viral and nonviral pathogens that caused illness and poor health and compromised the immune systems, none of the cats developed clinical signs of influenza. There was no evidence of horizontal transmission to other cats because only 2 cats developed antibodies against H5N1 virus. PMID:17479886

  16. Molecular epidemiology of influenza A (H5N1) viruses, Bangladesh, 2007-2011.

    PubMed

    Hoque, Md Ahasanul; Tun, Hein Min; Hassan, Mohammad Mahmudul; Khan, Shahneaz Ali; Islam, Skm Azizul; Islam, Md Nurul; Giasuddin, Md; Osmani, Tabm Muzaffar Goni; Islam, Ariful; Thornton, Ronald Norman; Burgess, Graham William; Skerratt, Lee Francis; Selleck, Paul; Brun, Edgar; Debnath, Nitish Chandra; Leung, Frederick Chi-Ching

    2013-09-01

    To investigate the origins, evolution and patterns of spread of HPAI H5N1 outbreaks in Bangladesh, we performed a phylogenetic reconstruction analysis using Bayesian methods. The analysis was conducted using 81 hemagglutinin (HA) gene sequences from the H5N1 viruses isolated in Bangladesh from 2007 to 2011, together with 264 publicly available HA sequences of clade 2.2, 2.3.2 and 2.3.4 retrieved from GenBank. Our study provides evidence that clade 2.2.2 viruses that caused outbreaks in Bangladesh were lineages independent from the viruses introduced earlier into India. Furthermore, the Bangladesh clade 2.2.2 descendents subsequently spread to India and Bhutan. This has implications for avian influenza control in southern Asia suggesting multiple routes of entry of the virus including one pathway that spread to neighboring countries via Bangladesh. PMID:23820377

  17. Evolution of highly pathogenic avian influenza H5N1 viruses in Egypt indicating progressive adaptation.

    PubMed

    Arafa, A; Suarez, D; Kholosy, S G; Hassan, M K; Nasef, S; Selim, A; Dauphin, G; Kim, M; Yilma, J; Swayne, D; Aly, M M

    2012-10-01

    Highly pathogenic avian influenza (HPAI) virus of the H5N1 subtype was first diagnosed in poultry in Egypt in 2006, and since then the disease became enzootic in poultry throughout the country, affecting the poultry industry and village poultry as well as infecting humans. Vaccination has been used as a part of the control strategy to help to control the disease. Epidemiological data with sequence analysis of H5N1 viruses is important to link the mechanism of virus evolution in Egypt. This study describes the evolutionary pattern of Egyptian H5N1 viruses based on molecular characterization for the isolates collected from commercial poultry farms and village poultry from 2006 to 2011. Genetic analysis of the hemagglutinin (HA) gene was done by sequencing of the full-length H5 gene. The epidemiological pattern of disease outbreaks in Egyptian poultry farms seems to be seasonal with no specific geographic distribution across the country. The molecular epidemiological data revealed that there are two major groups of viruses: the classic group of subclade 2.2.1 and a variant group of 2.2.1.1. The classic group is prevailing mainly in village poultry and had fewer mutations compared to the originally introduced virus in 2006. Since 2009, this group has started to be transmitted back to commercial sectors. The variant group emerged by late 2007, was prevalent mainly in vaccinated commercial poultry, mutated continuously at a higher rate until 2010, and started to decline in 2011. Genetic analysis of the neuraminidase (NA) gene and the other six internal genes indicates a grouping of the Egyptian viruses similar to that obtained using the HA gene, with no obvious reassortments. The results of this study indicate that HPAI-H5N1 viruses are progressively evolving and adapting in Egypt and continue to acquire new mutations every season. PMID:22760662

  18. Protection of pregnant mice, fetuses and neonates from lethality of H5N1 influenza viruses by maternal vaccination.

    PubMed

    Hwang, Seon Do; Shin, Jin Soo; Ku, Keun Bon; Kim, Hyun Soo; Cho, Sung Whan; Seo, Sang Heui

    2010-04-01

    The highly pathogenic H5N1 influenza viruses are one of candidates for the next pandemic. Information on protective immunity for pregnant animals by vaccination against the H5N1 influenza virus is limited. Here, we show that the immunization of pregnant mice with inactivated H5N1 influenza vaccine protects them, their fetuses, and their infant mice from H5N1 influenza viruses. Pregnant mice immunized with two doses of H5N1 influenza vaccine were protected from homologous infections of H5N1 influenza viruses with no viruses detected in fetuses, and that they were protected upto 30% from heterologous infections of H5N1 influenza viruses with viruses detected in fetuses. The infant mice born to mothers immunized with H5N1 influenza vaccine were fully protected from infections of H5N1 influenza viruses for upto 4 weeks of age. The protection of infant mice was closely related to the presence of IgG2a antibody in lung, heart, and rectum tissues. Our results suggest that maternal vaccination may be critical for protecting pregnant animals, their fetuses, and their infant mice from lethal infections of H5N1 influenza viruses.

  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.

    PubMed

    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. PMID:27304069

  20. 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

    PubMed Central

    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. PMID:27304069

  1. Avian influenza A H5N1 virus: a continuous threat to humans.

    PubMed

    To, Kelvin Kw; Ng, Kenneth Hl; Que, Tak-Lun; Chan, Jacky Mc; Tsang, Kay-Yan; Tsang, Alan Kl; Chen, Honglin; Yuen, Kwok-Yung

    2012-09-01

    We report the first case of severe pneumonia due to co-infection with the emerging avian influenza A (H5N1) virus subclade 2.3.2.1 and Mycoplasma pneumoniae. The patient was a returning traveller who had visited a poultry market in South China. We then review the epidemiology, virology, interspecies barrier limiting poultry-to-human transmission, clinical manifestation, laboratory diagnosis, treatment and control measures of H5N1 clades that can be transmitted to humans. The recent controversy regarding the experiments involving aerosol transmission of recombinant H5N1 virus between ferrets is discussed. We also review the relative contribution of the poor response to antiviral treatment and the virus-induced hyperinflammatory damage to the pathogenesis and the high mortality of this infection. The factors related to the host, virus or medical intervention leading to the difference in disease mortality of different countries remain unknown. Because most developing countries have difficulty in instituting effective biosecurity measures, poultry vaccination becomes an important control measure. The rapid evolution of the virus would adversely affect the efficacy of poultry vaccination unless a correctly matched vaccine was chosen, manufactured and administered in a timely manner. Vigilant surveillance must continue to allow better preparedness for another poultry or human pandemic due to new viral mutants.

  2. Avian influenza A H5N1 virus: a continuous threat to humans

    PubMed Central

    To, Kelvin KW; Ng, Kenneth HL; Que, Tak-Lun; Chan, Jacky MC; Tsang, Kay-Yan; Tsang, Alan KL; Chen, Honglin; Yuen, Kwok-Yung

    2012-01-01

    We report the first case of severe pneumonia due to co-infection with the emerging avian influenza A (H5N1) virus subclade 2.3.2.1 and Mycoplasma pneumoniae. The patient was a returning traveller who had visited a poultry market in South China. We then review the epidemiology, virology, interspecies barrier limiting poultry-to-human transmission, clinical manifestation, laboratory diagnosis, treatment and control measures of H5N1 clades that can be transmitted to humans. The recent controversy regarding the experiments involving aerosol transmission of recombinant H5N1 virus between ferrets is discussed. We also review the relative contribution of the poor response to antiviral treatment and the virus-induced hyperinflammatory damage to the pathogenesis and the high mortality of this infection. The factors related to the host, virus or medical intervention leading to the difference in disease mortality of different countries remain unknown. Because most developing countries have difficulty in instituting effective biosecurity measures, poultry vaccination becomes an important control measure. The rapid evolution of the virus would adversely affect the efficacy of poultry vaccination unless a correctly matched vaccine was chosen, manufactured and administered in a timely manner. Vigilant surveillance must continue to allow better preparedness for another poultry or human pandemic due to new viral mutants. PMID:26038430

  3. An impedance immunosensor based on low-cost microelectrodes and specific monoclonal antibodies for rapid detection of avian influenza virus H5N1 in chicken swabs.

    PubMed

    Lin, Jianhan; Wang, Ronghui; Jiao, Peirong; Li, Yuntao; Li, Yanbin; Liao, Min; Yu, Yude; Wang, Maohua

    2015-05-15

    Early screening of suspected cases is the key to control the spread of avian influenza (AI) H5N1. In our previous studies, an impedance biosensor with an interdigitated array microelectrode based biochip was developed and validated with pure AI H5 virus, but had limitations in cost and reliability of the biochip, specificity of the antibody against Asian in-field H5N1 virus and detection of H5N1 virus in real samples. The purpose of this study is to develop a low-cost impedance immunosensor for rapid detection of Asian in-field AI H5N1 virus in chicken swabs within 1h and validate it with the H5N1 virus. Specific monoclonal antibodies against AI H5N1 virus were developed by fusion of mouse myeloma cells with spleen cells isolated from an H5N1-virus-immunized mouse. Dot-ELISA analysis demonstrated that the developed antibodies had good affinity and specificity with the H5N1 virus. The microelectrodes were redesigned with compact size, fabricated using an improved wet-etching micro-fabrication process with a higher qualified production rate of 70-80%, and modified with the antibodies by the Protein A method. Equivalent circuit analysis indicated that electron transfer resistor was effective with the increase in impedance after capturing of the H5N1 viruses. Linear relationship between impedance change and logarithmic value of H5N1 virus at the concentrations from 2(-1) to 2(4) HAU/50 μl was found and the lower limit of detection was 2(-1) HAU/50 μl. No obvious interferences from non-target viruses such as H6N2, H9N2, Newcastle disease virus, and infectious bronchitis virus were found. Chicken swab tests showed that the impedance immunosensor had a comparable accuracy with real-time RT-PCR compared to viral isolation. PMID:25263315

  4. Bronchointerstitial pneumonia in guinea pigs following inoculation with H5N1 high pathogenicity avian influenza virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The H5N1 high pathogenicity avian influenza (HPAI) viruses have caused widespread disease of poultry in Asia, Africa and the Middle East, and sporadic human infections. The guinea pig model has been used to study human H3N2 and H1N1 influenza viruses, but knowledge is lacking on H5N1 HPAI virus inf...

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

    SciTech Connect

    DuBois, Rebecca M.; Zaraket, Hassan; Reddivari, Muralidhar; Heath, Richard J.; White, Stephen W.; Russell, Charles J.

    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.

  6. Birds and Influenza H5N1 Virus Movement to and within North America

    PubMed Central

    Hubálek, Zdenek

    2006-01-01

    Highly pathogenic avian influenza (HPAI) H5N1 expanded considerably during 2005 and early 2006 in both avian host species and geographic distribution. Domestic waterfowl and migratory birds are reservoirs, but lethality of this subtype appeared to initially limit migrant effectiveness as introductory hosts. This situation may have changed, as HPAI H5N1 has recently expanded across Eurasia and into Europe and Africa. Birds could introduce HPAI H5N1 to the Western Hemisphere through migration, vagrancy, and importation by people. Vagrants and migratory birds are not likely interhemispheric introductory hosts; import of infected domestic or pet birds is more probable. If reassortment or mutation were to produce a virus adapted for rapid transmission among humans, birds would be unlikely introductory hosts because of differences in viral transmission mechanisms among major host groups (i.e., gastrointestinal for birds, respiratory for humans). Another possible result of reassortment would be a less lethal form of avian influenza, more readily spread by birds. PMID:17176561

  7. Serosurveillance study on transmission of H5N1 virus during a 2006 avian influenza epidemic.

    PubMed

    Ceyhan, M; Yildirim, I; Ferraris, O; Bouscambert-Duchamp, M; Frobert, E; Uyar, N; Tezer, H; Oner, A F; Buzgan, T; Torunoglu, M A; Ozkan, B; Yilmaz, R; Kurtoglu, M G; Laleli, Y; Badur, S; Lina, B

    2010-09-01

    In 2006 an outbreak of avian influenza A(H5N1) in Turkey caused 12 human infections, including four deaths. We conducted a serological survey to determine the extent of subclinical infection caused by the outbreak. Single serum samples were collected from five individuals with avian influenza whose nasopharyngeal swabs tested positive for H5 RNA by polymerase chain reaction, 28 family contacts of the cases, 95 poultry cullers, 75 individuals known to have had contact with diseased chickens and 81 individuals living in the region with no known contact with infected chickens and/or patients. Paired serum samples were collected from 97 healthcare workers. All sera were tested for the presence of neutralizing antibodies by enzyme-linked immunoassay, haemagglutination inhibition and microneutralization assays. Only one serum sample, from a parent of an avian influenza patient, tested positive for H5N1 by microneutralization assay. This survey shows that there was minimal subclinical H5N1 infection among contacts of human cases and infected poultry in Turkey in 2006. Further, the low rate of subclinical infection following contact with diseased poultry gave further support to the reported low infectivity of the virus.

  8. Genetic analysis of influenza A virus (H5N1) derived from domestic cat and dog in Thailand.

    PubMed

    Amonsin, A; Songserm, T; Chutinimitkul, S; Jam-On, R; Sae-Heng, N; Pariyothorn, N; Payungporn, S; Theamboonlers, A; Poovorawan, Y

    2007-01-01

    Complete genome sequences of H5N1 viruses derived from a domestic cat "A/Cat/Thailand/KU-02/04" and dog "A/Dog/Thailand/KU-08/04" were comprehensively analyzed and compared with H5N1 isolates obtained during the 2004 and 2005 outbreaks. Phylogenetic analysis of both cat and dog viruses revealed that they are closely related to the H5N1 viruses recovered from avian influenza outbreaks of the same period. Genetic analysis of 8 viral gene segments showed some evidence of virulence in mammalian species. In summary, the H5N1 viruses that infected a domestic cat and dog are highly pathogenic avian influenza viruses that are virulent in mammalian species, potentially indicating transmission of H5N1 viruses from domestic animals to humans.

  9. Highly Pathogenic Avian Influenza Virus Subtype H5N1 Escaping Neutralization: More than HA Variation

    PubMed Central

    Höper, Dirk; Kalthoff, Donata; Hoffmann, Bernd

    2012-01-01

    Influenza A viruses are one of the major threats in modern health care. Novel viruses arise due to antigenic drift and antigenic shift, leading to escape from the immune system and resulting in a serious problem for disease control. In order to investigate the escape process and to enable predictions of escape, we serially passaged influenza A H5N1 virus in vitro 100 times under immune pressure. The generated escape viruses were characterized phenotypically and in detail by full-genome deep sequencing. Mutations already found in natural isolates were detected, evidencing the in vivo relevance of the in vitro-induced amino acid substitutions. Additionally, several novel alterations were triggered. Altogether, the results imply that our in vitro system is suitable to study influenza A virus evolution and that it might even be possible to predict antigenic changes of influenza A viruses circulating in vaccinated populations. PMID:22090121

  10. Global spatiotemporal and genetic footprint of the H5N1 avian influenza virus

    PubMed Central

    2014-01-01

    Background Since 2005, the Qinghai-like lineage of the highly pathogenic avian influenza A virus H5N1 has rapidly spread westward to Europe, the Middle East and Africa, reaching a dominant level at a global scale in 2006. Methods Based on a combination of genetic sequence data and H5N1 outbreak information from 2005 to 2011, we use an interdisciplinary approach to improve our understanding of the transmission pattern of this particular clade 2.2, and present cartography of global spatiotemporal transmission footprints with genetic characteristics. Results Four major viral transmission routes were derived with three sources— Russia, Mongolia, and the Middle East (Kuwait and Saudi Arabia)—in the three consecutive years 2005, 2006 and 2007. With spatiotemporal transmission along each route, genetic distances to isolate A/goose/Guangdong/1996 are becoming significantly larger, leading to a more challenging situation in certain regions like Korea, India, France, Germany, Nigeria and Sudan. Europe and India have had at least two incursions along multiple routes, causing a mixed virus situation. In addition, spatiotemporal distribution along the routes showed that 2007/2008 was a temporal separation point for the infection of different host species; specifically, wild birds were the main host in 2005–2007/2008 and poultry was responsible for the genetic mutation in 2009–2011. “Global-to-local” and “high-to-low latitude” transmission footprints have been observed. Conclusions Our results suggest that both wild birds and poultry play important roles in the transmission of the H5N1 virus clade, but with different spatial, temporal, and genetic dominance. These characteristics necessitate that special attention be paid to countries along the transmission routes. PMID:24885233

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

    PubMed Central

    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 synthetic sialosaccharides with α2,3 or α2,6 linkages) on transmissibility were assessed. A/Vietnam/1203/04 and A/Vietnam/JP36-2/05 viruses, which possess “avian-like” α2,3-linked sialic acid (SA) receptor specificity, caused neurological symptoms and death in ferrets inoculated with 103 50% tissue culture infectious doses. A/Hong Kong/213/03 and A/Turkey/65-596/06 viruses, which show binding affinity for “human-like” α2,6-linked SA receptors in addition to their affinity for α2,3-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. PMID:17459930

  12. Adenovirus-based vaccines against avian-origin H5N1 influenza viruses.

    PubMed

    He, Biao; Zheng, Bo-jian; Wang, Qian; Du, Lanying; Jiang, Shibo; Lu, Lu

    2015-02-01

    Since 1997, human infection with avian H5N1, having about 60% mortality, has posed a threat to public health. In this review, we describe the epidemiology of H5N1 transmission, advantages and disadvantages of different influenza vaccine types, and characteristics of adenovirus, finally summarizing advances in adenovirus-based H5N1 systemic and mucosal vaccines.

  13. Evolutionary Dynamics of Multiple Sublineages of H5N1 Influenza Viruses in Nigeria from 2006 to 2008 ▿ †

    PubMed Central

    Fusaro, Alice; Nelson, Martha I.; Joannis, Tony; Bertolotti, Luigi; Monne, Isabella; Salviato, Annalisa; Olaleye, Olufemi; Shittu, Ismaila; Sulaiman, Lanre; Lombin, Lami H.; Capua, Ilaria; Holmes, Edward C.; Cattoli, Giovanni

    2010-01-01

    Highly pathogenic A/H5N1 avian influenza (HPAI H5N1) viruses have seriously affected the Nigerian poultry industry since early 2006. Previous studies have identified multiple introductions of the virus into Nigeria and several reassortment events between cocirculating lineages. To determine the spatial, evolutionary, and population dynamics of the multiple H5N1 lineages cocirculating in Nigeria, we conducted a phylogenetic analysis of whole-genome sequences from 106 HPAI H5N1 viruses isolated between 2006 and 2008 and representing all 25 Nigerian states and the Federal Capital Territory (FCT) reporting outbreaks. We identified a major new subclade in Nigeria that is phylogenetically distinguishable from all previously identified sublineages, as well as two novel reassortment events. A detailed analysis of viral phylogeography identified two major source populations for the HPAI H5N1 virus in Nigeria, one in a major commercial poultry area (southwest region) and one in northern Nigeria, where contact between wild birds and backyard poultry is frequent. These findings suggested that migratory birds from Eastern Europe or Russia may serve an important role in the introduction of HPAI H5N1 viruses into Nigeria, although virus spread through the movement of poultry and poultry products cannot be excluded. Our study provides new insight into the genesis and evolution of H5N1 influenza viruses in Nigeria and has important implications for targeting surveillance efforts to rapidly identify the spread of the virus into and within Nigeria. PMID:20071565

  14. Characterizing wild bird contact and seropositivity to highly pathogenic avian influenza A (H5N1) virus in Alaskan residents

    PubMed Central

    Reed, Carrie; Bruden, Dana; Byrd, Kathy K; Veguilla, Vic; Bruce, Michael; Hurlburt, Debby; Wang, David; Holiday, Crystal; Hancock, Kathy; Ortiz, Justin R; Klejka, Joe; Katz, Jacqueline M; Uyeki, Timothy M

    2014-01-01

    Background Highly pathogenic avian influenza A (HPAI) H5N1 viruses have infected poultry and wild birds on three continents with more than 600 reported human cases (59% mortality) since 2003. Wild aquatic birds are the natural reservoir for avian influenza A viruses, and migratory birds have been documented with HPAI H5N1 virus infection. Since 2005, clade 2.2 HPAI H5N1 viruses have spread from Asia to many countries. Objectives We conducted a cross-sectional seroepidemiological survey in Anchorage and western Alaska to identify possible behaviors associated with migratory bird exposure and measure seropositivity to HPAI H5N1. Methods We enrolled rural subsistence bird hunters and their families, urban sport hunters, wildlife biologists, and a comparison group without bird contact. We interviewed participants regarding their exposures to wild birds and collected blood to perform serologic testing for antibodies against a clade 2.2 HPAI H5N1 virus strain. Results Hunters and wildlife biologists reported exposures to wild migratory birds that may confer risk of infection with avian influenza A viruses, although none of the 916 participants had evidence of seropositivity to HPAI H5N1. Conclusions We characterized wild bird contact among Alaskans and behaviors that may influence risk of infection with avian influenza A viruses. Such knowledge can inform surveillance and risk communication surrounding HPAI H5N1 and other influenza viruses in a population with exposure to wild birds at a crossroads of intercontinental migratory flyways. PMID:24828535

  15. Molecular Determinants of Virulence and Stability of a Reporter-Expressing H5N1 Influenza A Virus

    PubMed Central

    Zhao, Dongming; Fukuyama, Satoshi; Yamada, Shinya; Lopes, Tiago J. S.; Maemura, Tadashi; Katsura, Hiroaki; Ozawa, Makoto; Watanabe, Shinji; Neumann, Gabriele

    2015-01-01

    ABSTRACT We previously reported that an H5N1 virus carrying the Venus reporter gene, which was inserted into the NS gene segment from the A/Puerto Rico/8/1934(H1N1) virus (Venus-H5N1 virus), became more lethal to mice, and the reporter gene was stably maintained after mouse adaptation compared with the wild-type Venus-H5N1 (WT-Venus-H5N1) virus. However, the basis for this difference in virulence and Venus stability was unclear. Here, we investigated the molecular determinants behind this virulence and reporter stability by comparing WT-Venus-H5N1 virus with a mouse-adapted Venus-H5N1 (MA-Venus-H5N1) virus. To determine the genetic basis for these differences, we used reverse genetics to generate a series of reassortants of these two viruses. We found that reassortants with PB2 from MA-Venus-H5N1 (MA-PB2), MA-PA, or MA-NS expressed Venus more stably than did WT-Venus-H5N1 virus. We also found that a single mutation in PB2 (V25A) or in PA (R443K) increased the virulence of the WT-Venus-H5N1 virus in mice and that the presence of both of these mutations substantially enhanced the pathogenicity of the virus. Our results suggest roles for PB2 and PA in the stable maintenance of a foreign protein as an NS1 fusion protein in influenza A virus. IMPORTANCE The ability to visualize influenza viruses has far-reaching benefits in influenza virus research. Previously, we reported that an H5N1 virus bearing the Venus reporter gene became more pathogenic to mice and that its reporter gene was more highly expressed and more stably maintained after mouse adaptation. Here, we investigated the molecular determinants behind this enhanced virulence and reporter stability. We found that mutations in PB2 (V25A) and PA (R443K) play crucial roles in the stable maintenance of a foreign protein as an NS1 fusion protein in influenza A virus and in the virulence of influenza virus in mice. Our findings further our knowledge of the pathogenicity of influenza virus in mammals and will help

  16. Satellite tracking on the flyways of brown-headed gulls and their potential role in the spread of highly pathogenic avian influenza H5N1 virus.

    PubMed

    Ratanakorn, Parntep; Wiratsudakul, Anuwat; Wiriyarat, Witthawat; Eiamampai, Krairat; Farmer, Adrian H; Webster, Robert G; Chaichoune, Kridsada; Suwanpakdee, Sarin; Pothieng, Duangrat; Puthavathana, Pilaipan

    2012-01-01

    Brown-headed gulls (Larus brunnicephalus), winter visitors of Thailand, were tracked by satellite telemetry during 2008-2011 for investigating their roles in the highly pathogenic avian influenza (HPAI) H5N1 virus spread. Eight gulls negative for influenza virus infection were marked with solar-powered satellite platform transmitters at Bang Poo study site in Samut Prakarn province, Thailand; their movements were monitored by the Argos satellite tracking system, and locations were mapped. Five gulls completed their migratory cycles, which spanned 7 countries (China, Bangladesh, India, Myanmar, Thailand, Cambodia, and Vietnam) affected by the HPAI H5N1 virus. Gulls migrated from their breeding grounds in China to stay overwinter in Thailand and Cambodia; while Bangladesh, India, Myanmar, and Vietnam were the places of stopovers during migration. Gulls traveled an average distance of about 2400 km between Thailand and China and spent 1-2 weeks on migration. Although AI surveillance among gulls was conducted at the study site, no AI virus was isolated and no H5N1 viral genome or specific antibody was detected in the 75 gulls tested, but 6.6% of blood samples were positive for pan-influenza A antibody. No AI outbreaks were reported in areas along flyways of gulls in Thailand during the study period. Distance and duration of migration, tolerability of the captive gulls to survive the HPAI H5N1 virus challenge and days at viral shedding after the virus challenging suggested that the Brown-headed gull could be a potential species for AI spread, especially among Southeast Asian countries, the epicenter of H5N1 AI outbreak.

  17. Anti-H5N1 virus flavonoids from Capparis sinaica Veill.

    PubMed

    Ibrahim, Amany K; Youssef, Ahmed I; Arafa, Abdel Satar; Ahmed, Safwat A

    2013-01-01

    Methanolic extract of Capparis sinaica Veill was tested for its in vitro antiviral activity against highly pathogenic avian influenza strain H5N1 using plaque inhibition assay in Madin-Darby canine kidney. The results indicated that the extract possessed potent antiviral activity (100% inhibition at the concentration of 1 μg/ml). Based on this result, C. sinaica Veill was selected for further study by applying bioactivity-guided fractionation to isolate its antiviral principles. The fractions eluted with EtOAc and 25% MeOH in EtOAc were found to hold the antiviral activity. Further chromatographic separation of the fractions holding the antiviral activity led to the isolation of quercetin (1), isoquercetin (2) and rutin (3) for the first time from this species. The isolates showed reduction in the virus titre by 68.13%, 79.66% and 73.22% inhibition at a concentration of 1 ng/ml, respectively.

  18. Absence of neutralizing antibodies against influenza A/H5N1 virus among children in Kamphaeng Phet, Thailand

    PubMed Central

    Khuntirat, Benjawan; Love, Christopher S.; Buddhari, Darunee; Heil, Gary L.; Gibbons, Robert V.; Rothman, Alan L.; Srikiatkhachorn, Anon; Gray, Gregory C.; Yoon, In-Kyu

    2015-01-01

    Background Influenza A/H5N1 actively circulated in Kamphaeng Phet (KPP), Thailand from 2004–2006. A prospective longitudinal cohort study of influenza virus infection in 800 adults conducted during 2008 to 2010 in KPP suggested that subclinical or mild H5N1 infections had occurred among this adult cohort. However, this study was conducted after the peak of H5N1 activity in KPP. Coincidentally, banked serum samples were available from a prospective longitudinal cohort study of primary school children who had undergone active surveillance for febrile illnesses from 2004 to 2007 and lived in the same district of KPP as the adult cohort. Objectives We sought to investigate whether subclinical or mild H5N1 infections had occurred among KPP residents during the peak of H5N1 activity from 2004 to 2006. Study design H5N1 microneutralization (MN) assay was performed on banked serum samples from a prospective longitudinal cohort study of primary school children who had undergone active surveillance for febrile illnesses in KPP. Annual blood samples collected from 2004 to 2006 from 251 children were selected based on the criteria that they lived in villages with documented H5N1 infection. Result No H5N1 neutralizing antibodies were detected in 753 annual blood samples from 251 children. Conclusion During 2004 to 2006, very few subclinical or mild H5N1 infections occurred in KPP. Elevated H5N1 MN titers found in the adult cohort in 2008 were likely due to cross-reactivity from other influenza virus subtypes highlighting the complexities in interpreting influenza serological data. PMID:26209384

  19. Isolation and identification of highly pathogenic avian influenza virus subtype H5N1 in peafowl (Pavo cristatus).

    PubMed

    Ismail, Mahmoud Moussa; Khan, Owais Ahmed; Cattoli, Giovanni; Lu, Huaguang

    2010-03-01

    An outbreak of highly pathogenic avian influenza (HPAI) virus subtype H5N1 was first diagnosed in a "backyard" flock of peafowl (Pavo cristatus) raised on palace premises in the Kingdom of Saudi Arabia in December 3, 2007. The flock consisted of 40 peafowl, and their ages ranged from 3 to 5 years old. Affected birds suffered from depression, anorexia, and white diarrhea. Four dead birds were submitted for HPAI diagnosis at the Central Veterinary Diagnostic Laboratory in Riyadh. Brain and liver tissues and tracheal and cloacal swabs were taken from the dead birds and processed for a real-time reverse transcriptase (RT)-PCR test and virus isolation in specific-pathogen-free embryonating chicken eggs. The H5N1 subtype of avian influenza virus was isolated from the four dead birds and identified by a real-time RT-PCR before and after egg inoculation. The virus isolates were characterized as HPAI H5N1 virus by sequencing analysis. Phylogenetic comparisons revealed that the H5N1 viruses isolated from peafowl belong to the genetic clade 2.2 according to the World Health Organization nomenclature. The peafowl H5N1 virus falls into 2.2.2 sublineage II and clusters with the H5N1 viruses isolated from poultry in Saudi Arabia in 2007-08.

  20. Isolation and identification of highly pathogenic avian influenza virus subtype H5N1 in peafowl (Pavo cristatus).

    PubMed

    Ismail, Mahmoud Moussa; Khan, Owais Ahmed; Cattoli, Giovanni; Lu, Huaguang

    2010-03-01

    An outbreak of highly pathogenic avian influenza (HPAI) virus subtype H5N1 was first diagnosed in a "backyard" flock of peafowl (Pavo cristatus) raised on palace premises in the Kingdom of Saudi Arabia in December 3, 2007. The flock consisted of 40 peafowl, and their ages ranged from 3 to 5 years old. Affected birds suffered from depression, anorexia, and white diarrhea. Four dead birds were submitted for HPAI diagnosis at the Central Veterinary Diagnostic Laboratory in Riyadh. Brain and liver tissues and tracheal and cloacal swabs were taken from the dead birds and processed for a real-time reverse transcriptase (RT)-PCR test and virus isolation in specific-pathogen-free embryonating chicken eggs. The H5N1 subtype of avian influenza virus was isolated from the four dead birds and identified by a real-time RT-PCR before and after egg inoculation. The virus isolates were characterized as HPAI H5N1 virus by sequencing analysis. Phylogenetic comparisons revealed that the H5N1 viruses isolated from peafowl belong to the genetic clade 2.2 according to the World Health Organization nomenclature. The peafowl H5N1 virus falls into 2.2.2 sublineage II and clusters with the H5N1 viruses isolated from poultry in Saudi Arabia in 2007-08. PMID:20521659

  1. Molecular characterization of avian influenza H5N1 virus in Egypt and the emergence of a novel endemic subclade

    PubMed Central

    El-Shesheny, Rabeh; Kandeil, Ahmed; Bagato, Ola; Maatouq, Asmaa M.; Moatasim, Yassmin; Rubrum, Adam; Song, Min-Suk; Webby, Richard J.

    2014-01-01

    Clade 2.2 highly pathogenic H5N1 viruses have been in continuous circulation in Egyptian poultry since 2006. Their persistence caused significant genetic drift that led to the reclassification of these viruses into subclades 2.2.1 and 2.2.1.1. Here, we conducted full-genome sequence and phylogenetic analyses of 45 H5N1 isolated during 2006–2013 through systematic surveillance in Egypt, and 53 viruses that were sequenced previously and available in the public domain. Results indicated that H5N1 viruses in Egypt continue to evolve and a new distinct cluster has emerged. Mutations affecting viral virulence, pathogenicity, transmission, receptor-binding preference and drug resistance were studied. In light of our findings that H5N1 in Egypt continues to evolve, surveillance and molecular studies need to be sustained. PMID:24722680

  2. [Engineering by reverse genetics and characterization of the new reassortant influenza virus strain H5N1].

    PubMed

    Zeberezhnyĭ, A D; Grebennikova, T V; Vorkunova, G K; Yuzhakov, A G; Kostina, L V; Norkina, S N; Aliper, T I; Nepoklonov, E A; Lvov, D K

    2014-01-01

    Reverse genetics was applied to engineering of the reassortantvaccine candidate strain against highly pathogenic avian influenza viruses (HPAIVs) of the H5 subtype. The new strain recPR8-H5N1 contains the HA gene from the Russian HPAIV A/Kurgan/05/2005 (H5N1), the NA and internal genes from A/Puerto Rico/8/34 (H1N1). The strain recPR8-H5N1 demonstrated the antigenic specificity (H5), high proliferation rate in 12 days chicken embryos, and was lethal for the embryos in 36 hours. An inactivated emulsified vaccine based on the strain recPR8-H5N1 elicited high antibody titers and protected 6-week-old chickens from lethal challenge with the HPAIV A/Kurgan/05/2005 (H5N1) on day 21 after single immunization. Infection of non-vaccinated birds with the strain recPR8-H5N1 did not cause any pathology, and the virus was not detected using PCR in blood and cloacal swabs on day 7 p.i. Specific weak seroconversion caused by infection with the strain recPR8-H5N1 was detected on day 14 p.i. As a result, a new influenza virus strain was obtained with modified properties.

  3. Fatal influenza A (H5N1) virus Infection in zoo-housed Tigers in Yunnan Province, China

    PubMed Central

    Hu, Tingsong; Zhao, Huanyun; Zhang, Yan; Zhang, Wendong; Kong, Qiang; Zhang, Zhixiao; Cui, Qinghua; Qiu, Wei; Deng, Bo; Fan, Quanshui; Zhang, Fuqiang

    2016-01-01

    From 2014 to 2015, three cases of highly pathogenic avian influenza infection occurred in zoo-housed north-east China tigers (Panthera tigris ssp.altaica) and four tigers died of respiratory distress in succession in Yunnan Province, China. We isolated and characterized three highly pathogenic avian influenza A(H5N1) viruses from these tigers. Phylogenetic analysis indicated that A/tiger /Yunnan /tig1404 /2014(H5N1) belongs to the provisional subclade 2.3.4.4e which were novel reassortant influenza A (H5N1) viruses with six internal genes from avian influenza A (H5N2) viruses. The HA gene of the isolated A/tiger /Yunnan /tig1412 /2014(H5N1) virus belongs to the subclade 2.3.2.1b. The isolated A/tiger /Yunnan /tig1508/2015 (H5N1) virus was a novel reassortant influenza A (H5N1) virus with three internal genes (PB2, PB1 and M) from H9N2 virus and belongs to the subclade 2.3.2.1c. PMID:27162026

  4. Fatal influenza A (H5N1) virus Infection in zoo-housed Tigers in Yunnan Province, China.

    PubMed

    Hu, Tingsong; Zhao, Huanyun; Zhang, Yan; Zhang, Wendong; Kong, Qiang; Zhang, Zhixiao; Cui, Qinghua; Qiu, Wei; Deng, Bo; Fan, Quanshui; Zhang, Fuqiang

    2016-01-01

    From 2014 to 2015, three cases of highly pathogenic avian influenza infection occurred in zoo-housed north-east China tigers (Panthera tigris ssp.altaica) and four tigers died of respiratory distress in succession in Yunnan Province, China. We isolated and characterized three highly pathogenic avian influenza A(H5N1) viruses from these tigers. Phylogenetic analysis indicated that A/tiger /Yunnan /tig1404 /2014(H5N1) belongs to the provisional subclade 2.3.4.4e which were novel reassortant influenza A (H5N1) viruses with six internal genes from avian influenza A (H5N2) viruses. The HA gene of the isolated A/tiger /Yunnan /tig1412 /2014(H5N1) virus belongs to the subclade 2.3.2.1b. The isolated A/tiger /Yunnan /tig1508/2015 (H5N1) virus was a novel reassortant influenza A (H5N1) virus with three internal genes (PB2, PB1 and M) from H9N2 virus and belongs to the subclade 2.3.2.1c. PMID:27162026

  5. Fatal influenza A (H5N1) virus Infection in zoo-housed Tigers in Yunnan Province, China.

    PubMed

    Hu, Tingsong; Zhao, Huanyun; Zhang, Yan; Zhang, Wendong; Kong, Qiang; Zhang, Zhixiao; Cui, Qinghua; Qiu, Wei; Deng, Bo; Fan, Quanshui; Zhang, Fuqiang

    2016-05-10

    From 2014 to 2015, three cases of highly pathogenic avian influenza infection occurred in zoo-housed north-east China tigers (Panthera tigris ssp.altaica) and four tigers died of respiratory distress in succession in Yunnan Province, China. We isolated and characterized three highly pathogenic avian influenza A(H5N1) viruses from these tigers. Phylogenetic analysis indicated that A/tiger /Yunnan /tig1404 /2014(H5N1) belongs to the provisional subclade 2.3.4.4e which were novel reassortant influenza A (H5N1) viruses with six internal genes from avian influenza A (H5N2) viruses. The HA gene of the isolated A/tiger /Yunnan /tig1412 /2014(H5N1) virus belongs to the subclade 2.3.2.1b. The isolated A/tiger /Yunnan /tig1508/2015 (H5N1) virus was a novel reassortant influenza A (H5N1) virus with three internal genes (PB2, PB1 and M) from H9N2 virus and belongs to the subclade 2.3.2.1c.

  6. Full genome sequence of a recombinant H5N1 influenza virus from a condor in southern China.

    PubMed

    Jiao, Peirong; Yuan, Runyu; Song, Yafen; Wei, Liangmeng; Ren, Tao; Liao, Ming; Luo, Kaijian

    2012-07-01

    In this study, we report the first genomic information on an H5N1 avian influenza virus (AIV) isolated from a condor in Guangdong Province in southern China in 2003. Full genome sequencing and phylogenetic analyses show that it is a recombinant virus containing genome segments derived from the Eurasia and North America gene pools. This will be useful for analyses of the evolution of H5N1 AIV in southern China.

  7. Full genome sequence of a recombinant H5N1 influenza virus from a condor in southern China.

    PubMed

    Jiao, Peirong; Yuan, Runyu; Song, Yafen; Wei, Liangmeng; Ren, Tao; Liao, Ming; Luo, Kaijian

    2012-07-01

    In this study, we report the first genomic information on an H5N1 avian influenza virus (AIV) isolated from a condor in Guangdong Province in southern China in 2003. Full genome sequencing and phylogenetic analyses show that it is a recombinant virus containing genome segments derived from the Eurasia and North America gene pools. This will be useful for analyses of the evolution of H5N1 AIV in southern China. PMID:22733885

  8. Highly Pathogenic Avian Influenza A(H5N1) Virus Struck Migratory Birds in China in 2015

    PubMed Central

    Bi, Yuhai; Zhang, Zhenjie; Liu, Wenjun; Yin, Yanbo; Hong, Jianmin; Li, Xiangdong; Wang, Haiming; Wong, Gary; Chen, Jianjun; Li, Yunfeng; Ru, Wendong; Gao, Ruyi; Liu, Di; Liu, Yingxia; Zhou, Boping; Gao, George F.; Shi, Weifeng; Lei, Fumin

    2015-01-01

    Approximately 100 migratory birds, including whooper swans and pochards, were found dead in the Sanmenxia Reservoir Area of China during January 2015. The causative agent behind this outbreak was identified as H5N1 highly pathogenic avian influenza virus (HPAIV). Genetic and phylogenetic analyses revealed that this Sanmenxia H5N1 virus was a novel reassortant, possessing a Clade 2.3.2.1c HA gene and a H9N2-derived PB2 gene. Sanmenxia Clade 2.3.2.1c-like H5N1 viruses possess the closest genetic identity to A/Alberta/01/2014 (H5N1), which recently caused a fatal respiratory infection in Canada with signs of meningoencephalitis, a highly unusual symptom with influenza infections in humans. Furthermore, this virus was shown to be highly pathogenic to both birds and mammals, and demonstrate tropism for the nervous system. Due to the geographical location of Sanmenxia, these novel H5N1 viruses also have the potential to be imported to other regions through the migration of wild birds, similar to the H5N1 outbreak amongst migratory birds in Qinghai Lake during 2005. Therefore, further investigation and monitoring is required to prevent this novel reassortant virus from becoming a new threat to public health. PMID:26259704

  9. Highly Pathogenic Avian Influenza A(H5N1) Virus Struck Migratory Birds in China in 2015.

    PubMed

    Bi, Yuhai; Zhang, Zhenjie; Liu, Wenjun; Yin, Yanbo; Hong, Jianmin; Li, Xiangdong; Wang, Haiming; Wong, Gary; Chen, Jianjun; Li, Yunfeng; Ru, Wendong; Gao, Ruyi; Liu, Di; Liu, Yingxia; Zhou, Boping; Gao, George F; Shi, Weifeng; Lei, Fumin

    2015-01-01

    Approximately 100 migratory birds, including whooper swans and pochards, were found dead in the Sanmenxia Reservoir Area of China during January 2015. The causative agent behind this outbreak was identified as H5N1 highly pathogenic avian influenza virus (HPAIV). Genetic and phylogenetic analyses revealed that this Sanmenxia H5N1 virus was a novel reassortant, possessing a Clade 2.3.2.1c HA gene and a H9N2-derived PB2 gene. Sanmenxia Clade 2.3.2.1c-like H5N1 viruses possess the closest genetic identity to A/Alberta/01/2014 (H5N1), which recently caused a fatal respiratory infection in Canada with signs of meningoencephalitis, a highly unusual symptom with influenza infections in humans. Furthermore, this virus was shown to be highly pathogenic to both birds and mammals, and demonstrate tropism for the nervous system. Due to the geographical location of Sanmenxia, these novel H5N1 viruses also have the potential to be imported to other regions through the migration of wild birds, similar to the H5N1 outbreak amongst migratory birds in Qinghai Lake during 2005. Therefore, further investigation and monitoring is required to prevent this novel reassortant virus from becoming a new threat to public health.

  10. Fluorescence biosensor based on CdTe quantum dots for specific detection of H5N1 avian influenza virus

    NASA Astrophysics Data System (ADS)

    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.

  11. Evolutionary features of influenza A/H5N1 virus populations in Egypt: poultry and human health implications.

    PubMed

    Naguib, Mahmoud M; Abdelwhab, E M; Harder, Timm C

    2016-07-01

    Since 2006, in Egypt, highly pathogenic avian influenza virus (HPAIV) H5N1 has established endemic status in poultry. Bayesian evolutionary analysis sampling trees suggested an introduction date in the third quarter of 2005. Evolutionary dynamics using Bayesian analysis showed that H5N1 viruses of clade 2.2.1.1 evolved at higher rates than those of clade 2.2.1.2. Bayesian skyline plot analysis of the HA gene of 840 and NA gene of 401 Egyptian H5N1 viruses from 2006-2015 identified two waves of viral population expansion correlating with the stepwise emergence of the 2.2.1.1 variant lineage in 2008 and with the newly emerging 2.2.1.2 cluster in late 2014. H5N1 infections in human hosts in 2014-2015 were statistically linked to a contemporary poultry outbreak. PMID:27068161

  12. Pathogenicity and vaccine efficacy of different clades of Asian H5N1 avian influenza A viruses in domestic ducks.

    PubMed

    Kim, Jeong-Ki; Seiler, Patrick; Forrest, Heather L; Khalenkov, Alexey M; Franks, John; Kumar, Mahesh; Karesh, William B; Gilbert, Martin; Sodnomdarjaa, R; Douangngeun, Bounlom; Govorkova, Elena A; Webster, Robert G

    2008-11-01

    Waterfowl represent the natural reservoir of all subtypes of influenza A viruses, including H5N1. Ducks are especially considered major contributors to the spread of H5N1 influenza A viruses because they exhibit diversity in morbidity and mortality. Therefore, as a preventive strategy against endemic as well as pandemic influenza, it is important to reduce the spread of H5N1 influenza A viruses in duck populations. Here, we describe the pathogenicity of dominant clades (clades 1 and 2) of H5N1 influenza A viruses circulating in birds in Asia. Four representatives of dominant clades of the viruses cause symptomatic infection but lead to different profiles of lethality in domestic ducks. We also demonstrate the efficacy, cross-protectiveness, and immunogenicity of three different inactivated oil emulsion whole-virus H5 influenza vaccines (derived by implementing reverse genetics) to the viruses in domestic ducks. A single dose of the vaccines containing 1 mug of hemagglutinin protein provides complete protection against a lethal A/Duck/Laos/25/06 (H5N1) influenza virus challenge, with no evidence of morbidity, mortality, or shedding of the challenge virus. Moreover, two of the three vaccines achieved complete cross-clade or cross-subclade protection against the heterologous avian influenza virus challenge. Interestingly, the vaccines induce low or undetectable titers of hemagglutination inhibition (HI), cross-HI, and/or virus neutralization antibodies. The mechanism of complete protection in the absence of detectable antibody responses remains an open question.

  13. Environment: a potential source of animal and human infection with influenza A (H5N1) virus

    PubMed Central

    Horm, Srey V.; Gutiérrez, Ramona A.; Sorn, San; Buchy, Philippe

    2012-01-01

    Please cite this paper as: Horm et al. (2012) Environment: a potential source of animal and human infection with influenza A (H5N1) virus. Influenza and Other Respiratory Viruses 6(6), 442–448. Background  Very little is known regarding the persistence of highly pathogenic avian influenza H5N1 viruses in natural settings during outbreaks in tropical countries, although environmental factors may well play a role in the persistence and in the transmission of H5N1 virus. Objective  To investigate various environmental compartments surrounding outbreak areas as potential sources for H5N1 virus transmission. Methods  Environmental specimens were collected following outbreaks of avian influenza in Cambodia between April 2007 and February 2010. The methods used to concentrate H5N1 virus from water samples were based either on agglutination of the virus with chicken red blood cells or on adsorption on glass wool, followed by an elution‐concentration step. An elution‐concentration method was used for mud specimens. All samples that tested positive by real‐time RT‐PCRs (qRT‐PCRs) targeting the HA5, M and NA1 genes were inoculated into embryonated hen eggs for virus isolation. Results  Of a total of 246 samples, 46 (19%) tested positive for H5N1 by qRT‐PCRs. Viral RNA was frequently detected in dust, mud and soil samples from the farms’ environment (respectively, 46%, 31% and 15%). Samples collected from ponds gave a lower proportion of positive samples (6%) as compared to those collected from the farms (24%). In only one sample, infectious virus particles were successfully isolated. Conclusion  During H5N1 virus outbreaks, numerous environmental samples surrounding outbreak areas are contaminated by the virus and may act as potential sources for human and/or animal contamination. PMID:22340982

  14. Survival of H5N1 influenza virus in water and its inactivation by chemical methods.

    PubMed

    Mihai, Maria Elena; Tecu, Cristina; Ivanciuc, Alina Elena; Necula, Gheorghe; Lupulescu, Emilia; Onu, Adrian

    2011-01-01

    The ability of H5N1 Avian Influenza Virus (AIV) to survive in surface water has been assessed in experimental laboratory conditions, based on non-pathogenic avian reassortant model, by titration of infectivity (TCID50) at different time intervals, in three different types of water. The effect of different chemicals on AIV's survival was assessed using the same type of experimental model. After exposure to the chemical, followed by growth on a suitable substrate, the AIV was quantified by a real-time quantitative reverse transcriptase PCR (qRT-PCR). The reassortant virus persisted, and remained infective in aquatic environments, for 12 days at 22-35 degrees C and up to 20 days at 4 degrees C, irrespective of the type of water, supporting the hypothesis of a potential risk for transmitting the virus among birds and contaminating the household water via common sources of water. A significant decrease for AIV persistence models was recorded for sea water, after 12 days, at 35 degrees C. An effective inactivation has been shown when using commercially available products based on glutaraldehyde and penta potassium bis (peroxy mono sulphate) bis(sulphate), respectively. This rapid and safe method for decontamination, developed in this study, might be helpful in implementation of biosafety measures in laboratory and farms against AIV.

  15. Human H7N9 and H5N1 Influenza Viruses Differ in Induction of Cytokines and Tissue Tropism

    PubMed Central

    Meliopoulos, Victoria A.; Karlsson, Erik A.; Kercher, Lisa; Cline, Troy; Freiden, Pamela; Duan, Susu; Vogel, Peter; Webby, Richard J.; Guan, Yi; Peiris, Malik; Thomas, Paul G.

    2014-01-01

    ABSTRACT Since emerging in 2013, the avian-origin H7N9 influenza viruses have resulted in over 400 human infections, leading to 115 deaths to date. Although the epidemiology differs from human highly pathogenic avian H5N1 influenza virus infections, there is a similar rapid progression to acute respiratory distress syndrome. The aim of these studies was to compare the pathological and immunological characteristics of a panel of human H7N9 and H5N1 viruses in vitro and in vivo. Although there were similarities between particular H5N1 and H7N9 viruses, including association between lethal disease and spread to the alveolar spaces and kidney, there were also strain-specific differences. Both H5N1 and H7N9 viruses are capable of causing lethal infections, with mortality correlating most strongly with wider distribution of viral antigen in the lungs, rather than with traditional measures of virus titer and host responses. Strain-specific differences included hypercytokinemia in H5N1 infections that was not seen with the H7N9 infections regardless of lethality. Conversely, H7N9 viruses showed a greater tropism for respiratory epithelium covering nasal passages and nasopharynx-associated lymphoid tissue than H5N1 viruses, which may explain the enhanced transmission in ferret models. Overall, these studies highlight some distinctive properties of H5N1 and H7N9 viruses in different in vitro and in vivo models. IMPORTANCE The novel avian-origin H7N9 pandemic represents a serious threat to public health. The ability of H7N9 to cause serious lung pathology, leading in some cases to the development of acute respiratory distress syndrome, is of particular concern. Initial reports of H7N9 infection compared them to infections caused by highly pathogenic avian (HPAI) H5N1 viruses. Thus, it is of critical importance to understand the pathology and immunological response to infection with H7N9 compared to HPAI H5N1 viruses. We compared these responses in both in vitro and in vivo

  16. Lethal infection by a novel reassortant H5N1 avian influenza A virus in a zoo-housed tiger.

    PubMed

    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.

  17. Lethal infection by a novel reassortant H5N1 avian influenza A virus in a zoo-housed tiger.

    PubMed

    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. PMID:25461468

  18. Genetic and antigenic characterization of H5N1 viruses of clade 2.3.2.1 isolated in India.

    PubMed

    Bhat, Sushant; Bhatia, Sandeep; Pillai, Aravind S; Sood, Richa; Singh, Vikas Kumar; Shrivas, Om Prakash; Mishra, Suchitra K; Mawale, Namrata

    2015-11-01

    The recurrent circulation of highly pathogenic avian influenza (HPAI) H5N1 in Indian poultry since 2006 resulted in emergence of the viruses of distinct antigenic clades of haemagglutinin (HA) with the majority of the H5N1 outbreaks since 2011 belonging to clade 2.3.2.1. The present study was aimed to characterize the antigenic profile of a collection of H5N1 HPAI viruses of clade 2.3.2.1 isolated in India by applying antigenic cartography, serological data and phylogenetic analysis. Eleven H5N1 viruses (2 of clade 2.2 and 9 of clade 2.3.2.1) were selected based on genetic analysis and were further characterized by antigenic cartography analysis based on cross HI (hemagglutination inhibition) data. This study highlights the intercladal antigenic differences between clades 2.3.2.1 and 2.2 and the intracladal antigenic divergence among the clade 2.3.2.1 viruses. Five viruses of clade 2.3.2.1 were also studied for analysis of glycosylation pattern of Hemagglutinin (HA) gene and the growth kinetics analysis in MDCK cells in which the viruses CL03485/H5N1 and 03CL488/H5N1 showed better replication kinetics than other viruses. The study presents a baseline data of antigenicity and other factors that can be used in the selection of suitable H5 vaccine strains or HA donor viruses to develop H5 vaccine strains by reverse genetics or other methods for control of currently circulating H5N1 viruses in Indian region.

  19. Proposed lead molecules against Hemagglutinin of avian influenza virus (H5N1)

    PubMed Central

    Nandi, Tannistha

    2008-01-01

    Human infection with avian influenza H5N1 is an emerging infectious disease characterized by respiratory symptoms and a high fatality rate. Hemagglutinin and neuraminidase are the two surface proteins responsible for infection by influenza virus. Till date, neuraminidase has been the major target for antiviral drugs. In the present study we chose hemagglutinin protein as it mediates the binding of the virus to target cells through sialic acid residues on the host cell-surface. Hemagglutinin of H5 avian influenza (PDB ID: 1JSN) was used as the receptor protein. Ligands were generated by structure-based de novo approach and virtual screening of ZINC database. A total of 11,104 conformers were generated and docked into the receptor binding site using ‘High Throughput Virtual Screening’. We proposed potential lead molecules against the receptor binding site of hemagglutinin based on the results obtained from in silico docking and hydrogen bond interaction between the ligand and the 1JSN protein molecule. We found sialic acid derivative 1 to be the lead molecules amongst the ligands generated by structure based de novo approach. However the molecules obtained from ZINC database were showing better docking scores as well as conserved hydrogen bond interactions. Thus we proposed ZINC00487720 and ZINC00046810 as potential lead molecules that could be used as an inhibitor to the receptor binding site of hemagglutinin. They could now be studied in vivo to validate the in silico results. PMID:18317572

  20. Reproducibility of Serologic Assays for Influenza Virus A (H5N1)

    PubMed Central

    Heath, Alan; Major, Diane; Newman, Robert W.; Hoschler, Katja; Junzi, Wang; Katz, Jacqueline M.; Weir, Jerry P.; Zambon, Maria C.; Wood, John M.

    2009-01-01

    Hemagglutination-inhibition (HI) and neutralization are used to evaluate vaccines against influenza virus A (H5N1); however, poor standardization leads to interlaboratory variation of results. A candidate antibody standard (07/150) was prepared from pooled plasma of persons given clade 1 A/Vietnam/1194/2004 vaccine. To test human and sheep antiserum, 15 laboratories used HI and neutralization and reassortant A/Vietnam/1194/2004, A/turkey/Turkey/1/2005 (clade 2.2), and A/Anhui/1/2005 (clade 2.3.4) viruses. Interlaboratory variation was observed for both assays, but when titers were expressed relative to 07/150, overall percentage geometric coefficient of variation for A/Vietnam/1194/2004 was reduced from 125% to 61% for HI and from 183% to 81% for neutralization. Lack of reduced variability to clade 2 antigens suggested the need for clade-specific standards. Sheep antiserum as a standard did not reliably reduce variability. The World Health Organization has established 07/150 as an international standard for antibody to clade 1 subtype H5 and has an assigned potency of 1,000 IU/ampoule. PMID:19751587

  1. Protection and virus shedding of falcons vaccinated against highly pathogenic avian influenza A virus (H5N1).

    PubMed

    Lierz, Michael; Hafez, Hafez M; Klopfleisch, Robert; Lüschow, Dörte; Prusas, Christine; Teifke, Jens P; Rudolf, Miriam; Grund, Christian; Kalthoff, Donata; Mettenleiter, Thomas; Beer, Martin; Hardert, Timm

    2007-11-01

    Because fatal infections with highly pathogenic avian influenza A (HPAI) virus subtype H5N1 have been reported in birds of prey, we sought to determine detailed information about the birds' susceptibility and protection after vaccination. Ten falcons vaccinated with an inactivated influenza virus (H5N2) vaccine seroconverted. We then challenged 5 vaccinated and 5 nonvaccinated falcons with HPAI (H5N1). All vaccinated birds survived; all unvaccinated birds died within 5 days. For the nonvaccinated birds, histopathologic examination showed tissue degeneration and necrosis, immunohistochemical techniques showed influenza virus antigen in affected tissues, and these birds shed high levels of infectious virus from the oropharynx and cloaca. Vaccinated birds showed no influenza virus antigen in tissues and shed virus at lower titers from the oropharynx only. Vaccination could protect these valuable birds and, through reduced virus shedding, reduce risk for transmission to other avian species and humans.

  2. First reported incursion of highly pathogenic notifiable avian influenza A H5N1 viruses from clade 2.3.2 into European poultry.

    PubMed

    Reid, S M; Shell, W M; Barboi, G; Onita, I; Turcitu, M; Cioranu, R; Marinova-Petkova, A; Goujgoulova, G; Webby, R J; Webster, R G; Russell, C; Slomka, M J; Hanna, A; Banks, J; Alton, B; Barrass, L; Irvine, R M; Brown, I H

    2011-02-01

    This study reports the first incursion into European poultry of H5N1 highly pathogenic notifiable avian influenza A (HPNAI) viruses from clade 2.3.2 that affected domestic poultry and wild birds in Romania and Bulgaria, respectively. Previous occurrences in Europe of HPNAI H5N1 in these avian populations have involved exclusively viruses from clade 2.2. This represents the most westerly spread of clade 2.3.2 viruses, which have shown an apparently expanding range of geographical dispersal since mid-2009 following confirmation of infections in wild waterfowl species in Mongolia and Eastern Russia. During March 2010, AI infection was suspected at post-mortem examination of two hens from two backyard flocks in Tulcea Country, Romania. HPNAI of H5N1 subtype was confirmed by reverse transcription polymerase chain reaction (RT-PCR). A second outbreak was confirmed 2 weeks later by RT-PCR, affecting all hens from another flock located 55 km east of the first cluster. On the same day, an H5N1 HPNAI virus was detected from a pooled tissue sample collected from a dead Common Buzzard found on the Black Sea coast in Bulgaria. Detailed genetic characterization of the haemagglutinin gene revealed the cleavage site of the isolates to be consistent with viruses of high pathogenicity belonging to clade 2.3.2 of the contemporary Eurasian H5N1 lineage. Viruses from a clade other than 2.2 have apparently spread to wild birds, with potential maintenance and spread through such populations. Whilst the scale of threat posed by the apparent westward spread of the clade 2.3.2 viruses remains uncertain, ongoing vigilance for clinical signs of disease as part of existing passive surveillance frameworks for AI, and the prompt reporting of suspect cases in poultry is advised.

  3. Antigenic Variation in H5N1 clade 2.1 Viruses in Indonesia From 2005 to 2011.

    PubMed

    Setiawaty, Vivi; Pratiwi, Eka; Pawestri, Hana A; Ibrahim, Fera; Soebandrio, Amin

    2013-01-01

    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. PMID:25512692

  4. Molecular evolution of H5N1 highly pathogenic avian influenza viruses in Bangladesh between 2007 and 2012.

    PubMed

    Haque, M E; Giasuddin, M; Chowdhury, E H; Islam, M R

    2014-01-01

    In Bangladesh, highly pathogenic avian influenza (HPAI) virus subtype H5N1 was first detected in February 2007. Since then the virus has become entrenched in poultry farms of Bangladesh. There have so far been seven human cases of H5N1 HPAI infection in Bangladesh with one death. The objective of the present study was to investigate the molecular evolution of H5N1 HPAI viruses during 2007 to 2012. Partial or complete nucleotide sequences of all eight gene segments of two chicken isolates, five gene segments of a duck isolate and the haemagglutinin gene segment of 18 isolates from Bangladesh were established in the present study and subjected to molecular analysis. In addition, full-length sequences of different gene segments of other Bangladeshi H5N1 isolates available in GenBank were included in the analysis. The analysis revealed that the first introduction of clade 2.2 virus in Bangladesh in 2007 was followed by the introduction of clade 2.3.2.1 and 2.3.4 viruses in 2011. However, only clade 2.3.2.1 viruses could be isolated in 2012, indicating progressive replacement of clade 2.2 and 2.3.4 viruses. There has been an event of segment re-assortment between H5N1 and H9N2 viruses in Bangladesh, where H5N1 virus acquired the PB1 gene from a H9N2 virus. Point mutations have accumulated in Bangladeshi isolates over the last 5 years with potential modification of receptor binding site and antigenic sites. Extensive and continuous molecular epidemiological studies are necessary to monitor the evolution of circulating avian influenza viruses in Bangladesh.

  5. Efficacy of Parainfluenza Virus 5 Mutants Expressing Hemagglutinin from H5N1 Influenza A Virus in Mice

    PubMed Central

    Li, Zhuo; Gabbard, Jon D.; Mooney, Alaina; Chen, Zhenhai; Tompkins, S. Mark

    2013-01-01

    Parainfluenza virus 5 (PIV5) is a promising viral vector for vaccine development. PIV5 is safe, stable, efficacious, cost-effective to produce and, most interestingly, it overcomes preexisting antivector immunity. We have recently reported that PIV5 expressing the hemagglutinin (HA) from highly pathogenic avian influenza (HPAI) virus H5N1 (PIV5-H5) provides sterilizing immunity against lethal doses of HPAI H5N1 infection in mice. It is thought that induction of apoptosis can lead to enhanced antigen presentation. Previously, we have shown that deleting the SH gene and the conserved C terminus of the V gene in PIV5 results in mutant viruses (PIV5ΔSH and PIV5VΔC) that enhance induction of apoptosis. In this study, we inserted the HA gene of H5N1 into PIV5ΔSH (PIV5ΔSH-H5) or PIV5VΔC (PIV5VΔC-H5) and compared their efficacies as vaccine candidates to PIV5-H5. We have found that PIV5ΔSH-H5 induced the highest levels of anti-HA antibodies, the strongest T cell responses, and the best protection against an H5N1 lethal challenge in mice. These results suggest that PIV5ΔSH is a better vaccine vector than wild-type PIV5. PMID:23804633

  6. Vaccination with virus-like particles containing H5 antigens from three H5N1 clades protects chickens from H5N1 and H5N8 influenza viruses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Highly pathogenic avian influenza (HPAI) viruses, especially H5N1 strains, represent a public health threat and cause widespread morbidity and mortality in domestic poultry. Recombinant virus-like particles (VLPs) represent a promising novel vaccine approach to control avian influenza including HPAI...

  7. Antigenic analysis of highly pathogenic avian influenza virus H5N1 sublineages co-circulating in Egypt.

    PubMed

    Watanabe, Yohei; Ibrahim, Madiha S; Ellakany, Hany F; Kawashita, Norihito; Daidoji, Tomo; Takagi, Tatsuya; Yasunaga, Teruo; Nakaya, Takaaki; Ikuta, Kazuyoshi

    2012-10-01

    Highly pathogenic avian influenza virus H5N1 has spread across Eurasia and Africa, and outbreaks are now endemic in several countries, including Indonesia, Vietnam and Egypt. Continuous circulation of H5N1 virus in Egypt, from a single infected source, has led to significant genetic diversification with phylogenetically separable sublineages, providing an opportunity to study the impact of genetic evolution on viral phenotypic variation. In this study, we analysed the phylogeny of H5 haemagglutinin (HA) genes in influenza viruses isolated in Egypt from 2006 to 2011 and investigated the effect of conserved amino acid mutations in the HA genes in each of the sublineages on their antigenicity. The analysis showed that viruses in at least four sublineages still persisted in poultry in Egypt as of 2011. Using reverse genetics to generate HA-reassortment viruses with specific HA mutations, we found antigenic drift in the HA in two influenza virus sublineages, compared with the other currently co-circulating influenza virus sublineages in Egypt. Moreover, the two sublineages with significant antigenic drift were antigenically distinguishable. Our findings suggested that phylogenetically divergent H5N1 viruses, which were not antigenically cross-reactive, were co-circulating in Egypt, indicating that there was a problem in using a single influenza virus strain as seed virus to produce influenza virus vaccine in Egypt and providing data for designing more efficacious control strategies in H5N1-endemic areas.

  8. Bronchointerstitial pneumonia in guinea pigs following inoculation with H5N1 high pathogenicity avian influenza virus.

    PubMed

    Kwon, Y K; Lipatov, A S; Swayne, D E

    2009-01-01

    The H5N1 high-pathogenicity avian influenza (HPAI) viruses have caused widespread disease of poultry in Asia, Africa and the Middle East, and sporadic human infections. The guinea pig model has been used to study human H3N2 and H1N1 influenza viruses, but knowledge is lacking on H5N1 HPAI virus infections. Guinea pigs were inoculated intranasally or intragastrically with A/Vietnam/1203/04 (VN/04) or A/Muscovy duck/Vietnam/209/05 (MDk/VN/05) viruses. Mild listlessness was seen at 2 and 3 days postinoculation (DPI) in guinea pigs inoculated intranasally with VN/04 virus. At 5 DPI, the guinea pigs had bronchointerstitial pneumonia and virus was identified in bronchiolar epithelium and alveolar macrophages. Virus was isolated from the lungs but was lacking from other organs. Minimal lung lesions were seen in intranasal MDk/VN/06 group and virus was not detected, but serologic evidence of infection was observed. Intragastric exposure failed to produce infection or lesions with either virus. The localized respiratory disease in guinea pigs with H5N1 viruses was very similar to that of H3N2 and H1N1 influenza in humans and was less severe than reported for H5N1 human cases.

  9. Increased Pathogenicity of a Reassortant 2009 Pandemic H1N1 Influenza Virus Containing an H5N1 Hemagglutinin▿

    PubMed Central

    Cline, Troy D.; Karlsson, Erik A.; Freiden, Pamela; Seufzer, Bradley J.; Rehg, Jerold E.; Webby, Richard J.; Schultz-Cherry, Stacey

    2011-01-01

    A novel H1N1 influenza virus emerged in 2009 (pH1N1) to become the first influenza pandemic of the 21st century. This virus is now cocirculating with highly pathogenic H5N1 avian influenza viruses in many parts of the world, raising concerns that a reassortment event may lead to highly pathogenic influenza strains with the capacity to infect humans more readily and cause severe disease. To investigate the virulence of pH1N1-H5N1 reassortant viruses, we created pH1N1 (A/California/04/2009) viruses expressing individual genes from an avian H5N1 influenza strain (A/Hong Kong/483/1997). Using several in vitro models of virus replication, we observed increased replication for a reassortant CA/09 virus expressing the hemagglutinin (HA) gene of HK/483 (CA/09-483HA) relative to that of either parental CA/09 virus or reassortant CA/09 expressing other HK/483 genes. This increased replication correlated with enhanced pathogenicity in infected mice similar to that of the parental HK/483 strain. The serial passage of the CA/09 parental virus and the CA/09-483HA virus through primary human lung epithelial cells resulted in increased pathogenicity, suggesting that these viruses easily adapt to humans and become more virulent. In contrast, serial passage attenuated the parental HK/483 virus in vitro and resulted in slightly reduced morbidity in vivo, suggesting that sustained replication in humans attenuates H5N1 avian influenza viruses. Taken together, these data suggest that reassortment between cocirculating human pH1N1 and avian H5N1 influenza strains will result in a virus with the potential for increased pathogenicity in mammals. PMID:21917948

  10. Characaterization of H5N1 highly pathogenic avian influenza viruses isolated from poultry in Pakistan 2006-2008

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Nine avian influenza viruses (AIV), H5N1 subtype, were isolated from dead poultry in the Karachi region of Pakistan from 2006-2008. The intravenous pathogenicity indices and HA protein cleavage sites of all nine viruses were consistent with highly pathogenic AIV. Based on phylogenetic analysis of ...

  11. Complete Genome Sequence of the First H5N1 Avian Influenza Virus Isolated from Chickens in Lebanon in 2016

    PubMed Central

    Ibrahim, Elias; Sirawan, Abeer; El-Bazzal, Bassel; El Hage, Jeanne; Abi Said, Mounir; Kandeil, Ahmed; Ali, Mohamed A.

    2016-01-01

    We generated the full genome of a highly pathogenic H5N1 avian influenza virus that caused an outbreak on a chicken farm in Lebnaon in April 2016. Analysis revealed that the virus belonged to clade 2.3.2.1c that recently caused outbreaks in West Africa and the United Arab Emirates. PMID:27795243

  12. Potency of a recombinant NDV-H5 vaccine against various HPAI H5N1 virus challenges in SPF chickens.

    PubMed

    Lardinois, Amélyne; Steensels, Mieke; Lambrecht, Bénédicte; Desloges, Nathalie; Rahaus, Markus; Rebeski, Dierk; van den Berg, Thierry

    2012-12-01

    For the past decade, several recombinant Newcastle disease viruses (rNDV) have been used as a vector to express native or modified avian influenza (AI) hemagglutinins (HA) in order to give preventive protection against highly pathogenic avian influenza (HPAI) H5N1 viruses. Obtained protections were dependent on the age of the chickens, on the constructs and, in particular, on the homology between the HA that was inserted and the challenge strains. The objective of this study was to investigate the vaccine efficacy of a recombinant NDV La Sota-vectored vaccine expressing an Asian clade 1 H5 ectodomain (rNDV-H5) vaccine expressing a modified H5 ectodomain from an HPAI clade 1 H5N1 isolate as vaccine for 1-day-old specific-pathogen-free chickens. The inoculation route (oculonasal vs. drinking water), the dose-effect, and the protective range of this rNDV-H5 vaccine were studied. Both routes of vaccination induced an H5 serologic response and afforded a high degree of clinical protection against an Asian clade 1 HPAI H5N1 (AsH5N1) challenge without a significant difference between inoculation routes. A clear dose-effect could be demonstrated. Furthermore, when evaluating the protective range against antigenically divergent descendants of the Asian dade 1 HPAI H5N1 lineage, namely two Egyptian clade 2.2.1 H5N1 strains, the vaccine efficacy was less satisfactory. The rNDV-H5 vaccine provided good clinical protection and reduced viral shedding against Egyptian 2007 challenge but was unable to provide a similar protection against the more antigenically divergent Egyptian 2008 strain.

  13. Sparse serological evidence of H5N1 avian influenza virus infections in domestic cats, northeastern China.

    PubMed

    Sun, Lingshuang; Zhou, Pei; He, Shuyi; Luo, Yongfeng; Jia, Kun; Fu, Cheng; Sun, Yao; He, Huamei; Tu, Liqing; Ning, Zhangyong; Yuan, Ziguo; Wang, Heng; Li, Shoujun; Yuan, Liguo

    2015-05-01

    Today the cross-species transmission of avian influenza viruses (AIV) are a great concern. A number of AIV strains are now enzootic among poultry, with H9N2 and highly pathogenic H5N1 AIV strains prevalent in China. H5N1 strains have been recognized to infect zoo and domestic feline species. In this serological study we sought to examine evidence that H5N1 strains have infected domestic cats in northeastern China. In 2013, we conducted a cross-sectional serological study of 916 healthy cats in Heilongjian, Jilin, and Liaonin Provinces. Sera were screened with a hemagglutinin inhibition (HI) assay and seropositive specimens (HI ≥ 1:20) were further evaluated with a microneutralization (MN) assay against a clade 2.3.2 H5N1 AIV, a H9N2 AIV, A (H1N1)pdm09, and a canine H3N2 virus. While ∼2% of cats had elevated HI assays against H5N1, no elevations were confirmed (MN ≥ 1:80). These data serve as baseline for future surveillance for AIV infections among domestic cats. Conducting such surveillance seems important for geographical areas recognized as endemic for AIVs. This is especially true for countries such as China where domestic cats and poultry are often in close contact.

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

    PubMed Central

    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 was developed to identify antigenicity associated sites in influenza A viruses on the basis of immunologic datasets (i.e., from hemagglutination inhibition and microneutralization assays) and HA protein sequences. Twenty-one potential antigenicity associated sites were identified. A total of seventeen H5N1 mutants were used to validate the effects of eleven of these predicted sites on H5N1’s antigenicity, including seven newly identified sites not located in reported antibody binding sites. The experimental data confirmed that mutations of these tested sites lead to changes in viral antigenicity, validating our method. PMID:22609437

  15. Risk Distribution of Human Infections with Avian Influenza H7N9 and H5N1 virus in China

    PubMed Central

    Li, Xin-Lou; Yang, Yang; Sun, Ye; Chen, Wan-Jun; Sun, Ruo-Xi; Liu, Kun; Ma, Mai-Juan; Liang, Song; Yao, Hong-Wu; Gray, Gregory C.; Fang, Li-Qun; Cao, Wu-Chun

    2015-01-01

    It has been documented that the epidemiological characteristics of human infections with H7N9 differ significantly between H5N1. However, potential factors that may explain the different spatial distributions remain unexplored. We use boosted regression tree (BRT) models to explore the association of agro-ecological, environmental and meteorological variables with the occurrence of human cases of H7N9 and H5N1, and map the probabilities of occurrence of human cases. Live poultry markets, density of human, coverage of built-up land, relative humidity and precipitation were significant predictors for both. In addition, density of poultry, coverage of shrub and temperature played important roles for human H7N9 infection, whereas human H5N1 infection was associated with coverage of forest and water body. Based on the risks and distribution of ecological characteristics which may facilitate the circulation of the two viruses, we found Yangtze River Delta and Pearl River Delta, along with a few spots on the southeast coastline, to be the high risk areas for H7N9 and H5N1. Additional, H5N1 risk spots were identified in eastern Sichuan and southern Yunnan Provinces. Surveillance of the two viruses needs to be enhanced in these high risk areas to reduce the risk of future epidemics of avian influenza in China. PMID:26691585

  16. Risk Distribution of Human Infections with Avian Influenza H7N9 and H5N1 virus in China.

    PubMed

    Li, Xin-Lou; Yang, Yang; Sun, Ye; Chen, Wan-Jun; Sun, Ruo-Xi; Liu, Kun; Ma, Mai-Juan; Liang, Song; Yao, Hong-Wu; Gray, Gregory C; Fang, Li-Qun; Cao, Wu-Chun

    2015-01-01

    It has been documented that the epidemiological characteristics of human infections with H7N9 differ significantly between H5N1. However, potential factors that may explain the different spatial distributions remain unexplored. We use boosted regression tree (BRT) models to explore the association of agro-ecological, environmental and meteorological variables with the occurrence of human cases of H7N9 and H5N1, and map the probabilities of occurrence of human cases. Live poultry markets, density of human, coverage of built-up land, relative humidity and precipitation were significant predictors for both. In addition, density of poultry, coverage of shrub and temperature played important roles for human H7N9 infection, whereas human H5N1 infection was associated with coverage of forest and water body. Based on the risks and distribution of ecological characteristics which may facilitate the circulation of the two viruses, we found Yangtze River Delta and Pearl River Delta, along with a few spots on the southeast coastline, to be the high risk areas for H7N9 and H5N1. Additional, H5N1 risk spots were identified in eastern Sichuan and southern Yunnan Provinces. Surveillance of the two viruses needs to be enhanced in these high risk areas to reduce the risk of future epidemics of avian influenza in China. PMID:26691585

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

    SciTech Connect

    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

    2009-12-20

    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{sup +} 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.

  18. Persistence of highly pathogenic avian influenza H5N1 virus defined by agro-ecological niche

    USGS Publications Warehouse

    Hogerwerf, Lenny; Wallace, Rob G.; Ottaviani, Daniela; Slingenbergh, Jan; Prosser, Diann; Bergmann, Luc; Gilbert, Marius

    2010-01-01

    The highly pathogenic avian influenza (HPAI) H5N1 virus has spread across Eurasia and into Africa. Its persistence in a number of countries continues to disrupt poultry production, impairs smallholder livelihoods, and raises the risk a genotype adapted to human-to-human transmission may emerge. While previous studies identified domestic duck reservoirs as a primary risk factor associated with HPAI H5N1 persistence in poultry in Southeast Asia, little is known of such factors in countries with different agro-ecological conditions, and no study has investigated the impact of such conditions on HPAI H5N1 epidemiology at the global scale. This study explores the patterns of HPAI H5N1 persistence worldwide, and for China, Indonesia, and India includes individual provinces that have reported HPAI H5N1 presence during the 2004–2008 period. Multivariate analysis of a set of 14 agricultural, environmental, climatic, and socio-economic factors demonstrates in quantitative terms that a combination of six variables discriminates the areas with human cases and persistence: agricultural population density, duck density, duck by chicken density, chicken density, the product of agricultural population density and chicken output/input ratio, and purchasing power per capita. The analysis identifies five agro-ecological clusters, or niches, representing varying degrees of disease persistence. The agro-ecological distances of all study areas to the medoid of the niche with the greatest number of human cases are used to map HPAI H5N1 risk globally. The results indicate that few countries remain where HPAI H5N1 would likely persist should it be introduced.

  19. Persistence of highly pathogenic avian influenza H5N1 virus defined by agro-ecological niche.

    PubMed

    Hogerwerf, Lenny; Wallace, Rob G; Ottaviani, Daniela; Slingenbergh, Jan; Prosser, Diann; Bergmann, Luc; Gilbert, Marius

    2010-06-01

    The highly pathogenic avian influenza (HPAI) H5N1 virus has spread across Eurasia and into Africa. Its persistence in a number of countries continues to disrupt poultry production, impairs smallholder livelihoods, and raises the risk a genotype adapted to human-to-human transmission may emerge. While previous studies identified domestic duck reservoirs as a primary risk factor associated with HPAI H5N1 persistence in poultry in Southeast Asia, little is known of such factors in countries with different agro-ecological conditions, and no study has investigated the impact of such conditions on HPAI H5N1 epidemiology at the global scale. This study explores the patterns of HPAI H5N1 persistence worldwide, and for China, Indonesia, and India includes individual provinces that have reported HPAI H5N1 presence during the 2004-2008 period. Multivariate analysis of a set of 14 agricultural, environmental, climatic, and socio-economic factors demonstrates in quantitative terms that a combination of six variables discriminates the areas with human cases and persistence: agricultural population density, duck density, duck by chicken density, chicken density, the product of agricultural population density and chicken output/input ratio, and purchasing power per capita. The analysis identifies five agro-ecological clusters, or niches, representing varying degrees of disease persistence. The agro-ecological distances of all study areas to the medoid of the niche with the greatest number of human cases are used to map HPAI H5N1 risk globally. The results indicate that few countries remain where HPAI H5N1 would likely persist should it be introduced.

  20. Persistence of avian influenza virus (H5N1) in feathers detached from bodies of infected domestic ducks.

    PubMed

    Yamamoto, Yu; Nakamura, Kikuyasu; Yamada, Manabu; Mase, Masaji

    2010-08-01

    Asian lineage highly pathogenic avian influenza virus (H5N1) continues to cause mortality in poultry and wild bird populations at a panzootic scale. However, little is known about its persistence in contaminated tissues derived from infected birds. We investigated avian influenza virus (H5N1) persistence in feathers detached from bodies of infected ducks to evaluate their potential risk for environmental contamination. Four-week-old domestic ducks were inoculated with different clades of avian influenza virus (H5N1). Feathers, drinking water, and feces were collected on day 3 postinoculation and stored at 4 degrees C or 20 degrees C. Viral persistence in samples was investigated for 360 days by virus isolation and reverse transcription-PCR. Infectious viruses persisted for the longest period in feathers, compared with drinking water and feces, at both 4 degrees C and 20 degrees C. Viral infectivity persisted in the feathers for 160 days at 4 degrees C and for 15 days at 20 degrees C. Viral titers of 10(4.3) 50% egg infectious doses/ml or greater were detected for 120 days in feathers stored at 4 degrees C. Viral RNA in feathers was more stable than the infectivity. These results indicate that feathers detached from domestic ducks infected with highly pathogenic avian influenza virus (H5N1) can be a source of environmental contamination and may function as fomites with high viral loads in the environment.

  1. Surveillance on A/H5N1 virus in domestic poultry and wild birds in Egypt

    PubMed Central

    2013-01-01

    Background The endemic H5N1 high pathogenicity avian influenza virus (A/H5N1) in poultry in Egypt continues to cause heavy losses in poultry and poses a significant threat to human health. Methods Here we describe results of A/H5N1 surveillance in domestic poultry in 2009 and wild birds in 2009–2010. Tracheal and cloacal swabs were collected from domestic poultry from 22024 commercial farms, 1435 backyards and 944 live bird markets (LBMs) as well as from 1297 wild birds representing 28 different types of migratory birds. Viral RNA was extracted from a mix of tracheal and cloacal swabs media. Matrix gene of avian influenza type A virus was detected using specific real-time reverse-transcription polymerase chain reaction (RT-qPCR) and positive samples were tested by RT-qPCR for simultaneous detection of the H5 and N1 genes. Results In this surveillance, A/H5N1 was detected from 0.1% (n = 23/) of examined commercial poultry farms, 10.5% (n = 151) of backyard birds and 11.4% (n = 108) of LBMs but no wild bird tested positive for A/H5N1. The virus was detected from domestic poultry year-round with higher incidence in the warmer months of summer and spring particularly in backyard birds. Outbreaks were recorded mostly in Lower Egypt where 95.7% (n = 22), 68.9% (n = 104) and 52.8% (n = 57) of positive commercial farms, backyards and LBMs were detected, respectively. Higher prevalence (56%, n = 85) was reported in backyards that had mixed chickens and waterfowl together in the same vicinity and LBMs that had waterfowl (76%, n = 82). Conclusion Our findings indicated broad circulation of the endemic A/H5N1 among poultry in 2009 in Egypt. In addition, the epidemiology of A/H5N1 has changed over time with outbreaks occurring in the warmer months of the year. Backyard waterfowl may play a role as a reservoir and/or source of A/H5N1 particularly in LBMs. The virus has been established in poultry in the Nile Delta where major metropolitan areas

  2. Changes in the hemagglutinin of H5N1 viruses during human infection – Influence on receptor binding☆

    PubMed Central

    Crusat, Martin; Liu, Junfeng; Palma, Angelina S.; Childs, Robert A.; Liu, Yan; Wharton, Stephen A.; Lin, Yi Pu; Coombs, Peter J.; Martin, Stephen R.; Matrosovich, Mikhail; Chen, Zi; Stevens, David J.; Hien, Vo Minh; Thanh, Tran Tan; Nhu, Le Nguyen Truc; Nguyet, Lam Anh; Ha, Do Quang; van Doorn, H.Rogier; Hien, Tran Tinh; Conradt, Harald S.; Kiso, Makoto; Gamblin, Steve J.; Chai, Wengang; Skehel, John J.; Hay, Alan J.; Farrar, Jeremy; de Jong, Menno D.; Feizi, Ten

    2013-01-01

    As avian influenza A(H5N1) viruses continue to circulate in Asia and Africa, global concerns of an imminent pandemic persist. Recent experimental studies suggest that efficient transmission between humans of current H5N1 viruses only requires a few genetic changes. An essential step is alteration of the virus hemagglutinin from preferential binding to avian receptors for the recognition of human receptors present in the upper airway. We have identified receptor-binding changes which emerged during H5N1 infection of humans, due to single amino acid substitutions, Ala134Val and Ile151Phe, in the hemagglutinin. Detailed biological, receptor-binding, and structural analyses revealed reduced binding of the mutated viruses to avian-like receptors, but without commensurate increased binding to the human-like receptors investigated, possibly reflecting a receptor-binding phenotype intermediate in adaptation to more human-like characteristics. These observations emphasize that evolution in nature of avian H5N1 viruses to efficient binding of human receptors is a complex multistep process. PMID:24050651

  3. Vaccine Protection of Turkeys Against H5N1 Highly Pathogenic Avian Influenza Virus with a Recombinant Turkey Herpesvirus Expressing the Hemagglutinin Gene of Avian Influenza.

    PubMed

    Kapczynski, Darrell R; Dorsey, Kristi; Chrzastek, Klaudia; Moraes, Mauro; Jackwood, Mark; Hilt, Debra; Gardin, Yannick

    2016-06-01

    Outbreaks of H5 highly pathogenic avian influenza (HPAI) in commercial poultry are a constant threat to animal health and food supplies. While vaccination can enhance protection and reduce the spread of disease, there is considerable evidence that the level of immunity required for protection varies by subtype and virulence of field virus. In this study, the efficacy of a recombinant turkey herpesvirus (rHVT) vector vaccine expressing the hemagglutinin gene from a clade 2.2 AI virus (A/Swan/Hungary/4999/2006) was evaluated in turkeys for protection against challenge with A/Whooper Swan/Mongolia/L244/2005 H5N1 HPAI clade 2.2. One-day-old turkeys received a single vaccination and were challenged at 4 wk postvaccination with 2 × 10(6) 50% embryo infectious dose per bird. The results demonstrate that following H5N1 HPAI challenge 96% protection was observed in rHVT-AI vaccinated turkeys. The oral and cloacal swabs taken from challenged birds demonstrated that vaccinated birds had lower incidence and titers of viral shedding compared with sham-vaccinated birds. From respiratory and gastrointestinal tracts, there was a greater than 6 log10 reduction in shedding in vaccinated birds as compared with the controls. This study provides support for the use of a commercially available rHVT-AI vaccine to protect turkeys against H5N1 HPAI. PMID:27309280

  4. Risk for infection with highly pathogenic influenza A virus (H5N1) in chickens, Hong Kong, 2002.

    PubMed

    Kung, Nina Y; Morris, Roger S; Perkins, Nigel R; Sims, Les D; Ellis, Trevor M; Bissett, Lucy; Chow, Mary; Shortridge, Ken F; Guan, Yi; Peiris, Malik J S

    2007-03-01

    We used epidemiologic evaluation, molecular epidemiology, and a case-control study to identify possible risk factors for the spread of highly pathogenic avian influenza A virus (subtype H5N1) in chicken farms during the first quarter of 2002 in Hong Kong. Farm profiles, including stock sources, farm management, and biosecurity measures, were collected from 16 case and 46 control chicken farms by using a pretested questionnaire and personal interviews. The risk for influenza A (H5N1) infection was assessed by using adjusted odds ratios based on multivariate logistic regression analysis. Retail marketing of live poultry was implicated as the main source of exposure to infection on chicken farms in Hong Kong during this period. Infection control measures should be reviewed and upgraded as necessary to reduce the spread of influenza A (H5N1) related to live poultry markets, which are commonplace across Asia.

  5. Understanding the potential dispersal of HPAI H5N1 virus by migratory wildfowl

    USGS Publications Warehouse

    Gaidet, Nicolas; Cappelle, Julien; Takekawa, John Y.; Prosser, Diann J.; Iverson, Samuel A.; Douglas, David C.; Perry, William M.; Mundkur, Taej; Newman, Scott H.

    2010-01-01

    We analysed wildfowl movements between 2006-2009, including 228 birds from 19 species, part of a larger international programme (see Figure 1) coordinated by the Food and Agricultural Organisation (FAO) of the United Nations aimed at understanding if there are temporal or spatial relationships between HPAI H5N1 outbreaks and movements of migratory wildfowl, the first large scale data set available for such an analysis.

  6. Increased Number of Human Cases of Influenza Virus A(H5N1) Infection, Egypt, 2014-15.

    PubMed

    Refaey, Samir; Azziz-Baumgartner, Eduardo; Amin, Marwa Mohamed; Fahim, Manal; Roguski, Katherine; Elaziz, Hanaa Abu Elsood Abd; Iuliano, A Danielle; Salah, Noha; Uyeki, Timothy M; Lindstrom, Steven; Davis, Charles Todd; Eid, Alaa; Genedy, Mohamed; Kandeel, Amr

    2015-12-01

    During November 2014-April 2015, a total of 165 case-patients with influenza virus A(H5N1) infection, including 6 clusters and 51 deaths, were identified in Egypt. Among infected persons, 99% reported poultry exposure: 19% to ill poultry and 35% to dead poultry. Only 1 person reported wearing personal protective equipment while working with poultry. PMID:26584397

  7. Increased Number of Human Cases of Influenza Virus A(H5N1) Infection, Egypt, 2014-15.

    PubMed

    Refaey, Samir; Azziz-Baumgartner, Eduardo; Amin, Marwa Mohamed; Fahim, Manal; Roguski, Katherine; Elaziz, Hanaa Abu Elsood Abd; Iuliano, A Danielle; Salah, Noha; Uyeki, Timothy M; Lindstrom, Steven; Davis, Charles Todd; Eid, Alaa; Genedy, Mohamed; Kandeel, Amr

    2015-12-01

    During November 2014-April 2015, a total of 165 case-patients with influenza virus A(H5N1) infection, including 6 clusters and 51 deaths, were identified in Egypt. Among infected persons, 99% reported poultry exposure: 19% to ill poultry and 35% to dead poultry. Only 1 person reported wearing personal protective equipment while working with poultry.

  8. Mechanisms of transmission and spread of H5N1 high pathogenicity avian influenza virus in birds and mammals

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Eurasian-African H5N1 high pathogenicity avian influenza (HPAI) virus has crossed multiple species barriers to infect poultry, captive and wild birds, carnivorous mammals and humans. The specific transmission mechanisms are unclear in most cases, but experimental studies and field data sug...

  9. Corneal Opacity in Domestic Ducks Experimentally Infected With H5N1 Highly Pathogenic Avian Influenza Virus.

    PubMed

    Yamamoto, Y; Nakamura, K; Yamada, M; Mase, M

    2016-01-01

    Domestic ducks can be a key factor in the regional spread of H5N1 highly pathogenic avian influenza (HPAI) virus in Asia. The authors performed experimental infections to examine the relationship between corneal opacity and H5N1 HPAI virus infection in domestic ducks (Anas platyrhyncha var domestica). A total of 99 domestic ducks, including 3 control birds, were used in the study. In experiment 1, when domestic ducks were inoculated intranasally with 2 H5N1 HPAI viruses, corneal opacity appeared more frequently than neurologic signs and mortality. Corneal ulceration and exophthalmos were rare findings. Histopathologic examinations of the eyes of domestic ducks in experiment 2 revealed that corneal opacity was due to the loss of corneal endothelial cells and subsequent keratitis with edema. Influenza viral antigen was detected in corneal endothelial cells and some other ocular cells by immunohistochemistry. Results suggest that corneal opacity is a characteristic and frequent finding in domestic ducks infected with the H5N1 HPAI virus. Confirming this ocular change may improve the detection rate of infected domestic ducks in the field.

  10. Molecular and antigenic evolution and geographical spread of H5N1 highly pathogenic avian influenza viruses in western Africa.

    PubMed

    Ducatez, M F; Olinger, C M; Owoade, A A; Tarnagda, Z; Tahita, M C; Sow, A; De Landtsheer, S; Ammerlaan, W; Ouedraogo, J B; Osterhaus, A D M E; Fouchier, R A M; Muller, C P

    2007-08-01

    In Africa, highly pathogenic avian influenza H5N1 virus was first detected in northern Nigeria and later also in other regions of the country. Since then, seven other African countries have reported H5N1 infections. This study reports a comparison of full-length genomic sequences of H5N1 isolates from seven chicken farms in Nigeria and chicken and hooded vultures in Burkina Faso with earlier H5N1 outbreaks worldwide. In addition, the antigenicity of Nigerian H5N1 isolates was compared with earlier strains. All African strains clustered within three sublineages denominated A (south-west Nigeria, Niger), B (south-west Nigeria, Egypt, Djibouti) and C (northern Nigeria, Burkina Faso, Sudan, Côte d'Ivoire), with distinct nucleotide and amino acid signatures and distinct geographical distributions within Africa. Probable non-African ancestors within the west Asian/Russian/European lineage distinct from the south-east Asian lineages were identified for each sublineage. All reported human cases in Africa were caused by sublineage B. Substitution rates were calculated on the basis of sequences from 11 strains from a single farm in south-west Nigeria. As H5N1 emerged essentially at the same time in the north and south-west of Nigeria, the substitution rates confirmed that the virus probably did not spread from the north to the south, given the observed sequence diversity, but that it entered the country via three independent introductions. The strains from Burkina Faso seemed to originate from northern Nigeria. At least two of the sublineages also circulated in Europe in 2006 as seen in Germany, further suggesting that the sublineages had already emerged outside of Africa and seemed to have followed the east African/west Asian and Black Sea/Mediterranean flyways of migratory birds. PMID:17622635

  11. Characterization of duck H5N1 influenza viruses with differing pathogenicity in mallard (Anas platyrhynchos) ducks.

    PubMed

    Tang, Yinghua; Wu, Peipei; Peng, Daxin; Wang, Xiaobo; Wan, Hongquan; Zhang, Pinghu; Long, Jinxue; Zhang, Wenjun; Li, Yanfang; Wang, Wenbin; Zhang, Xiaorong; Liu, Xiufan

    2009-12-01

    A number of H5N1 influenza outbreaks have occurred in aquatic birds in Asia. As aquatic birds are the natural reservoir of influenza A viruses and do not usually show clinical disease upon infection, the repeated H5N1 outbreaks have highlighted the importance of continuous surveillance on H5N1 viruses in aquatic birds. In the present study we characterized the biological properties of four H5N1 avian influenza viruses, which had been isolated from ducks, in different animal models. In specific pathogen free (SPF) chickens, all four isolates were highly pathogenic. In SPF mice, the S and Y isolates were moderately pathogenic. However, in mallard ducks, two isolates had low pathogenicity, while the other two were highly pathogenic and caused lethal infection. A representative isolate with high pathogenicity in ducks caused systemic infection and replicated effectively in all 10 organs tested in challenged ducks, whereas a representative isolate with low pathogenicity in ducks was only detected in some organs in a few challenged ducks. Comparison of complete genomic sequences from the four isolates showed that the same amino acid residues that have been reported to be associated with virulence and host adaption/restriction of influenza viruses were present in the PB2, HA, NA, M and NS genes, while the amino acid residues at the HA cleavage site were diverse. From these results it appeared that the virulence of H5N1 avian influenza viruses was increased for ducks and that amino acid substitutions at the HA cleavage site might have contributed to the differing pathogenicity of these isolates in mallards. A procedure for the intravenous pathogenicity index test in a mallard model for assessing the virulence of H5/H7 subtype avian influenza viruses in waterfowl is described.

  12. H5N1 Influenza Virus Pathogenesis in Genetically Diverse Mice Is Mediated at the Level of Viral Load

    PubMed Central

    Boon, Adrianus C. M.; Finkelstein, David; Zheng, Ming; Liao, Guochun; Allard, John; Klumpp, Klaus; Webster, Robert; Peltz, Gary; Webby, Richard J.

    2011-01-01

    ABSTRACT The genotype of the host is one of several factors involved in the pathogenesis of an infectious disease and may be a key parameter in the epidemiology of highly pathogenic H5N1 influenza virus infection in humans. Gene polymorphisms may affect the viral replication rate or alter the host’s immune response to the virus. In humans, it is unclear which aspect dictates the severity of H5N1 virus disease. To identify the mechanism underlying differential responses to H5N1 virus infection in a genetically diverse population, we assessed the host responses and lung viral loads in 21 inbred mouse strains upon intranasal inoculation with A/Hong Kong/213/03 (H5N1). Resistant mouse strains survived large inocula while susceptible strains succumbed to infection with 1,000- to 10,000-fold-lower doses. Quantitative analysis of the viral load after inoculation with an intermediate dose found significant associations with lethality as early as 2 days postinoculation, earlier than any other disease indicator. The increased viral titers in the highly susceptible strains mediated a hyperinflamed environment, indicated by the distinct expression profiles and increased production of inflammatory mediators on day 3. Supporting the hypothesis that viral load rather than an inappropriate response to the virus was the key severity-determining factor, we performed quantitative real-time PCR measuring the cytokine/viral RNA ratio. No significant differences between susceptible and resistant mouse strains were detected, confirming that it is the host genetic component controlling viral load, and therefore replication dynamics, that is primarily responsible for a host’s susceptibility to a given H5N1 virus. PMID:21896679

  13. Multiple reassortment events among highly pathogenic avian influenza A(H5N1) viruses detected in Bangladesh.

    PubMed

    Gerloff, Nancy A; Khan, Salah Uddin; Balish, Amanda; Shanta, Ireen S; Simpson, Natosha; Berman, Lashondra; Haider, Najmul; Poh, Mee Kian; Islam, Ausraful; Gurley, Emily; Hasnat, Md Abdul; Dey, T; Shu, Bo; Emery, Shannon; Lindstrom, Stephen; Haque, Ainul; Klimov, Alexander; Villanueva, Julie; Rahman, Mahmudur; Azziz-Baumgartner, Eduardo; Ziaur Rahman, Md; Luby, Stephen P; Zeidner, Nord; Donis, Ruben O; Sturm-Ramirez, Katharine; Davis, C Todd

    2014-02-01

    In Bangladesh, little is known about the genomic composition and antigenicity of highly pathogenic avian influenza A(H5N1) viruses, their geographic distribution, temporal patterns, or gene flow within the avian host population. Forty highly pathogenic avian influenza A(H5N1) viruses isolated from humans and poultry in Bangladesh between 2008 and 2012 were analyzed by full genome sequencing and antigenic characterization. The analysis included viruses collected from avian hosts and environmental sampling in live bird markets, backyard poultry flocks, outbreak investigations in wild birds or poultry and from three human cases. Phylogenetic analysis indicated that the ancestors of these viruses reassorted (1) with other gene lineages of the same clade, (2) between different clades and (3) with low pathogenicity avian influenza A virus subtypes. Bayesian estimates of the time of most recent common ancestry, combined with geographic information, provided evidence of probable routes and timelines of virus spread into and out of Bangladesh.

  14. An Anti-H5N1 Influenza Virus FcDART Antibody Is a Highly Efficacious Therapeutic Agent and Prophylactic against H5N1 Influenza Virus Infection

    PubMed Central

    Zanin, Mark; Keck, Zhen-Yong; Rainey, G. Jonah; Lam, Chia-Ying Kao; Boon, Adrianus C. M.; Rubrum, Adam; Darnell, Daniel; Wong, Sook-San; Griffin, Yolanda; Xia, Jinming; Webster, Robert G.; Johnson, Syd; Foung, Steven

    2015-01-01

    ABSTRACT Highly pathogenic H5N1 avian influenza viruses are associated with severe disease in humans and continue to be a pandemic threat. While vaccines are available, other approaches are required for patients that typically respond poorly to vaccination, such as the elderly and the immunocompromised. To produce a therapeutic agent that is highly efficacious at low doses and is broadly specific against antigenically drifted H5N1 influenza viruses, we developed two neutralizing monoclonal antibodies and combined them into a single bispecific Fc fusion protein (the Fc dual-affinity retargeting [FcDART] molecule). In mice, a single therapeutic or prophylactic dose of either monoclonal antibody at 2.5 mg/kg of body weight provided 100% protection against challenge with A/Vietnam/1203/04 (H5N1) or the antigenically drifted strain A/Whooper swan/Mongolia/244/05 (H5N1). In ferrets, a single 1-mg/kg prophylactic dose provided 100% protection against A/Vietnam/1203/04 challenge. FcDART was also effective, as a single 2.5-mg/kg therapeutic or prophylactic dose in mice provided 100% protection against A/Vietnam/1203/04 challenge. Antibodies bound to conformational epitopes in antigenic sites on the globular head of the hemagglutinin protein, on the basis of analysis of mutants with antibody escape mutations. While it was possible to generate escape mutants in vitro, they were neutralized by the antibodies in vivo, as mice infected with escape mutants were 100% protected after only a single therapeutic dose of the antibody used to generate the escape mutant in vitro. In summary, we have combined the antigen specificities of two highly efficacious anti-H5N1 influenza virus antibodies into a bispecific FcDART molecule, which represents a strategy to produce broadly neutralizing antibodies that are effective against antigenically diverse influenza viruses. IMPORTANCE Highly pathogenic H5N1 avian influenza viruses are associated with severe disease in humans and are a pandemic

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

    PubMed Central

    Lum, Jacob; Wang, Ronghui; Hargis, Billy; Tung, Steve; Bottje, Walter; Lu, Huaguang; Li, Yanbin

    2015-01-01

    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. PMID:26230699

  16. Determination of efficacious vaccine seed strains for use against Egyptian H5N1 highly pathogenic avian influenza viruses through antigenic cartography and in vivo challenge studies

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Since 2006, there have been reported outbreaks of H5N1 highly pathogenic avian influenza (HPAI) in vaccinated chickens in Africa and Asia. This study provides experimental data for selection of efficacious H5N1 vaccine seed strains against recently circulating strains of H5N1 HPAI viruses in Egypt....

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

    PubMed Central

    Balish, Amanda; Hoang, Anh Nguyen; Gustin, Kortney M.; Nhung, Pham Thi; Jones, Joyce; Thu, Ngoc Nguyen; Davis, William; Ngoc, Thao Nguyen Thi; Jang, Yunho; Sleeman, Katrina; Villanueva, Julie; Kile, James; Gubareva, Larisa V.; Lindstrom, Stephen; Tumpey, Terrence M.; Davis, C. Todd; Long, Nguyen Thanh

    2015-01-01

    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. PMID:26244768

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

    PubMed

    Thor, Sharmi W; Nguyen, Hieu; Balish, Amanda; Hoang, Anh Nguyen; Gustin, Kortney M; Nhung, Pham Thi; Jones, Joyce; Thu, Ngoc Nguyen; Davis, William; Ngoc, Thao Nguyen Thi; Jang, Yunho; Sleeman, Katrina; Villanueva, Julie; Kile, James; Gubareva, Larisa V; Lindstrom, Stephen; Tumpey, Terrence M; Davis, C Todd; Long, Nguyen Thanh

    2015-01-01

    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. PMID:26244768

  19. Spread of Influenza Virus A (H5N1) Clade 2.3.2.1 to Bulgaria in Common Buzzards

    PubMed Central

    Marinova-Petkova, Atanaska; Georgiev, Georgi; Seiler, Patrick; Darnell, Daniel; Franks, John; Krauss, Scott; Webby, Richard J.

    2012-01-01

    On March 15, 2010, a highly pathogenic avian influenza virus was isolated from the carcass of a common buzzard (Buteo buteo) in Bulgaria. Phylogenetic analyses of the virus showed a close genetic relationship with influenza virus A (H5N1) clade 2.3.2.1 viruses isolated from wild birds in the Tyva Republic and Mongolia during 2009–2010. Designated A/common buzzard/Bulgaria/38WB/2010, this strain was highly pathogenic in chickens but had low pathogenicity in mice and ferrets and no molecular markers of increased pathogenicity in mammals. The establishment of clade 2.3.2.1 highly pathogenic avian influenza viruses of the H5N1 subtype in wild birds in Europe would increase the likelihood of health threats to humans and poultry in the region. PMID:23017273

  20. Isolation of highly pathogenic avian influenza H5N1 virus from Saker falcons (Falco cherrug) in the Middle East.

    PubMed

    Marjuki, Henju; Wernery, Ulrich; Yen, Hui-Ling; Franks, John; Seiler, Patrick; Walker, David; Krauss, Scott; Webster, Robert G

    2009-01-01

    There is accumulating evidence that birds of prey are susceptible to fatal infection with highly pathogenic avian influenza (HPAI) virus. We studied the antigenic, molecular, phylogenetic, and pathogenic properties of 2 HPAI H5N1 viruses isolated from dead falcons in Saudi Arabia and Kuwait in 2005 and 2007, respectively. Phylogenetic and antigenic analyses grouped both isolates in clade 2.2 (Qinghai-like viruses). However, the viruses appeared to have spread westward via different flyways. It remains unknown how these viruses spread so rapidly from Qinghai after the 2005 outbreak and how they were introduced into falcons in these two countries. The H5N1 outbreaks in the Middle East are believed by some to be mediated by wild migratory birds. However, sporting falcons may be at additional risk from the illegal import of live quail to feed them.

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

    SciTech Connect

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

    2008-09-05

    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 {alpha}-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.

  2. Pathology of highly pathogenic avian influenza virus (H5N1) infection in Canada geese (Branta canadensis): preliminary studies.

    PubMed

    Neufeld, J L; Embury-Hyatt, C; Berhane, Y; Manning, L; Ganske, S; Pasick, J

    2009-09-01

    Susceptibility of Canada geese (Branta canadensis) to highly pathogenic avian influenza (HPAI) virus (H5N1) infection was studied by inoculating 10 naïve (antibody-negative) animals (5 adults and 5 juveniles) with A/chicken/Vietnam/14/05 (H5N1) virus. In the adults, 1 of 5 became infected, and 4 of 5 remained normal; in the juvenile group, 5 of 5 became infected. The pathology observed in the affected animals was similar to that reported in natural occurrences. Peripheral and parasympathetic nervous systems were examined and found infected, as well as cerebrospinal fluid-contacting neurons. In some locations with significant virus infection in cells, the expected inflammatory reaction was absent or very mild. Immunohistochemistry was used to locate influenza A virus nucleoprotein in brain, spinal cord, respiratory and digestive systems, pancreas, heart, and peripheral and parasympathetic nervous systems. Further studies are needed to explain age-related differences in susceptibility.

  3. Oseltamivir-Ribavirin Combination Therapy for Highly Pathogenic H5N1 Influenza Virus Infection in Mice▿

    PubMed Central

    Ilyushina, Natalia A.; Hay, Alan; Yilmaz, Neziha; Boon, Adrianus C. M.; Webster, Robert G.; Govorkova, Elena A.

    2008-01-01

    We studied the effects of a neuraminidase inhibitor (oseltamivir) and an inhibitor of influenza virus polymerases (ribavirin) against two highly pathogenic H5N1 influenza viruses. In vitro, A/Vietnam/1203/04 virus (clade 1) was highly susceptible to oseltamivir carboxylate (50% inhibitory concentration [IC50] = 0.3 nM), whereas A/Turkey/15/06 virus (clade 2.2) had reduced susceptibility (IC50 = 5.5 nM). In vivo, BALB/c mice were treated with oseltamivir (1, 10, 50, or 100 mg/kg of body weight/day), ribavirin (37.5, 55, or 75 mg/kg/day), or the combination of both drugs for 8 days, starting 4 h before virus inoculation. Monotherapy produced a dose-dependent antiviral effect against the two H5N1 viruses in vivo. Three-dimensional analysis of the drug-drug interactions revealed that oseltamivir and ribavirin interacted principally in an additive manner, with several exceptions of marginal synergy or marginal antagonism at some concentrations. The combination of ribavirin at 37.5 mg/kg/day and oseltamivir at 1 mg/kg/day and the combination of ribavirin at 37.5 mg/kg/day and oseltamivir at 10 mg/kg/day were synergistic against A/Vietnam/1203/04 and A/Turkey/15/06 viruses, respectively. These optimal oseltamivir-ribavirin combinations significantly inhibited virus replication in mouse organs, prevented the spread of H5N1 viruses beyond the respiratory tract, and abrogated the cytokine response (P < 0.01). Importantly, we observed clear differences between the efficacies of the drug combinations against two H5N1 viruses: higher doses were required for the protection of mice against A/Turkey/15/06 virus than for the protection of mice against A/Vietnam/1203/04 virus. Our preliminary results suggest that oseltamivir-ribavirin combinations can have a greater or lesser antiviral effect than monotherapy, depending on the H5N1 virus and the concentrations used. PMID:18725448

  4. Dryocrassin ABBA, a novel active substance for use against amantadine-resistant H5N1 avian influenza virus.

    PubMed

    Ou, Changbo; Zhang, Qiang; Wu, Guojiang; Shi, Ningning; He, Cheng

    2015-01-01

    The occurrence of multi-drug resistant highly pathogenic avian influenza virus (HPAIV) strains highlights the urgent need for strategies for the prevention and control of avian influenza virus. The aim of our current study is to evaluate the antiviral activity of dryocrassin ABBA isolated from Rhizoma Dryopteridis Crassirhizomatis (RDC) against an amantadine-resistant H5N1 (A/Chicken/Hebei/706/2005) strain in a mouse model. Post inoculation with HPAIV H5N1 virus in mice, the survival rate was 87, 80, and 60% respectively in the 33, 18, and 12.5 mg/kg dryocrassin ABBA-treated groups. On the other hand, the survival rate was 53 and 20%, respectively in the amantadine-treated group and untreated group. Mice administered with dryocrassin ABBA or amantadine showed a significant weight increase compared to the untreated group. Moreover, 33 and 18 mg/kg dryocrassin ABBA have decreased lung index (P >0.05) and virus loads (P <0.01) compared to the untreated group on day 7. Also, on day 7 bronchoalveolar lavage fluid pro-inflammatory cytokines (IL-6, TNF-α, and IFN-γ) decreased significantly (P <0.01) while anti-inflammatory cytokines (IL-10 and MCP-1) were increased significantly (P <0.01) in the 33 and 18 mg/kg dryocrassin ABBA-treated groups compared to the amantadine group and the untreated group. Moreover, the concentrations of IL-12 in drug-treated groups were significantly (P < 0.01) lowered compared with the untreated group. Based on the above we conclude that orally administered dryocrassin ABBA provided mice protection against avian influenza virus H5N1 by inhibiting inflammation and reducing virus loads. Dryocrassin ABBA is a potential novel lead compound which had antiviral effects on amantadine-resistant avian influenza virus H5N1 infection.

  5. The PA protein directly contributes to the virulence of H5N1 avian influenza viruses in domestic ducks.

    PubMed

    Song, Jiasheng; Feng, Huapeng; Xu, Jing; Zhao, Dongming; Shi, Jianzhong; Li, Yanbing; Deng, Guohua; Jiang, Yongping; Li, Xuyong; Zhu, Pengyang; Guan, Yuntao; Bu, Zhigao; Kawaoka, Yoshihiro; Chen, Hualan

    2011-03-01

    During their circulation in nature, H5N1 avian influenza viruses (AIVs) have acquired the ability to kill their natural hosts, wild birds and ducks. The genetic determinants for this increased virulence are largely unknown. In this study, we compared two genetically similar H5N1 AIVs, A/duck/Hubei/49/05 (DK/49) and A/goose/Hubei/65/05 (GS/65), that are lethal for chickens but differ in their virulence levels in ducks. To explore the genetic basis for this difference in virulence, we generated a series of reassortants and mutants of these two viruses. The virulence of the reassortant bearing the PA gene from DK/49 in the GS/65 background increased 10(5)-fold relative to that of the GS/65 virus. Substitution of two amino acids, S224P and N383D, in PA contributed to the highly virulent phenotype. The amino acid 224P in PA increased the replication of the virus in duck embryo fibroblasts, and the amino acid 383D in PA increased the polymerase activity in duck embryo fibroblasts and delayed the accumulation of the PA and PB1 polymerase subunits in the nucleus of virus-infected cells. Our results provide strong evidence that the polymerase PA subunit is a virulence factor for H5N1 AIVs in ducks.

  6. X-ray structure of NS1 from a highly pathogenic H5N1 influenza virus.

    PubMed

    Bornholdt, Zachary A; Prasad, B V Venkataram

    2008-12-18

    The recent emergence of highly pathogenic avian (H5N1) influenza viruses, their epizootic and panzootic nature, and their association with lethal human infections have raised significant global health concerns. Several studies have underlined the importance of non-structural protein NS1 in the increased pathogenicity and virulence of these strains. NS1, which consists of two domains-a double-stranded RNA (dsRNA) binding domain and the effector domain, separated through a linker-is an antagonist of antiviral type-I interferon response in the host. Here we report the X-ray structure of the full-length NS1 from an H5N1 strain (A/Vietnam/1203/2004) that was associated with 60% of human deaths in an outbreak in Vietnam. Compared to the individually determined structures of the RNA binding domain and the effector domain from non-H5N1 strains, the RNA binding domain within H5N1 NS1 exhibits modest structural changes, while the H5N1 effector domain shows significant alteration, particularly in the dimeric interface. Although both domains in the full-length NS1 individually participate in dimeric interactions, an unexpected finding is that these interactions result in the formation of a chain of NS1 molecules instead of distinct dimeric units. Three such chains in the crystal interact with one another extensively to form a tubular organization of similar dimensions to that observed in the cryo-electron microscopy images of NS1 in the presence of dsRNA. The tubular oligomeric organization of NS1, in which residues implicated in dsRNA binding face a 20-A-wide central tunnel, provides a plausible mechanism for how NS1 sequesters varying lengths of dsRNA, to counter cellular antiviral dsRNA response pathways, while simultaneously interacting with other cellular ligands during an infection.

  7. X-ray structure of NS1 from a highly pathogenic H5N1 influenza virus

    SciTech Connect

    Bornholdt, Zachary A.; Prasad, B.V. Venkataram

    2009-04-08

    The recent emergence of highly pathogenic avian (H5N1) influenza viruses, their epizootic and panzootic nature, and their association with lethal human infections have raised significant global health concerns. Several studies have underlined the importance of non-structural protein NS1 in the increased pathogenicity and virulence of these strains. NS1, which consists of two domains - a double-stranded RNA (dsRNA) binding domain and the effector domain, separated through a linker - is an antagonist of antiviral type-I interferon response in the host. Here we report the X-ray structure of the full-length NS1 from an H5N1 strain (A/Vietnam/1203/2004) that was associated with 60% of human deaths in an outbreak in Vietnam. Compared to the individually determined structures of the RNA binding domain and the effector domain from non-H5N1 strains, the RNA binding domain within H5N1 NS1 exhibits modest structural changes, while the H5N1 effector domain shows significant alteration, particularly in the dimeric interface. Although both domains in the full-length NS1 individually participate in dimeric interactions, an unexpected finding is that these interactions result in the formation of a chain of NS1 molecules instead of distinct dimeric units. Three such chains in the crystal interact with one another extensively to form a tubular organization of similar dimensions to that observed in the cryo-electron microscopy images of NS1 in the presence of dsRNA. The tubular oligomeric organization of NS1, in which residues implicated in dsRNA binding face a 20-{angstrom}-wide central tunnel, provides a plausible mechanism for how NS1 sequesters varying lengths of dsRNA, to counter cellular antiviral dsRNA response pathways, while simultaneously interacting with other cellular ligands during an infection.

  8. Protective Efficacy of an H5N1 Inactivated Vaccine Against Challenge with Lethal H5N1, H5N2, H5N6, and H5N8 Influenza Viruses in Chickens.

    PubMed

    Zeng, Xianying; Chen, Pucheng; Liu, Liling; Deng, Guohua; Li, Yanbing; Shi, Jianzhong; Kong, Huihui; Feng, Huapeng; Bai, Jie; Li, Xin; Shi, Wenjun; Tian, Guobin; Chen, Hualan

    2016-05-01

    The Goose/Guangdong-lineage H5 viruses have evolved into diverse clades and subclades based on their hemagglutinin (HA) gene during their circulation in wild birds and poultry. Since late 2013, the clade 2.3.4.4 viruses have become widespread in poultry and wild bird populations around the world. Different subtypes of the clade 2.3.4.4 H5 viruses, including H5N1, H5N2, H5N6, and H5N8, have caused vast disease outbreaks in poultry in Asia, Europe, and North America. In this study, we developed a new H5N1 inactivated vaccine by using a seed virus (designated as Re-8) that contains the HA and NA genes from a clade 2.3.4.4 virus, A/chicken/Guizhou/4/13(H5N1) (CK/GZ/4/13), and its six internal genes from the high-growth A/Puerto Rico/8/1934 (H1N1) virus. We evaluated the protective efficacy of this vaccine in chickens challenged with one H5N1 clade 2.3.2.1b virus and six different subtypes of clade 2.3.4.4 viruses, including H5N1, H5N2, H5N6, and H5N8 strains. In the clade 2.3.2.1b virus DK/GX/S1017/13-challenged groups, half of the vaccinated chickens shed virus through the oropharynx and two birds (20%) died during the observation period. All of the control chickens shed viruses and died within 6 days of infection with challenge virus. All of the vaccinated chickens remained healthy following challenge with the six clade 2.3.4.4 viruses, and virus shedding was not detected from any of these birds; however, all of the control birds shed viruses and died within 4 days of challenge with the clade 2.3.4.4 viruses. Our results indicate that the Re-8 vaccine provides protection against different subtypes of clade 2.3.4.4 H5 viruses. PMID:27309064

  9. In vitro inactivation of two Egyptian A/H5N1 viruses by four commercial chemical disinfectants.

    PubMed

    Marzouk, Eman; Abd El-Hamid, Hatem S; Awad, Ashraf M; Zessin, Karl-Hans; Abdelwhab, E M; Hafez, Hafez M

    2014-09-01

    The highly pathogenic H5N1 avian influenza virus (A/H5N1) devastated the poultry industry and posed a serious health threat. Cleaning and disinfection are essential parts of preventative and postoutbreak management of A/H5N1 infections in poultry. In this preliminary study, we used suspension and carrier tests to evaluate the impact of concentration, time of exposure, surface porosity, and organic matter on the ability of four commercial chemical disinfectants to inactivate two A/H5N1 viruses of clade 2.2.1 isolated in 2006 and 2010 from broiler flocks in Egypt. Viruses were incubated with 0.5%, 1%, and 2% of formalin, glutaraldehyde, TH4, and Virkon S for 15, 30, 60, and 120 min at room temperature (22 +/- 2 C). In suspension tests, in the absence of organic matter, all disinfectants, at each concentration, except Virkon S 0.5%, effectively inactivated virus suspensions after a 15-min exposure time. In the presence of organic matter, the use of low concentrations of formalin (0.5%), glutaraldehyde (0.5%), or Virkon S (0.5%) was not sufficient to inactivate the viruses after 15 min. In gauze carrier tests, only formalin at any concentration for 15 min was sufficient to inactivate the viruses, whereas different concentrations or exposure times were required for glutaraldehyde (0.5% for 60 min), TH4 (0.5% for 30 min), and Virkon S (0.5% for 60 min or 1% for 30 min). In wood carrier tests, total inactivation of the virus was obtained at concentrations of 0.5% for 30 min (formalin and TH4) or 60 min (glutaraldehyde and Virkon S). This study emphasizes the need to use high concentrations of and/or extended time of exposure to disinfectants for efficient inactivation of A/H5N1, particularly in the presence of organic matter or different surfaces, which are common in poultry operations. In addition, it seemed that the virus isolated in 2010 was more resistant to disinfectants than the isolate from 2006 when wood was used as a carrier.

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

    PubMed

    Halbherr, Stefan J; Brostoff, Terza; Tippenhauer, Merve; Locher, Samira; Berger Rentsch, Marianne; Zimmer, Gert

    2013-01-01

    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.

  11. Vaccination with Recombinant RNA Replicon Particles Protects Chickens from H5N1 Highly Pathogenic Avian Influenza Virus

    PubMed Central

    Halbherr, Stefan J.; Brostoff, Terza; Tippenhauer, Merve; Locher, Samira; Berger Rentsch, Marianne; Zimmer, Gert

    2013-01-01

    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. PMID:23762463

  12. Vaccination with virus-like particles containing H5 antigens from three H5N1 clades protects chickens from H5N1 and H5N8 influenza viruses.

    PubMed

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

    2016-03-18

    Highly pathogenic avian influenza (HPAI) viruses, especially H5N1 strains, represent a public health threat and cause widespread morbidity and mortality in domestic poultry. Recombinant virus-like particles (VLPs) represent a promising novel vaccine approach to control avian influenza including HPAI strains. Influenza VLPs contain viral hemagglutinin (HA), which can be expressed in cell culture within highly immunogenic VLPs that morphologically and antigenically resemble influenza virions, except VLPs are non-infectious. Here we describe a recombinant VLP containing HA proteins derived from three distinct clades of H5N1 viruses as an experimental, broadly protective H5 avian influenza vaccine. A baculovirus vector was configured to co-express the H5 genes from recent H5N1 HPAI isolates A/chicken/Germany/2014 (clade 2.3.4.4), A/chicken/West Java/Subang/29/2007 (clade 2.1.3) and A/chicken/Egypt/121/2012 (clade 2.2.1). Co-expression of these genes in Sf9 cells along with influenza neuraminidase (NA) and retrovirus gag genes resulted in production of triple-clade H555 VLPs that exhibited hemagglutination activity and morphologically resembled influenza virions. Vaccination of chickens with these VLPs resulted in induction of serum antibody responses and efficient protection against experimental challenges with three different viruses including the recent U.S. H5N8 HPAI isolate. We conclude that these novel triple-clade VLPs represent a feasible strategy for simultaneously evoking protective antibodies against multiple variants of H5 influenza virus.

  13. Effect of species, breed and route of virus inoculation on the pathogenicity of H5N1 highly pathogenic influenza (HPAI) viruses in domestic ducks

    Technology Transfer Automated Retrieval System (TEKTRAN)

    H5N1 highly pathogenic avian influenza (HPAI) viruses continue to be a threat to poultry in many regions of the world. Domestic ducks have been recognized as one of the primary factors in the spread of H5N1 HPAI. To improve the control of this disease it’s necessary to better understand the pathog...

  14. Comparisons of highly virulent H5N1 influenza A viruses isolated from humans and chickens from Hong Kong.

    PubMed

    Suarez, D L; Perdue, M L; Cox, N; Rowe, T; Bender, C; Huang, J; Swayne, D E

    1998-08-01

    Genes of an influenza A (H5N1) virus from a human in Hong Kong isolated in May 1997 were sequenced and found to be all avian-like (K. Subbarao et al., Science 279:393-395, 1998). Gene sequences of this human isolate were compared to those of a highly pathogenic chicken H5N1 influenza virus isolated from Hong Kong in April 1997. Sequence comparisons of all eight RNA segments from the two viruses show greater than 99% sequence identity between them. However, neither isolate's gene sequence was closely (>95% sequence identity) related to any other gene sequences found in the GenBank database. Phylogenetic analysis demonstrated that the nucleotide sequences of at least four of the eight RNA segments clustered with Eurasian origin avian influenza viruses. The hemagglutinin gene phylogenetic analysis also included the sequences from an additional three human and two chicken H5N1 virus isolates from Hong Kong, and the isolates separated into two closely related groups. However, no single amino acid change separated the chicken origin and human origin isolates, but they all contained multiple basic amino acids at the hemagglutinin cleavage site, which is associated with a highly pathogenic phenotype in poultry. In experimental intravenous inoculation studies with chickens, all seven viruses were highly pathogenic, killing most birds within 24 h. All infected chickens had virtually identical pathologic lesions, including moderate to severe diffuse edema and interstitial pneumonitis. Viral nucleoprotein was most frequently demonstrated in vascular endothelium, macrophages, heterophils, and cardiac myocytes. Asphyxiation from pulmonary edema and generalized cardiovascular collapse were the most likely pathogenic mechanisms responsible for illness and death. In summary, a small number of changes in hemagglutinin gene sequences defined two closely related subgroups, with both subgroups having human and chicken members, among the seven viruses examined from Hong Kong, and all

  15. Host immune responses of ducks infected with H5N1 highly pathogenic avian influenza viruses of different pathogenicities.

    PubMed

    Wei, Liangmeng; Jiao, Peirong; Song, Yafen; Cao, Lan; Yuan, Runyu; Gong, Lang; Cui, Jin; Zhang, Shuo; Qi, Wenbao; Yang, Su; Liao, Ming

    2013-10-25

    Our previous studies have illustrated three strains of duck-origin H5N1 highly pathogenic avian influenza viruses (HPAIVs) had varying levels of pathogenicity in ducks (Sun et al., 2011). However, the host immune response of ducks infected with those of H5N1 HPAIVs was unclear. Here, we compared viral distribution and mRNA expression of immune-related genes in ducks following infection with the two HPAIV (A/Duck/Guangdong/212/2004, DK212 and A/Duck/Guangdong/383/2008, DK383). DK383 could replicate in the tested tissue of ducks (brain, spleen, lungs, cloacal bursa, kidney, and pancreas) more rapid and efficiently than DK212 at 1 and 2 days post-inoculation. Quantitative real-time PCR analysis showed that the expression levels of TLR3, IL-6, IL-8, and MHC class II in brains were higher than those of respective genes in lungs during the early stage of post infection. Furthermore, the expression levels of IL-6 and IL-8 in the brain of ducks following infection with DK383 were remarkably higher than those of ducks infected with DK212, respectively. Our results suggest that the shift in the H5N1 HPAIVs to increased virulence in ducks may be associated with efficient and rapid replication of the virus, accompanied by early destruction of host immune responses. These data are helpful to understand the underlying mechanism of the different outcome of H5N1 HPAIVs infection in ducks.

  16. Seroprevalence of Antibodies to Avian Influenza Virus A (H5N1) among Residents of Villages with Human Cases, Thailand, 20051

    PubMed Central

    Laosiritaworn, Yongjua; Phuthavathana, Pilaipan; Uyeki, Timothy M.; O’Reilly, Michael; Yampikulsakul, Nattaphon; Phurahong, Sumreung; Poorak, Phisanu; Prasertsopon, Jarunee; Kularb, Rumporn; Nateerom, Kannika; Sawanpanyalert, Narumol; Jiraphongsa, Chuleeporn

    2009-01-01

    In 2005, we assessed the seroprevalence of neutralizing antibodies to avian influenza virus A (H5N1) among 901 residents of 4 villages in Thailand where at least 1 confirmed human case of influenza (H5N1) had occurred during 2004. Although 68.1% of survey participants (median age 40 years) were exposed to backyard poultry and 25.7% were exposed to sick or dead chickens, all participants were seronegative for influenza virus (H5N1). PMID:19402962

  17. Cross-Reactive, Cell-Mediated Immunity and Protection of Chickens from Lethal H5N1 Influenza Virus Infection in Hong Kong Poultry Markets

    PubMed Central

    Seo, Sang Heui; Webster, Robert G.

    2001-01-01

    In 1997, avian H5N1 influenza virus transmitted from chickens to humans resulted in 18 confirmed infections. Despite harboring lethal H5N1 influenza viruses, most chickens in the Hong Kong poultry markets showed no disease signs. At this time, H9N2 influenza viruses were cocirculating in the markets. We investigated the role of H9N2 influenza viruses in protecting chickens from lethal H5N1 influenza virus infections. Sera from chickens infected with an H9N2 influenza virus did not cross-react with an H5N1 influenza virus in neutralization or hemagglutination inhibition assays. Most chickens primed with an H9N2 influenza virus 3 to 70 days earlier survived the lethal challenge of an H5N1 influenza virus, but infected birds shed H5N1 influenza virus in their feces. Adoptive transfer of T lymphocytes or CD8+ T cells from inbred chickens (B2/B2) infected with an H9N2 influenza virus to naive inbred chickens (B2/B2) protected them from lethal H5N1 influenza virus. In vitro cytotoxicity assays showed that T lymphocytes or CD8+ T cells from chickens infected with an H9N2 influenza virus recognized target cells infected with either an H5N1 or H9N2 influenza virus in a dose-dependent manner. Our findings indicate that cross-reactive cellular immunity induced by H9N2 influenza viruses protected chickens from lethal infection with H5N1 influenza viruses in the Hong Kong markets in 1997 but permitted virus shedding in the feces. Our findings are the first to suggest that cross-reactive cellular immunity can change the outcome of avian influenza virus infection in birds in live markets and create a situation for the perpetuation of H5N1 influenza viruses. PMID:11222674

  18. Cross-reactive, cell-mediated immunity and protection of chickens from lethal H5N1 influenza virus infection in Hong Kong poultry markets.

    PubMed

    Seo, S H; Webster, R G

    2001-03-01

    In 1997, avian H5N1 influenza virus transmitted from chickens to humans resulted in 18 confirmed infections. Despite harboring lethal H5N1 influenza viruses, most chickens in the Hong Kong poultry markets showed no disease signs. At this time, H9N2 influenza viruses were cocirculating in the markets. We investigated the role of H9N2 influenza viruses in protecting chickens from lethal H5N1 influenza virus infections. Sera from chickens infected with an H9N2 influenza virus did not cross-react with an H5N1 influenza virus in neutralization or hemagglutination inhibition assays. Most chickens primed with an H9N2 influenza virus 3 to 70 days earlier survived the lethal challenge of an H5N1 influenza virus, but infected birds shed H5N1 influenza virus in their feces. Adoptive transfer of T lymphocytes or CD8(+) T cells from inbred chickens (B(2)/B(2)) infected with an H9N2 influenza virus to naive inbred chickens (B(2)/B(2)) protected them from lethal H5N1 influenza virus. In vitro cytotoxicity assays showed that T lymphocytes or CD8(+) T cells from chickens infected with an H9N2 influenza virus recognized target cells infected with either an H5N1 or H9N2 influenza virus in a dose-dependent manner. Our findings indicate that cross-reactive cellular immunity induced by H9N2 influenza viruses protected chickens from lethal infection with H5N1 influenza viruses in the Hong Kong markets in 1997 but permitted virus shedding in the feces. Our findings are the first to suggest that cross-reactive cellular immunity can change the outcome of avian influenza virus infection in birds in live markets and create a situation for the perpetuation of H5N1 influenza viruses.

  19. Recombinant human interferon reduces titer of the 1918 pandemic and H5N1 influenza viruses in a guinea pig model

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Although H5N1 subtype influenza viruses have yet to acquire the ability to transmit efficiently among humans, the geographic expansion, genetic diversity and persistence of H5N1 viruses in birds indicates that pandemic potential of these viruses remains high. Vaccination remains the primary means f...

  20. High-yield production of a stable Vero cell-based vaccine candidate against the highly pathogenic avian influenza virus H5N1

    SciTech Connect

    Zhou, Fangye; Zhou, Jian; Ma, Lei; Song, Shaohui; Zhang, Xinwen; Li, Weidong; Jiang, Shude; Wang, Yue; Liao, Guoyang

    2012-05-18

    Highlights: Black-Right-Pointing-Pointer Vero cell-based HPAI H5N1 vaccine with stable high yield. Black-Right-Pointing-Pointer Stable high yield derived from the YNVa H3N2 backbone. Black-Right-Pointing-Pointer H5N1/YNVa has a similar safety and immunogenicity to H5N1delta. -- Abstract: Highly pathogenic avian influenza (HPAI) viruses pose a global pandemic threat, for which rapid large-scale vaccine production technology is critical for prevention and control. Because chickens are highly susceptible to HPAI viruses, the supply of chicken embryos for vaccine production might be depleted during a virus outbreak. Therefore, developing HPAI virus vaccines using other technologies is critical. Meeting vaccine demand using the Vero cell-based fermentation process has been hindered by low stability and yield. In this study, a Vero cell-based HPAI H5N1 vaccine candidate (H5N1/YNVa) with stable high yield was achieved by reassortment of the Vero-adapted (Va) high growth A/Yunnan/1/2005(H3N2) (YNVa) virus with the A/Anhui/1/2005(H5N1) attenuated influenza vaccine strain (H5N1delta) using the 6/2 method. The reassorted H5N1/YNVa vaccine maintained a high hemagglutination (HA) titer of 1024. Furthermore, H5N1/YNVa displayed low pathogenicity and uniform immunogenicity compared to that of the parent virus.

  1. Risk-based surveillance for H5N1 avian influenza virus in wild birds in Great Britain.

    PubMed

    Snow, L C; Newson, S E; Musgrove, A J; Cranswick, P A; Crick, H Q P; Wilesmith, J W

    2007-12-01

    Recent outbreaks of the H5N1 strain of avian influenza in Europe have highlighted the need for continuous surveillance and early detection to reduce the likelihood of a major outbreak in the commercial poultry industry. In Great Britain (gb), one possible route by which H5N1 could be introduced into domestic poultry is through migratory wild birds from Europe and Asia. Extensive monitoring data on the 24 wild bird species considered most likely to introduce the virus into GB, and analyses of local poultry populations, were used to develop a risk profile to identify the areas where H5N1 is most likely to enter and spread to commercial poultry. The results indicate that surveillance would be best focused on areas of Norfolk, Suffolk, Lancashire, Lincolnshire, south-west England and the Welsh borders, with areas of lower priority in Anglesey, south-west Wales, north-east Aberdeenshire and the Firth of Forth area of Scotland. These areas have significant poultry populations including a large number of free-range flocks, and a high abundance of the 24 wild bird species.

  2. Pathobiological Characterization of a Novel Reassortant Highly Pathogenic H5N1 Virus Isolated in British Columbia, Canada, 2015

    PubMed Central

    Berhane, Yohannes; Kobasa, Darwyn; Embury-Hyatt, Carissa; Pickering, Brad; Babiuk, Shawn; Joseph, Tomy; Bowes, Victoria; Suderman, Mathew; Leung, Anders; Cottam-Birt, Colleen; Hisanaga, Tamiko; Pasick, John

    2016-01-01

    In the current study, we describe the pathobiologic characteristics of a novel reassortant virus - A/chicken/BC/FAV-002/2015 (H5N1) belonging to clade 2.3.4.4 that was isolated from backyard chickens in British Columbia, Canada. Sequence analyses demonstrate PB1, PA, NA and NS gene segments were of North American lineage while PB2, HA, NP and M were derived from a Eurasian lineage H5N8 virus. This novel virus had a 19 amino acid deletion in the neuraminidase stalk. We evaluated the pathogenic potential of this isolate in various animal models. The virus was highly pathogenic to mice with a LD50 of 10 plaque forming units (PFU), but had limited tissue tropism. It caused only subclinical infection in pigs which did result in seroconversion. This virus was highly pathogenic to chickens, turkeys, juvenile Muscovy ducks (Cairnia moschata foma domestica) and adult Chinese geese (Anser cynoides domesticus) causing a systemic infection in all species. The virus was also efficiently transmitted and resulted in mortality in naïve contact ducks, geese and chickens. Our findings indicate that this novel H5N1 virus has a wide host range and enhanced surveillance of migratory waterfowl may be necessary in order to determine its potential to establish itself in the wild bird reservoir. PMID:26988892

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

    PubMed Central

    Arai, Yasuha; Kawashita, Norihito; Daidoji, Tomo; Ibrahim, Madiha S.; El-Gendy, Emad M.; Takagi, Tatsuya; Takahashi, Kazuo; Suzuki, Yasuo; Ikuta, Kazuyoshi; Nakaya, Takaaki; Shioda, Tatsuo; Watanabe, Yohei

    2016-01-01

    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. PMID:27097026

  4. Pathobiological Characterization of a Novel Reassortant Highly Pathogenic H5N1 Virus Isolated in British Columbia, Canada, 2015.

    PubMed

    Berhane, Yohannes; Kobasa, Darwyn; Embury-Hyatt, Carissa; Pickering, Brad; Babiuk, Shawn; Joseph, Tomy; Bowes, Victoria; Suderman, Mathew; Leung, Anders; Cottam-Birt, Colleen; Hisanaga, Tamiko; Pasick, John

    2016-01-01

    In the current study, we describe the pathobiologic characteristics of a novel reassortant virus - A/chicken/BC/FAV-002/2015 (H5N1) belonging to clade 2.3.4.4 that was isolated from backyard chickens in British Columbia, Canada. Sequence analyses demonstrate PB1, PA, NA and NS gene segments were of North American lineage while PB2, HA, NP and M were derived from a Eurasian lineage H5N8 virus. This novel virus had a 19 amino acid deletion in the neuraminidase stalk. We evaluated the pathogenic potential of this isolate in various animal models. The virus was highly pathogenic to mice with a LD50 of 10 plaque forming units (PFU), but had limited tissue tropism. It caused only subclinical infection in pigs which did result in seroconversion. This virus was highly pathogenic to chickens, turkeys, juvenile Muscovy ducks (Cairnia moschata foma domestica) and adult Chinese geese (Anser cynoides domesticus) causing a systemic infection in all species. The virus was also efficiently transmitted and resulted in mortality in naïve contact ducks, geese and chickens. Our findings indicate that this novel H5N1 virus has a wide host range and enhanced surveillance of migratory waterfowl may be necessary in order to determine its potential to establish itself in the wild bird reservoir. PMID:26988892

  5. Pathobiological Characterization of a Novel Reassortant Highly Pathogenic H5N1 Virus Isolated in British Columbia, Canada, 2015.

    PubMed

    Berhane, Yohannes; Kobasa, Darwyn; Embury-Hyatt, Carissa; Pickering, Brad; Babiuk, Shawn; Joseph, Tomy; Bowes, Victoria; Suderman, Mathew; Leung, Anders; Cottam-Birt, Colleen; Hisanaga, Tamiko; Pasick, John

    2016-03-18

    In the current study, we describe the pathobiologic characteristics of a novel reassortant virus - A/chicken/BC/FAV-002/2015 (H5N1) belonging to clade 2.3.4.4 that was isolated from backyard chickens in British Columbia, Canada. Sequence analyses demonstrate PB1, PA, NA and NS gene segments were of North American lineage while PB2, HA, NP and M were derived from a Eurasian lineage H5N8 virus. This novel virus had a 19 amino acid deletion in the neuraminidase stalk. We evaluated the pathogenic potential of this isolate in various animal models. The virus was highly pathogenic to mice with a LD50 of 10 plaque forming units (PFU), but had limited tissue tropism. It caused only subclinical infection in pigs which did result in seroconversion. This virus was highly pathogenic to chickens, turkeys, juvenile Muscovy ducks (Cairnia moschata foma domestica) and adult Chinese geese (Anser cynoides domesticus) causing a systemic infection in all species. The virus was also efficiently transmitted and resulted in mortality in naïve contact ducks, geese and chickens. Our findings indicate that this novel H5N1 virus has a wide host range and enhanced surveillance of migratory waterfowl may be necessary in order to determine its potential to establish itself in the wild bird reservoir.

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

    PubMed

    Arai, Yasuha; Kawashita, Norihito; Daidoji, Tomo; Ibrahim, Madiha S; El-Gendy, Emad M; Takagi, Tatsuya; Takahashi, Kazuo; Suzuki, Yasuo; Ikuta, Kazuyoshi; Nakaya, Takaaki; Shioda, Tatsuo; Watanabe, Yohei

    2016-04-01

    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.

  7. Tracking the Evolution in Phylogeny, Structure and Function of H5N1 Influenza Virus PA Gene.

    PubMed

    Wei, K; Lin, Y; Li, Y; Chen, Y

    2016-10-01

    Highly pathogenic avian influenza (HPAI) H5N1 viruses have severely affected the poultry industry of Vietnam and Indonesia. The outbreaks of HPAI H5N1 viruses continue to pose a serious threat to public health, which have profound impacts on public health. In this study, we presented phylogenetic evidences for five reassortants among HPAI H5N1 viruses sampled from Vietnam and Indonesia during 2003-2013 and found that reassortment events occurred more frequently in the three gene segments (PB1, PA and HA) than in the remaining five gene segments (PB2, NA, NP, NS and MP). The sequence-based analyses have revealed that the PA protein displays high levels of DNA sequence polymorphism and variability than other internal proteins. Seven positive selection sites were detected in PA proteins, which ranked second only to the surface glycoproteins. Structure-based comparative analysis of PA proteins showed a remarkable sequence conservation between the high-pathogenic, low-pathogenic and reassortant viruses, indicating that PA appears to be a potential antiviral target. Furthermore, by analysing the published data, we compared the differential expression of genes involved in RIG-I- and MAVS-mediated intracellular type I interferon (IFN)-inducing pathway between the VN3028IIcl2-infected, IDN3006-infected and IDN3006/PA-infected groups. Our analyses indicated that the inhibitory effect of the PA protein on MAVS was not strong. In addition, transcriptional levels of 33 mitochondrial proteins involved in the induction of apoptosis have significantly increased, suggesting that PA may play an important role in apoptosis signalling pathway.

  8. Variability in pathobiology of South Korean H5N1 high-pathogenicity avian influenza virus infection for 5 species of migratory waterfowl

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The biological outcome of H5N1 high pathogenicity avian influenza (HPAI) virus infection in wild waterfowl is poorly understood. This study examined infectivity and pathobiology of A/chicken/Korea/IS/06 (H5N1) HPAI virus infection in Mute swans (Cygnus olor), Greylag geese (Anser anser), Ruddy Sheld...

  9. High level of genetic compatibility between swine-origin H1N1 and highly pathogenic avian H5N1 influenza viruses.

    PubMed

    Octaviani, Cássio Pontes; Ozawa, Makoto; Yamada, Shinya; Goto, Hideo; Kawaoka, Yoshihiro

    2010-10-01

    Reassortment is an important mechanism for the evolution of influenza viruses. Here, we coinfected cultured cells with the pandemic swine-origin influenza virus (S-OIV) and a contemporary H5N1 virus and found that these two viruses have high genetic compatibility. Studies of human lung cell lines indicated that some reassortants had better growth kinetics than their parental viruses. We conclude that reassortment between these two viruses can occur and could create pandemic H5N1 viruses.

  10. Spatial Modeling of Wild Bird Risk Factors for Highly Pathogenic A(H5N1) Avian Influenza Virus Transmission.

    PubMed

    Prosser, Diann J; Hungerford, Laura L; Erwin, R Michael; Ottinger, Mary Ann; Takekawa, John Y; Newman, Scott H; Xiao, Xiangming; Ellis, Erle C

    2016-05-01

    One of the longest-persisting avian influenza viruses in history, highly pathogenic avian influenza virus (HPAIV) A(H5N1), continues to evolve after 18 yr, advancing the threat of a global pandemic. Wild waterfowl (family Anatidae) are reported as secondary transmitters of HPAIV and primary reservoirs for low-pathogenic avian influenza viruses, yet spatial inputs for disease risk modeling for this group have been lacking. Using geographic information software and Monte Carlo simulations, we developed geospatial indices of waterfowl abundance at 1 and 30 km resolutions and for the breeding and wintering seasons for China, the epicenter of H5N1. Two spatial layers were developed: cumulative waterfowl abundance (WAB), a measure of predicted abundance across species, and cumulative abundance weighted by H5N1 prevalence (WPR), whereby abundance for each species was adjusted based on prevalence values and then totaled across species. Spatial patterns of the model output differed between seasons, with higher WAB and WPR in the northern and western regions of China for the breeding season and in the southeast for the wintering season. Uncertainty measures indicated highest error in southeastern China for both WAB and WPR. We also explored the effect of resampling waterfowl layers from 1 to 30 km resolution for multiscale risk modeling. Results indicated low average difference (less than 0.16 and 0.01 standard deviations for WAB and WPR, respectively), with greatest differences in the north for the breeding season and southeast for the wintering season. This work provides the first geospatial models of waterfowl abundance available for China. The indices provide important inputs for modeling disease transmission risk at the interface of poultry and wild birds. These models are easily adaptable, have broad utility to both disease and conservation needs, and will be available to the scientific community for advanced modeling applications.

  11. Spatial Modeling of Wild Bird Risk Factors for Highly Pathogenic A(H5N1) Avian Influenza Virus Transmission.

    PubMed

    Prosser, Diann J; Hungerford, Laura L; Erwin, R Michael; Ottinger, Mary Ann; Takekawa, John Y; Newman, Scott H; Xiao, Xiangming; Ellis, Erle C

    2016-05-01

    One of the longest-persisting avian influenza viruses in history, highly pathogenic avian influenza virus (HPAIV) A(H5N1), continues to evolve after 18 yr, advancing the threat of a global pandemic. Wild waterfowl (family Anatidae) are reported as secondary transmitters of HPAIV and primary reservoirs for low-pathogenic avian influenza viruses, yet spatial inputs for disease risk modeling for this group have been lacking. Using geographic information software and Monte Carlo simulations, we developed geospatial indices of waterfowl abundance at 1 and 30 km resolutions and for the breeding and wintering seasons for China, the epicenter of H5N1. Two spatial layers were developed: cumulative waterfowl abundance (WAB), a measure of predicted abundance across species, and cumulative abundance weighted by H5N1 prevalence (WPR), whereby abundance for each species was adjusted based on prevalence values and then totaled across species. Spatial patterns of the model output differed between seasons, with higher WAB and WPR in the northern and western regions of China for the breeding season and in the southeast for the wintering season. Uncertainty measures indicated highest error in southeastern China for both WAB and WPR. We also explored the effect of resampling waterfowl layers from 1 to 30 km resolution for multiscale risk modeling. Results indicated low average difference (less than 0.16 and 0.01 standard deviations for WAB and WPR, respectively), with greatest differences in the north for the breeding season and southeast for the wintering season. This work provides the first geospatial models of waterfowl abundance available for China. The indices provide important inputs for modeling disease transmission risk at the interface of poultry and wild birds. These models are easily adaptable, have broad utility to both disease and conservation needs, and will be available to the scientific community for advanced modeling applications. PMID:27309075

  12. Susceptibility of openbill storks (Anastomius oscitans) to highly pathogenic avian influenza virus subtype H5N1.

    PubMed

    Chaichoun, Kridsada; Wiriyarat, Withawat; Phonaknguen, Rassmeepen; Sariya, Ladawan; Taowan, Nam-aoy; Chakritbudsabong, Warunya; Chaisilp, Natnapat; Eiam-ampai, Krirat; Phuttavatana, Pilaipan; Ratanakorn, Parntep

    2013-09-01

    This investigation detailed the clinical disease, gross and histologic lesions in juvenile openbill storks (Anastomus oscitans) intranasally inoculated with an avian influenza virus, A/chicken/Thailand/vsmu-3 (H5N1), which is highly pathogenic for chickens. High morbidity and mortality were observed in openbill storks inoculated with HPAI H5N1 virus. Gross lesions from infected birds were congestion and brain hemorrhage (10/20), pericardial effusions, pericarditis and focal necrosis of the cardiac muscle (2/20), pulmonary edema and pulmonary necrosis, serosanguineous fluid in the bronchis (16/20), liver congestion (6/20), bursitis (5/20), subcutaneous hemorrhages (2/20) and pinpoint proventiculus hemorrhage (2/20). Real time RT-PCR demonstrated the presence of viral RNA in organs associated with the lesions: brain, trachea, lungs, liver, spleen and intestines. Similar to viral genome detection, virus was also isolated from these vital organs. Antibodies to influenza virus detected with a hemagglutination inhibition test, were found only in the openbill storks who died 8 days post-inoculation.

  13. The Creation of a Contagious H5N1 Influenza Virus: Implications for the Education of Life Scientists

    PubMed Central

    Novossiolova, Tatyana; Minehata, Masamichi; Dando, Malcolm

    2012-01-01

    The paper contends that the ongoing controversy surrounding the creation of a contagious H5N1 influenza virus has already exposed the severe limitations of the possibility of preventing the hostile misuse of the life sciences by dint of oversight of proposals and publications. It further argues that in order to prevent the potential wholesale militarisation of the life sciences, it is essential that life scientists become aware of their responsibilities within the context of the Biological and Toxin Weapons Convention (BTWC) and actively contribute their expertise to strengthening the biological weapons non-proliferation regime . PMID:22984642

  14. The Creation of a Contagious H5N1 Influenza Virus: Implications for the Education of Life Scientists.

    PubMed

    Novossiolova, Tatyana; Minehata, Masamichi; Dando, Malcolm

    2012-01-01

    The paper contends that the ongoing controversy surrounding the creation of a contagious H5N1 influenza virus has already exposed the severe limitations of the possibility of preventing the hostile misuse of the life sciences by dint of oversight of proposals and publications. It further argues that in order to prevent the potential wholesale militarisation of the life sciences, it is essential that life scientists become aware of their responsibilities within the context of the Biological and Toxin Weapons Convention (BTWC) and actively contribute their expertise to strengthening the biological weapons non-proliferation regime .

  15. Extrapolating theoretical efficacy of inactivated influenza A/H5N1 virus vaccine from human immunogenicity studies.

    PubMed

    Feldstein, Leora R; Matrajt, Laura; Elizabeth Halloran, M; Keitel, Wendy A; Longini, Ira M

    2016-07-19

    Influenza A virus subtype H5N1 has been a public health concern for almost 20years due to its potential ability to become transmissible among humans. Phase I and II clinical trials have assessed safety, reactogenicity and immunogenicity of inactivated influenza A/H5N1 virus vaccines. A shortage of vaccine is likely to occur during the first months of a pandemic. Hence, determining whether to give one dose to more people or two doses to fewer people to best protect the population is essential. We use hemagglutination-inhibition antibody titers as an immune correlate for avian influenza vaccines. Using an established relationship to obtain a theoretical vaccine efficacy from immunogenicity data from thirteen arms of six phase I and phase II clinical trials of inactivated influenza A/H5N1 virus vaccines, we assessed: (1) the proportion of theoretical vaccine efficacy achieved after a single dose (defined as primary response level), and (2) whether theoretical efficacy increases after a second dose, with and without adjuvant. Participants receiving vaccine with AS03 adjuvant had higher primary response levels (range: 0.48-0.57) compared to participants receiving vaccine with MF59 adjuvant (range: 0.32-0.47), with no observed trends in primary response levels by antigen dosage. After the first and second doses, vaccine with AS03 at dosage levels 3.75, 7.5 and 15mcg had the highest estimated theoretical vaccine efficacy: Dose (1) 45% (95% CI: 36-57%), 53% (95% CI: 42-63%) and 55% (95% CI: 44-64%), respectively and Dose (2) 93% (95% CI: 89-96%), 97% (95% CI: 95-98%) and 97% (95% CI: 96-100%), respectively. On average, the estimated theoretical vaccine efficacy of lower dose adjuvanted vaccines (AS03 and MF59) was 17% higher than that of higher dose unadjuvanted vaccines, suggesting that including an adjuvant is dose-sparing. These data indicate adjuvanted inactivated influenza A/H5N1 virus vaccine produces high theoretical efficacy after two doses to protect individuals

  16. Chimeric neuraminidase and mutant PB1 gene constellation improves growth and yield of H5N1 vaccine candidate virus.

    PubMed

    Plant, Ewan P; Ye, Zhiping

    2015-04-01

    We previously showed that a mutated PB1 gene improved the growth kinetics of a H3N2 influenza reassortant. Here, we showed that the same mutations improved the growth kinetics of a virus containing the A/Vietnam/1203/2004 (H5N1) haemagglutinin and neuraminidase (NA). Total protein yield and NA activity were increased when a chimeric NA was included. These increases indicated that the synergistic effect was due to the gene constellation containing both the altered PB1 gene and the chimeric NA gene.

  17. H5N1 Avian Flu (H5N1 Bird Flu)

    MedlinePlus

    ... H5N1 - Avian/Bird Flu H5N1 Avian Flu - H5N1 Bird Flu H5N1 is a highly pathogenic avian (bird) flu virus that has caused serious outbreaks in ... been no reported infections with these viruses in birds, poultry, or people in the United States. You ...

  18. Satellite tracking of Northern Pintail Anas acuta during outbreaks of the H5N1 virus in Japan: implications for virus spread

    USGS Publications Warehouse

    Yamaguchi, Noriyuki; Hupp, Jerry W.; Higuchi, Hiroyoshi; Flint, Paul L.; Pearce, John M.

    2010-01-01

    We fitted Northern Pintail Anas acuta in Japan with satellite transmitters and monitored their spring migration movements relative to locations where the highly pathogenic H5N1 avian influenza virus was detected in Whooper Swans Cygnus cygnus in 2008. Pintails were assumed not to be infected with the H5N1 virus at the time they were marked because capture occurred between 2 and 5 months before reported outbreaks of the virus in Japan. We assessed spatial and temporal overlap between marked birds and occurrence of the virus and tracked Pintails after they departed outbreak locations. Eight of 66 (12.1%) Northern Pintails marked with satellite transmitters used wetlands in Japan where the H5N1 virus was detected in Whooper Swans. Apparent survival did not differ between Pintails that used H5N1 sites and those that did not. However, the proportion of Pintails that migrated from Japan was significantly lower among birds that used H5N1 sites compared with those that did not (0.50 vs. 0.79). Northern Pintails were present at the H5N1 sites from 1 to 88 days, with five birds present at the sites from 0 to 7 days prior to detection of the virus in Swans. The six Pintails observed to depart H5N1 sites did so within 2–77 days of the reported outbreaks and moved between 6 and 1200 km within 4 days of departure. Four Pintails migrated to eastern Russia. After their departure from outbreak sites, Northern Pintails made long-distance migrations within the period when newly infected ducks would shed the H5N1 virus. This supports a hypothesized mechanism by which a highly pathogenic avian influenza virus could be spread by migratory birds.

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

    PubMed

    Hagag, Ibrahim Thabet; Mansour, Shimaa M G; Zhang, Zerui; Ali, Ahmed A H; Ismaiel, El-Bakry M; Salama, Ali A; Cardona, Carol J; Collins, James; Xing, Zheng

    2015-01-01

    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 of the HPAIVH5N

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

    PubMed Central

    Hagag, Ibrahim Thabet; Mansour, Shimaa M. G.; Zhang, Zerui; Ali, Ahmed A. H.; Ismaiel, El-Bakry M.; Salama, Ali A.; Cardona, Carol J.; Collins, James; Xing, Zheng

    2015-01-01

    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 of the HPAIVH5N

  1. The multigenic nature of the differences in pathogenicity of H5N1 highly pathogenic avian influenza viruses in domestic ducks

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Eurasian H5N1 highly pathogenic avian influenza (HPAI) viruses have evolved into many genetic lineages. The divergent strains that have arisen express distinct pathobiological features and increased virulence for many bird species including domestic waterfowl. The pathogenicity of H5N1 HPAI vi...

  2. A computationally optimized broadly reactive H5 hemagglutinin vaccine provides protection against homologous and heterologous H5N1 highly pathogenic avian influenza virus infection in chickens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Since its emergence in 1996 in China, H5N1 highly pathogenic avian influenza (HPAI) virus has continuously evolved into different genetic clades that have created challenges to maintaining antigenically relevant H5N1 vaccine seeds. Therefore, a universal (multi-hemagglutinin [HA] subtype) or more c...

  3. Issues encountered in development of enzyme-linked immunosorbent assay for use in detecting influenza A virus subtype H5N1 exposure in swine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A potential mechanism by which highly pathogenic avian influenza H5N1 viruses could become established in humans is through the infection of and adaptation in pigs. To detect the occurrence of such adaptation, monitoring of the pig populations in endemic H5N1 areas through serological screening woul...

  4. Host-specific exposure and fatal neurologic disease in wild raptors from highly pathogenic avian influenza virus H5N1 during the 2006 outbreak in Germany.

    PubMed

    van den Brand, Judith Ma; Krone, Oliver; Wolf, Peter U; van de Bildt, Marco W G; van Amerongen, Geert; Osterhaus, Albert D M E; Kuiken, Thijs

    2015-01-01

    Raptors may contract highly pathogenic avian influenza virus H5N1 by hunting or scavenging infected prey. However, natural H5N1 infection in raptors is rarely reported. Therefore, we tested raptors found dead during an H5N1 outbreak in wild waterbirds in Mecklenburg-Western Pomerania, Germany, in 2006 for H5N1-associated disease. We tested 624 raptors of nine species-common buzzard (385), Eurasian sparrowhawk (111), common kestrel (38), undetermined species of buzzard (36), white-tailed sea eagle (19), undetermined species of raptor (12), northern goshawk (10), peregrine falcon (6), red kite (3), rough-legged buzzard (3), and western marsh-harrier (1)-for H5N1 infection in tracheal or combined tracheal/cloacal swabs of all birds, and on major tissues of all white-tailed sea eagles. H5N1 infection was detected in two species: common buzzard (12 positive, 3.1%) and peregrine falcon (2 positive, 33.3%). In all necropsied birds (both peregrine falcons and the six freshest common buzzards), H5N1 was found most consistently and at the highest concentration in the brain, and the main H5N1-associated lesion was marked non-suppurative encephalitis. Other H5N1-associated lesions occurred in air sac, lung, oviduct, heart, pancreas, coelomic ganglion, and adrenal gland. Our results show that the main cause of death in H5N1-positive raptors was encephalitis. Our results imply that H5N1 outbreaks in wild waterbirds are more likely to lead to exposure to and mortality from H5N1 in raptors that hunt or scavenge medium-sized birds, such as common buzzards and peregrine falcons, than in raptors that hunt small birds and do not scavenge, such as Eurasian sparrowhawks and common kestrels. PMID:25879698

  5. Prevalence and distribution of avian influenza a(H5N1) virus clade variants in live bird markets of Vietnam, 2011-2013.

    PubMed

    Nguyen, Diep T; Bryant, Juliet E; Davis, C Todd; Nguyen, Long V; Pham, Long T; Loth, Leo; Inui, Ken; Nguyen, Tung; Jang, Yunho; To, Thanh L; Nguyen, Tho D; Hoang, Diep T; Do, Hoa T; Nguyen, Trang T; Newman, Scott; Jennifer Siembieda; Pham, Dong V

    2014-12-01

    Active surveillance for avian influenza (Al) viruses in poultry sold at live bird markets (LBMs) was conducted in 44 of 63 provinces throughout Vietnam over two periods from September 2011 to February 2012 and October 2012 to June 2013. The study objectives were to assess the prevalence of avian influenza type A, H5, and H5N1 subtype viruses and characterize the geographical and temporal distribution of H5N1 virus genetic variants across the country. Monthly sampling was conducted in 394 LBMs located in 372 communes. A total of 9790 oropharyngeal swabs from poultry were screened for influenza A virus by real-time reverse-transcriptase PCR Virus isolation was attempted on all positive samples in embryonated chicken eggs, and the HA1 region of each H5 virus isolate was sequenced. Market prevalence of H5 subtype virus was 32.2% (127/394) over the cumulative 15 mo of surveillance. Phylogenetic analyses indicated that clade 1.1 viruses persisted in the south, whereas three genetically distinct subgroups of dade 2.3.2.1 were found simultaneously in northern, central, and southern Vietnam. Clade 2.3.2.1c viruses first appeared in July 2012 and spread rapidly to the center and south of Vietnam in late 2012, where they were predominant among clade 2.3.2.1 viruses and were detected in both active LBM surveillance and poultry outbreaks. Given the overlapping geographic distribution of dade variants and the antigenic divergence previously described for these dades, current AI poultry vaccines used in Vietnam may require bivalent formulations containing representatives of both dade 1.1 and dade 2.3.2.1 viruses.

  6. Prevalence and distribution of avian influenza a(H5N1) virus clade variants in live bird markets of Vietnam, 2011-2013.

    PubMed

    Nguyen, Diep T; Bryant, Juliet E; Davis, C Todd; Nguyen, Long V; Pham, Long T; Loth, Leo; Inui, Ken; Nguyen, Tung; Jang, Yunho; To, Thanh L; Nguyen, Tho D; Hoang, Diep T; Do, Hoa T; Nguyen, Trang T; Newman, Scott; Jennifer Siembieda; Pham, Dong V

    2014-12-01

    Active surveillance for avian influenza (Al) viruses in poultry sold at live bird markets (LBMs) was conducted in 44 of 63 provinces throughout Vietnam over two periods from September 2011 to February 2012 and October 2012 to June 2013. The study objectives were to assess the prevalence of avian influenza type A, H5, and H5N1 subtype viruses and characterize the geographical and temporal distribution of H5N1 virus genetic variants across the country. Monthly sampling was conducted in 394 LBMs located in 372 communes. A total of 9790 oropharyngeal swabs from poultry were screened for influenza A virus by real-time reverse-transcriptase PCR Virus isolation was attempted on all positive samples in embryonated chicken eggs, and the HA1 region of each H5 virus isolate was sequenced. Market prevalence of H5 subtype virus was 32.2% (127/394) over the cumulative 15 mo of surveillance. Phylogenetic analyses indicated that clade 1.1 viruses persisted in the south, whereas three genetically distinct subgroups of dade 2.3.2.1 were found simultaneously in northern, central, and southern Vietnam. Clade 2.3.2.1c viruses first appeared in July 2012 and spread rapidly to the center and south of Vietnam in late 2012, where they were predominant among clade 2.3.2.1 viruses and were detected in both active LBM surveillance and poultry outbreaks. Given the overlapping geographic distribution of dade variants and the antigenic divergence previously described for these dades, current AI poultry vaccines used in Vietnam may require bivalent formulations containing representatives of both dade 1.1 and dade 2.3.2.1 viruses. PMID:25619005

  7. Avian influenza vaccines against H5N1 'bird flu'.

    PubMed

    Li, Chengjun; Bu, Zhigao; Chen, Hualan

    2014-03-01

    H5N1 avian influenza viruses (AIVs) have spread widely to more than 60 countries spanning three continents. To control the disease, vaccination of poultry is implemented in many of the affected countries, especially in those where H5N1 viruses have become enzootic in poultry and wild birds. Recently, considerable progress has been made toward the development of novel avian influenza (AI) vaccines, especially recombinant virus vector vaccines and DNA vaccines. Here, we will discuss the recent advances in vaccine development and use against H5N1 AIV in poultry. Understanding the properties of the available, novel vaccines will allow for the establishment of rational vaccination protocols, which in turn will help the effective control and prevention of H5N1 AI.

  8. H5N1 Whole-Virus Vaccine Induces Neutralizing Antibodies in Humans Which Are Protective in a Mouse Passive Transfer Model

    PubMed Central

    Howard, M. Keith; Sabarth, Nicolas; Savidis-Dacho, Helga; Portsmouth, Daniel; Kistner, Otfried; Kreil, Thomas R.; Ehrlich, Hartmut J.; Barrett, P. Noel

    2011-01-01

    Background Vero cell culture-derived whole-virus H5N1 vaccines have been extensively tested in clinical trials and consistently demonstrated to be safe and immunogenic; however, clinical efficacy is difficult to evaluate in the absence of wide-spread human disease. A lethal mouse model has been utilized which allows investigation of the protective efficacy of active vaccination or passive transfer of vaccine induced sera following lethal H5N1 challenge. Methods We used passive transfer of immune sera to investigate antibody-mediated protection elicited by a Vero cell-derived, non-adjuvanted inactivated whole-virus H5N1 vaccine. Mice were injected intravenously with H5N1 vaccine-induced rodent or human immune sera and subsequently challenged with a lethal dose of wild-type H5N1 virus. Results Passive transfer of H5N1 vaccine-induced mouse, guinea pig and human immune sera provided dose-dependent protection of recipient mice against lethal challenge with wild-type H5N1 virus. Protective dose fifty values for serum H5N1 neutralizing antibody titers were calculated to be ≤1∶11 for all immune sera, independently of source species. Conclusions These data underpin the confidence that the Vero cell culture-derived, whole-virus H5N1 vaccine will be effective in a pandemic situation and support the use of neutralizing serum antibody titers as a correlate of protection for H5N1 vaccines. PMID:21876771

  9. Effect of species, breed and route of virus inoculation on the pathogenicity of H5N1 highly pathogenic influenza (HPAI) viruses in domestic ducks

    PubMed Central

    2013-01-01

    H5N1 highly pathogenic avian influenza (HPAI) viruses continue to be a threat to poultry in many regions of the world. Domestic ducks have been recognized as one of the primary factors in the spread of H5N1 HPAI. In this study we examined the pathogenicity of H5N1 HPAI viruses in different species and breeds of domestic ducks and the effect of route of virus inoculation on the outcome of infection. We determined that the pathogenicity of H5N1 HPAI viruses varies between the two common farmed duck species, with Muscovy ducks (Cairina moschata) presenting more severe disease than various breeds of Anas platyrhynchos var. domestica ducks including Pekin, Mallard-type, Black Runners, Rouen, and Khaki Campbell ducks. We also found that Pekin and Muscovy ducks inoculated with two H5N1 HPAI viruses of different virulence, given by any one of three routes (intranasal, intracloacal, or intraocular), became infected with the viruses. Regardless of the route of inoculation, the outcome of infection was similar for each species but depended on the virulence of the virus used. Muscovy ducks showed more severe clinical signs and higher mortality than the Pekin ducks. In conclusion, domestic ducks are susceptible to H5N1 HPAI virus infection by different routes of exposure, but the presentation of the disease varied by virus strain and duck species. This information helps support the planning and implementation of H5N1 HPAI surveillance and control measures in countries with large domestic duck populations. PMID:23876184

  10. Effect of species, breed and route of virus inoculation on the pathogenicity of H5N1 highly pathogenic influenza (HPAI) viruses in domestic ducks.

    PubMed

    Pantin-Jackwood, Mary; Swayne, David E; Smith, Diane; Shepherd, Eric

    2013-07-22

    H5N1 highly pathogenic avian influenza (HPAI) viruses continue to be a threat to poultry in many regions of the world. Domestic ducks have been recognized as one of the primary factors in the spread of H5N1 HPAI. In this study we examined the pathogenicity of H5N1 HPAI viruses in different species and breeds of domestic ducks and the effect of route of virus inoculation on the outcome of infection. We determined that the pathogenicity of H5N1 HPAI viruses varies between the two common farmed duck species, with Muscovy ducks (Cairina moschata) presenting more severe disease than various breeds of Anas platyrhynchos var. domestica ducks including Pekin, Mallard-type, Black Runners, Rouen, and Khaki Campbell ducks. We also found that Pekin and Muscovy ducks inoculated with two H5N1 HPAI viruses of different virulence, given by any one of three routes (intranasal, intracloacal, or intraocular), became infected with the viruses. Regardless of the route of inoculation, the outcome of infection was similar for each species but depended on the virulence of the virus used. Muscovy ducks showed more severe clinical signs and higher mortality than the Pekin ducks. In conclusion, domestic ducks are susceptible to H5N1 HPAI virus infection by different routes of exposure, but the presentation of the disease varied by virus strain and duck species. This information helps support the planning and implementation of H5N1 HPAI surveillance and control measures in countries with large domestic duck populations.

  11. A cell culture (Vero)-derived H5N1 whole-virus vaccine induces cross-reactive memory responses.

    PubMed

    Ehrlich, Hartmut J; Müller, Markus; Fritsch, Sandor; Zeitlinger, Markus; Berezuk, Greg; Löw-Baselli, Alexandra; van der Velden, Maikel V W; Pöllabauer, Eva Maria; Maritsch, Friedrich; Pavlova, Borislava G; Tambyah, Paul A; Oh, Helen M L; Montomoli, Emanuele; Kistner, Otfried; Noel Barrett, P

    2009-10-01

    Novel strategies are required to provide rapid vaccine coverage in the event of an influenza pandemic. A phase I/II dose finding/formulation study was performed with a whole-virus H5N1 clade 1 A/Vietnam vaccine (2-dose priming regimen) to evaluate safety and immunogenicity. Seventy-seven of 141 subjects in this study received a booster (12-17 months after priming) with a 7.5-microg dose of a clade 2.1 A/Indonesia vaccine. The prime-boost regimen resulted in antibody responses against clade 1, 2.1, 2.2, and 2.3 viruses that were significantly higher than those after the priming regimen. These findings demonstrate that a prime-boost regimen may alleviate vaccine supply constraints in a pandemic.

  12. Effects of flavonoid-induced oxidative stress on anti-H5N1 influenza a virus activity exerted by baicalein and biochanin A

    PubMed Central

    2014-01-01

    Background Different flavonoids are known to interfere with influenza A virus replication. Recently, we showed that the structurally similar flavonoids baicalein and biochanin A inhibit highly pathogenic avian H5N1 influenza A virus replication by different mechanisms in A549 lung cells. Here, we investigated the effects of both compounds on H5N1-induced reactive oxygen species (ROS) formation and the role of ROS formation during H5N1 replication. Findings Baicalein and biochanin A enhanced H5N1-induced ROS formation in A549 cells and primary human monocyte-derived macrophages. Suppression of ROS formation induced by baicalein and biochanin A using the antioxidant N-acetyl-L-cysteine strongly increased the anti-H5N1 activity of both compounds in A549 cells but not in macrophages. Conclusions These findings emphasise that flavonoids induce complex pharmacological actions some of which may interfere with H5N1 replication while others may support H5N1 replication. A more detailed understanding of these actions and the underlying structure-activity relationships is needed to design agents with optimised anti-H5N1 activity. PMID:24958200

  13. Reassortant H5N1 avian influenza viruses containing PA or NP gene from an H9N2 virus significantly increase the pathogenicity in mice.

    PubMed

    Hao, Xiaoli; Hu, Jiao; Wang, Jiongjiong; Xu, Jing; Cheng, Hao; Xu, Yunpeng; Li, Qunhui; He, Dongchang; Liu, Xiaowen; Wang, Xiaoquan; Gu, Min; Hu, Shunlin; Xu, Xiulong; Liu, Huimou; Chen, Sujuan; Peng, Daxin; Liu, Xiufan

    2016-08-30

    Reassortment between different influenza viruses is a crucial way to generate novel influenza viruses with unpredictable virulence and transmissibility, which may threaten the public health. As currently in China, avian influenza viruses (AIVs) of H9N2 and H5N1 subtypes are endemic in poultry in many areas, while they are prone to reassort with each other naturally. In order to evaluate the risk of the reassortment to public health, A/Goose/Jiangsu/k0403/2010 [GS/10(H5N1)] virus was used as a backbone to generate a series of reassortants, each contained a single internal gene derived from the predominant S genotype of the A/Chicken/Jiangsu/WJ57/2012 [WJ/57(H9N2)]. We next assessed the biological characteristics of these assortments, including pathogenicity, replication efficiency and polymerase activity. We found that the parental WJ/57(H9N2) and GS/10(H5N1) viruses displayed high genetic compatibility. Notably, the H5N1 reassortants containing the PA or NP gene from WJ/57(H9N2) virus significantly increased virulence and replication ability in mice, as well as markedly enhanced polymerase activity. Our results indicate that the endemicity of H9N2 and H5N1 in domestic poultry greatly increases the possibility of generating new viruses by reassortment that may pose a great threat to poultry industry and public health. PMID:27527770

  14. A Unique and Conserved Neutralization Epitope in H5N1 Influenza Viruses Identified by an Antibody against the A/Goose/Guangdong/1/96 Hemagglutinin

    PubMed Central

    Zhu, Xueyong; Guo, Yong-Hui; Jiang, Tao; Wang, Ya-Di; Chan, Kwok-Hung; Li, Xiao-Feng; Yu, Wenli; McBride, Ryan; Paulson, James C.; Yuen, Kwok-Yung; Qin, Cheng-Feng

    2013-01-01

    Despite substantial efforts to control and contain H5N1 influenza viruses, bird flu viruses continue to spread and evolve. Neutralizing antibodies against conserved epitopes on the viral hemagglutinin (HA) could confer immunity to the diverse H5N1 virus strains and provide information for effective vaccine design. Here, we report the characterization of a broadly neutralizing murine monoclonal antibody, H5M9, to most H5N1 clades and subclades that was elicited by immunization with viral HA of A/Goose/Guangdong/1/96 (H5N1), the immediate precursor of the current dominant strains of H5N1 viruses. The crystal structures of the Fab′ fragment of H5M9 in complexes with H5 HAs of A/Vietnam/1203/2004 and A/Goose/Guangdong/1/96 reveal a conserved epitope in the HA1 vestigial esterase subdomain that is some distance from the receptor binding site and partially overlaps antigenic site C of H3 HA. Further epitope characterization by selection of escape mutants and epitope mapping by flow cytometry analysis of site-directed mutagenesis of HA with a yeast cell surface display identified four residues that are critical for H5M9 binding. D53, Y274, E83a, and N276 are all conserved in H5N1 HAs and are not in H5 epitopes identified by other mouse or human antibodies. Antibody H5M9 is effective in protection of H5N1 virus both prophylactically and therapeutically and appears to neutralize by blocking both virus receptor binding and postattachment steps. Thus, the H5M9 epitope identified here should provide valuable insights into H5N1 vaccine design and improvement, as well as antibody-based therapies for treatment of H5N1 infection. PMID:24049169

  15. Efficient strategy for constructing duck enteritis virus-based live attenuated vaccine against homologous and heterologous H5N1 avian influenza virus and duck enteritis virus infection.

    PubMed

    Zou, Zhong; Hu, Yong; Liu, Zhigang; Zhong, Wei; Cao, Hangzhou; Chen, Huanchun; Jin, Meilin

    2015-04-16

    Duck is susceptible to many pathogens, such as duck hepatitis virus, duck enteritis virus (DEV), duck tembusu virus, H5N1 highly pathogenic avian influenza virus (HPAIV) in particular. With the significant role of duck in the evolution of H5N1 HPAIV, control and eradication of H5N1 HPAIV in duck through vaccine immunization is considered an effective method in minimizing the threat of a pandemic outbreak. Consequently, a practical strategy to construct a vaccine against these pathogens should be determined. In this study, the DEV was examined as a candidate vaccine vector to deliver the hemagglutinin (HA) gene of H5N1, and its potential as a polyvalent vaccine was evaluated. A modified mini-F vector was inserted into the gB and UL26 gene junction of the attenuated DEV vaccine strain C-KCE genome to generate an infectious bacterial artificial chromosome (BAC) of C-KCE (vBAC-C-KCE). The HA gene of A/duck/Hubei/xn/2007 (H5N1) was inserted into the C-KCE genome via the mating-assisted genetically integrated cloning (MAGIC) to generate the recombinant vector pBAC-C-KCE-HA. A bivalent vaccine C-KCE-HA was developed by eliminating the BAC backbone. Ducks immunized with C-KCE-HA induced both the cross-reactive antibodies and T cell response against H5. Moreover, C-KCE-HA-immunized ducks provided rapid and long-lasting protection against homologous and heterologous HPAIV H5N1 and DEV clinical signs, death, and primary viral replication. In conclusion, our BAC-C-KCE is a promising platform for developing a polyvalent live attenuated vaccine.

  16. Identification and Structural Characterization of a Broadly Neutralizing Antibody Targeting a Novel Conserved Epitope on the Influenza Virus H5N1 Hemagglutinin

    PubMed Central

    Du, Lanying; Jin, Lei; Zhao, Guangyu; Sun, Shihui; Li, Junfeng; Yu, Hong; Li, Ye; Zheng, Bo-Jian; Liddington, Robert C.

    2013-01-01

    The unabated circulation of the highly pathogenic avian influenza A virus/H5N1 continues to be a serious threat to public health worldwide. Because of the high frequency of naturally occurring mutations, the emergence of H5N1 variants with high virulence has raised great concerns about the potential transmissibility of the virus in humans. Recent studies have shown that laboratory-mutated or reassortant H5N1 viruses could be efficiently transmitted among mammals, particularly ferrets, the best animal model for humans. Thus, it is critical to establish effective strategies to combat future H5N1 pandemics. In this study, we identified a broadly neutralizing monoclonal antibody (MAb), HA-7, that potently neutralized all tested strains of H5N1 covering clades 0, 1, 2.2, 2.3.4, and 2.3.2.1 and completely protected mice against lethal challenges of H5N1 viruses from clades 1 and 2.3.4. HA-7 specifically targeted the globular head of the H5N1 virus hemagglutinin (HA). Using electron microscopy technology with three-dimensional reconstruction (3D-EM), we discovered that HA-7 bound to a novel and highly conserved conformational epitope that was centered on residues 81 to 83 and 117 to 122 of HA1 (H5 numbering). We further demonstrated that HA-7 inhibited viral entry during postattachment events but not at the receptor-binding step, which is fully consistent with the 3D-EM result. Taken together, we propose that HA-7 could be humanized as an effective passive immunotherapeutic agent for antiviral stockpiling for future influenza pandemics caused by emerging unpredictable H5N1 strains. Our study also provides a sound foundation for the rational design of vaccines capable of inducing broad-spectrum immunity against H5N1. PMID:23221567

  17. Rapid detection of avian influenza virus H5N1 in chicken tracheal samples using an impedance aptasensor with gold nanoparticles for signal amplification.

    PubMed

    Karash, Sardar; Wang, Ronghui; Kelso, Lisa; Lu, Huaguang; Huang, Tony Jun; Li, Yanbin

    2016-10-01

    Highly pathogenic avian influenza virus H5N1 is a continuous threat to public health and poultry industry. The recurrence of the H5N1 led us to develop a robust, specific, and rapid detection method for the virus. In this study, an impedance aptasensor was developed for the virus detection using specific H5N1 aptamer and a gold interdigitated microelectrode. Streptavidin was immobilized on the microelectrode surface and biotin labeled H5N1 aptamer was bound to the immobilized streptavidin. The microelectrode was blocked with the polyethylene glycol and the bound aptamer captured the virus. The impedance change caused by the captured virus was measured using an impedance analyzer. To enhance impedance signal, a nanoparticle-based amplifier was designed and implemented by forming a network-like gold nanoparticles/H5N1-aptamer/thiocyanuric acid. The detection limit of the impedance aptasensor was 0.25 HAU for the pure virus and 1 HAU for the tracheal chicken swab samples spiked with the H5N1 virus. The detection time of aptasensor without employing the amplifier was less than an hour. The amplifier increased impedance by a 57-fold for the 1 HAU samples. Only negligible impedance change was observed for non-target viruses such as H5N2, H5N3, H7N2, H1N1, and H2N2. This aptasensor provides a foundation for the development of a portable aptasensor instrument. PMID:27452670

  18. Reassortment between Avian H5N1 and Human Influenza Viruses Is Mainly Restricted to the Matrix and Neuraminidase Gene Segments

    PubMed Central

    Schrauwen, Eefje J. A.; Bestebroer, Theo M.; Rimmelzwaan, Guus F.; Osterhaus, Albert D. M. E.; Fouchier, Ron A. M.; Herfst, Sander

    2013-01-01

    Highly pathogenic avian influenza H5N1 viruses have devastated the poultry industry in many countries of the eastern hemisphere. Occasionally H5N1 viruses cross the species barrier and infect humans, sometimes with a severe clinical outcome. When this happens, there is a chance of reassortment between H5N1 and human influenza viruses. To assess the potential of H5N1 viruses to reassort with contemporary human influenza viruses (H1N1, H3N2 and pandemic H1N1), we used an in vitro selection method to generate reassortant viruses, that contained the H5 hemagglutinin gene, and that have a replication advantage in vitro. We found that the neuraminidase and matrix gene segments of human influenza viruses were preferentially selected by H5 viruses. However, these H5 reassortant viruses did not show a marked increase in replication in MDCK cells and human bronchial epithelial cells. In ferrets, inoculation with a mixture of H5N1-pandemic H1N1 reassortant viruses resulted in outgrowth of reassortant H5 viruses that had incorporated the neuraminidase and matrix gene segment of pandemic 2009 H1N1. This virus was not transmitted via aerosols or respiratory droplets to naïve recipient ferrets. Altogether, these data emphasize the potential of avian H5N1 viruses to reassort with contemporary human influenza viruses. The neuraminidase and matrix gene segments of human influenza viruses showed the highest genetic compatibility with HPAI H5N1 virus. PMID:23527283

  19. Reassortment between Avian H5N1 and human influenza viruses is mainly restricted to the matrix and neuraminidase gene segments.

    PubMed

    Schrauwen, Eefje J A; Bestebroer, Theo M; Rimmelzwaan, Guus F; Osterhaus, Albert D M E; Fouchier, Ron A M; Herfst, Sander

    2013-01-01

    Highly pathogenic avian influenza H5N1 viruses have devastated the poultry industry in many countries of the eastern hemisphere. Occasionally H5N1 viruses cross the species barrier and infect humans, sometimes with a severe clinical outcome. When this happens, there is a chance of reassortment between H5N1 and human influenza viruses. To assess the potential of H5N1 viruses to reassort with contemporary human influenza viruses (H1N1, H3N2 and pandemic H1N1), we used an in vitro selection method to generate reassortant viruses, that contained the H5 hemagglutinin gene, and that have a replication advantage in vitro. We found that the neuraminidase and matrix gene segments of human influenza viruses were preferentially selected by H5 viruses. However, these H5 reassortant viruses did not show a marked increase in replication in MDCK cells and human bronchial epithelial cells. In ferrets, inoculation with a mixture of H5N1-pandemic H1N1 reassortant viruses resulted in outgrowth of reassortant H5 viruses that had incorporated the neuraminidase and matrix gene segment of pandemic 2009 H1N1. This virus was not transmitted via aerosols or respiratory droplets to naïve recipient ferrets. Altogether, these data emphasize the potential of avian H5N1 viruses to reassort with contemporary human influenza viruses. The neuraminidase and matrix gene segments of human influenza viruses showed the highest genetic compatibility with HPAI H5N1 virus.

  20. H1N1, but Not H3N2, Influenza A Virus Infection Protects Ferrets from H5N1 Encephalitis

    PubMed Central

    Wang, Guoji; Carter, Donald M.; Crevar, Corey J.; Ross, Ted M.; Wiley, Clayton A.

    2014-01-01

    ABSTRACT Seasonal influenza causes substantial morbidity and mortality because of efficient human-to-human spread. Rarely, zoonotic strains of influenza virus spread to humans, where they have the potential to mediate new pandemics with high mortality. We studied systemic viral spread after intranasal infection with highly pathogenic avian influenza virus (H5N1 [A/Viet Nam/1203/2004]) in ferrets with or without prior pandemic H1N1pdm09 (A/Mexico/4108/2009) or H3N2 (A/Victoria/361/2011) infection. After intranasal challenge with H5N1 influenza virus, naive ferrets rapidly succumbed to systemic infection. Animals challenged with H5N1 influenza virus greater than 3 months after recovering from an initial H1N1pdm09 infection survived H5N1 virus challenge and cleared virus from the respiratory tract 4 days after infection. However, a prolonged low-level infection of hematopoietic elements in the small bowel lamina propria, liver, and spleen was present for greater than 2 weeks postinfection, raising the potential for reassortment of influenza genes in a host infected with multiple strains of influenza. Animals previously infected with an H3N2 influenza virus succumbed to systemic disease and encephalitis after H5N1 virus challenge. These results indicate prior infection with different seasonal influenza strains leads to radically different protection from H5N1 challenge and fatal encephalitis. IMPORTANCE Seasonal influenza is efficiently transmitted from human to human, causing substantial morbidity and mortality. Rarely, zoonotic strains of influenza virus spread to humans, where they have the potential to mediate new pandemics with high mortality. Infection of naive ferrets with H5N1 avian influenza virus causes a rapid and lethal systemic disease. We studied systemic H5N1 viral spread after infection of ferrets with or without prior exposure to either of two seasonal influenza virus strains, H1N1 and H3N2. Ferrets previously infected with H1N1 survive H5N1 challenge

  1. Pathogenesis of H5N1 influenza virus infections in mice and ferret models differ between respiratory and digestive system exposure

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background. Epidemiological, clinical and laboratory data suggests H5N1 influenza viruses are transmitted through and predominantly affect the respiratory system of mammals. Some data suggests digestive system involvement. However, direct evidence of alimentary transmission and infection in mammal...

  2. Identification, Characterization, and Natural Selection of Mutations Driving Airborne Transmission of A/H5N1 virus

    PubMed Central

    Linster, Martin; van Boheemen, Sander; de Graaf, Miranda; Schrauwen, Eefje J. A.; Lexmond, Pascal; Mänz, Benjamin; Bestebroer, Theo M.; Baumann, Jan; van Riel, Debby; Rimmelzwaan, Guus F.; Osterhaus, Albert D.M.E.; Matrosovich, Mikhail; Fouchier, Ron A. M.; Herfst, Sander

    2014-01-01

    SUMMARY Recently, A/H5N1 influenza viruses were shown to acquire airborne transmissibility between ferrets upon targeted mutagenesis and virus passage. The critical genetic changes in airborne A/Indonesia/5/05 were not yet identified. Here, five substitutions proved to be sufficient to determine this airborne transmission phenotype. Substitutions in PB1 and PB2 collectively caused enhanced transcription and virus replication. One substitution increased HA thermostability and lowered the pH of membrane fusion. Two substitutions independently changed HA binding preference from α2,3 linked to α2,6 linked sialic acid receptors. The loss of a glycosylation site in HA enhanced overall binding to receptors. The acquired substitutions emerged early during ferret passage as minor variants and became dominant rapidly. Identification of substitutions that are essential for airborne transmission of avian influenza viruses between ferrets and their associated phenotypes advances our fundamental understanding of virus transmission and will increase the value of future surveillance programs and public health risk assessments. PMID:24725402

  3. Ferrets develop fatal influenza after inhaling small particle aerosols of highly pathogenic avian influenza virus A/Vietnam/1203/2004 (H5N1)

    PubMed Central

    2010-01-01

    Background There is limited knowledge about the potential routes for H5N1 influenza virus transmission to and between humans, and it is not clear whether humans can be infected through inhalation of aerosolized H5N1 virus particles. Ferrets are often used as a animal model for humans in influenza pathogenicity and transmissibility studies. In this manuscript, a nose-only bioaerosol inhalation exposure system that was recently developed and validated was used in an inhalation exposure study of aerosolized A/Vietnam/1203/2004 (H5N1) virus in ferrets. The clinical spectrum of influenza resulting from exposure to A/Vietnam/1203/2004 (H5N1) through intranasal verses inhalation routes was analyzed. Results Ferrets were successfully infected through intranasal instillation or through inhalation of small particle aerosols with four different doses of Influenza virus A/Vietnam/1203/2004 (H5N1). The animals developed severe influenza encephalomyelitis following intranasal or inhalation exposure to 101, 102, 103, or 104 infectious virus particles per ferret. Conclusions Aerosolized Influenza virus A/Vietnam/1203/2004 (H5N1) is highly infectious and lethal in ferrets. Clinical signs appeared earlier in animals infected through inhalation of aerosolized virus compared to those infected through intranasal instillation. PMID:20843329

  4. Characterization of human influenza A (H5N1) virus infection in mice: neuro-, pneumo- and adipotropic infection.

    PubMed

    Nishimura, H; Itamura, S; Iwasaki, T; Kurata, T; Tashiro, M

    2000-10-01

    Mice (ddY strain, 4 weeks old) were infected intranasally with the H5N1 influenza viruses A/Hong Kong/156/97 (HK156) and A/Hong Kong/483/97 (HK483) isolated from humans. HK156 and HK483 required 200 and 5 p.f.u. of virus, respectively, to give a 50% lethal dose to the mice when the volume of inoculum was set at 10 microl. Both viruses caused encephalitis and severe bronchopneumonia in infected mice. The severity of lung lesions caused by the viruses was essentially similar, whereas HK483 caused more extensive lesions in the brain than did HK156. This was supported by the results of virus titration of organ homogenates, which showed that the virus titres in brains of HK483-infected mice were more than 100-fold higher than those of HK156-infected mice, while those in lungs were almost equivalent. Both viruses were detected in homogenates of the heart, liver, spleen and kidney and blood of the infected mice. Virus antigen was detected by immunohistology in the heart and liver, albeit sporadically, but caused no degenerative change in these organs. The antigen was not detected in the thymus, spleen, pancreas, kidney or gastrointestinal tract. In contrast, virus antigen was found frequently in adipose tissues attached to those organs. The adipose tissues showed severe degenerative change and the virus titres in the tissues were high and comparable to those in lungs. Thus, infection of HK156 and HK483 in our mouse model was pneumo-, neuro- and adipotropic, but not pantropic. Furthermore, HK483 showed higher neurotropism than HK156, which may account for its higher lethality.

  5. Duck MDA5 functions in innate immunity against H5N1 highly pathogenic avian influenza virus infections.

    PubMed

    Wei, Liangmeng; Cui, Jin; Song, Yafen; Zhang, Shuo; Han, Fei; Yuan, Runyu; Gong, Lang; Jiao, Peirong; Liao, Ming

    2014-01-01

    Melanoma differentiation-associated gene 5 (MDA5) is an important intracellular receptor that recognizes long molecules of viral double-stranded RNA in innate immunity. To understand the mechanism of duck MDA5-mediated innate immunity, we cloned the MDA5 cDNA from the Muscovy duck (Cairina moschata). Quantitative real-time PCR analysis indicates that duck MDA5 mRNA was constitutively expressed in all sampled tissues. A significant increase of MDA5 mRNA was detected in the brain, spleen and lungs of ducks after infection with an H5N1 highly pathogenic avian influenza virus (HPAIV). We investigated the role of the predicted functional domains of MDA5. The results indicate the caspase activation and recruitment domain (CARD) of duck MDA5 had a signal transmission function through IRF-7-dependent signaling pathway. Overexpression of the CARD strongly activated the chicken IFN-β promoter and upregulated the mRNA expression of antiviral molecules (such as OAS, PKR and Mx), proinflammatory cytokines (such as IL-2, IL-6, IFN-α and IFN-γ, but not IL-1β and IL-8) and retinoic acid-inducible gene I (RIG-I)-like receptors (RLR) (RIG-I and LGP2) without exogenous stimulation. We also demonstrate the NS1 of the H5N1 HPAIV inhibited the duck MDA5-mediated signaling pathway in vitro. These results suggest that duck MDA5 is an important receptor for inducing antiviral activity in the host immune response of ducks.

  6. [Characterization of the H5N1 influenza virus isolated during an outbreak among wild birds in Russia (Tuva Republic) in 2010].

    PubMed

    Marchenko, V Iu; Sharshov, K A; Silko, N Iu; Susloparov, I M; Durymanov, A G; ZaĭkovskaIa, A V; Alekseev, A Iu; Smolovskaia, O V; Stefanenko, A P; Malkova, E M; Shestopalov, A M

    2011-01-01

    The study of basic biological properties of H5N1 subtype strain isolated during an outbreak among wild birds in Russia in 2010 was presented. The study was carried out using conventional methods according to the WHO recommendations. H5N1 influenza virus isolated in Siberia belonged to clade 2.3.2 of the hemagglutinin gene; the phylogenetic analysis was performed. The antigenic characteristics and the basic genetic markers of biological properties were studied. It was shown that all strains were highly pathogenic for chickens and white mice. Thus, it was shown that in Russia in the 2010 H5N1 virus phylogenetically closely related to Asian variants caused epizootic among wild birds. The potential danger of this variant of the virus for humans was confirmed by different methods. We discussed the possibility of formation of H5N1 influenza natural focus.

  7. Single-dose vaccination of a recombinant parainfluenza virus 5 expressing NP from H5N1 virus provides broad immunity against influenza A viruses.

    PubMed

    Li, Zhuo; Gabbard, Jon D; Mooney, Alaina; Gao, Xiudan; Chen, Zhenhai; Place, Ryan J; Tompkins, S Mark; He, Biao

    2013-05-01

    Influenza viruses often evade host immunity via antigenic drift and shift despite previous influenza virus infection and/or vaccination. Vaccines that match circulating virus strains are needed for optimal protection. Development of a universal influenza virus vaccine providing broadly cross-protective immunity will be of great importance. The nucleoprotein (NP) of influenza A virus is highly conserved among all strains of influenza A viruses and has been explored as an antigen for developing a universal influenza virus vaccine. In this work, we generated a recombinant parainfluenza virus 5 (PIV5) containing NP from H5N1 (A/Vietnam/1203/2004), a highly pathogenic avian influenza (HPAI) virus, between HN and L (PIV5-NP-HN/L) and tested its efficacy. PIV5-NP-HN/L induced humoral and T cell responses in mice. A single inoculation of PIV5-NP-HN/L provided complete protection against lethal heterosubtypic H1N1 challenge and 50% protection against lethal H5N1 HPAI virus challenge. To improve efficacy, NP was inserted into different locations within the PIV5 genome. Recombinant PIV5 containing NP between F and SH (PIV5-NP-F/SH) or between SH and HN (PIV5-NP-SH/HN) provided better protection against H5N1 HPAI virus challenge than did PIV5-NP-HN/L. These results suggest that PIV5 expressing NP from H5N1 has the potential to be utilized as a universal influenza virus vaccine.

  8. H5N1 hybrid viruses bearing 2009/H1N1 virus genes transmit in guinea pigs by respiratory droplet.

    PubMed

    Zhang, Ying; Zhang, Qianyi; Kong, Huihui; Jiang, Yongping; Gao, Yuwei; Deng, Guohua; Shi, Jianzhong; Tian, Guobin; Liu, Liling; Liu, Jinxiong; Guan, Yuntao; Bu, Zhigao; Chen, Hualan

    2013-06-21

    In the past, avian influenza viruses have crossed species barriers to trigger human pandemics by reassorting with mammal-infective viruses in intermediate livestock hosts. H5N1 viruses are able to infect pigs, and some of them have affinity for the mammalian type α-2,6-linked sialic acid airway receptor. Using reverse genetics, we systematically created 127 reassortant viruses between a duck isolate of H5N1, specifically retaining its hemagglutinin (HA) gene throughout, and a highly transmissible, human-infective H1N1 virus. We tested the virulence of the reassortants in mice as a correlate for virulence in humans and tested transmissibility in guinea pigs, which have both avian and mammalian types of airway receptor. Transmission studies showed that the H1N1 virus genes encoding acidic polymerase and nonstructural protein made the H5N1 virus transmissible by respiratory droplet between guinea pigs without killing them. Further experiments implicated other H1N1 genes in the enhancement of mammal-to-mammal transmission, including those that encode nucleoprotein, neuraminidase, and matrix, as well as mutations in H5 HA that improve affinity for humanlike airway receptors. Hence, avian H5N1 subtype viruses do have the potential to acquire mammalian transmissibility by reassortment in current agricultural scenarios.

  9. Short-Term Heat Shock Affects Host–Virus Interaction in Mice Infected with Highly Pathogenic Avian Influenza Virus H5N1

    PubMed Central

    Xue, Jia; Fan, Xiaoxu; Yu, Jing; Zhang, Shouping; Xiao, Jin; Hu, Yanxin; Wang, Ming

    2016-01-01

    Highly pathogenic avian influenza virus (HPAIV) H5N1 is a highly contagious virus that can cause acute respiratory infections and high human fatality ratio due to excessive inflammatory response. Short-term heat shock, as a stressful condition, could induce the expression of heat shock proteins that function as molecular chaperones to protect cells against multiple stresses. However, the protective effect of short-term heat shock in influenza infection is far from being understood. In this study, mice were treated at 39°C for 4 h before being infected with HPAIV H5N1. Interestingly, short-term heat shock significantly increased the levels of HSP70 and pro-inflammatory cytokines IL-6, TNF-α, IFN-β, and IFN-γ in the lung tissues of mice. Following HPAIV H5N1 infection, short-term heat shock alleviated immunopathology and viral replication in lung tissue and repressed the weight loss and increased the survival rate of H5N1-infected mice. Our data reported that short-term heat shock provided beneficial anti-HPAIV H5N1 properties in mice model, which offers an alternative strategy for non-drug prevention for influenza infection. PMID:27379054

  10. Experimental challenge and pathology of highly pathogenic avian influenza virus H5N1 in dunlin (Calidris alpina), an intercontinental migrant shorebird species

    USGS Publications Warehouse

    Hall, Jeffrey S.; Franson, J. Christian; Gill, Robert E.; Meteyer, Carol U.; TeSlaa, Joshua L.; Nashold, Sean; Dusek, Robert J.; Ip, Hon S.

    2011-01-01

    Conclusions Dunlin are highly susceptible to infection with HPAIV H5N1. They become infected after exposure to relatively small doses of the virus and if they become infected, they are most likely to suffer mortality within 3–5 days. These results have important implications regarding the risks of transport and transmission of HPAIV H5N1 to North America by this species and raises questions for further investigation.

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

    PubMed

    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

  12. Perpetuation of H5N1 and H9N2 avian influenza viruses in natural water bodies.

    PubMed

    Zhang, Hongbo; Li, Yan; Chen, Jianjun; Chen, Quanjiao; Chen, Ze

    2014-07-01

    Water bodies are an important route for the spread and transmission of avian influenza virus (AIV). The determining factor for an AIV to transmit through diffusion in water is the term of viability of the virus in the water body. To better understand the perpetuation of AIV in natural water bodies, and thus the risks of AIV spread and transmission via such bodies, we systematically studied the inactivation dynamics of two AIV strains (H5N1 and H9N2) at different temperatures in water bodies of important migratory bird habitats within China (Dongting Lake, Poyang Lake, the Hubei segment of the Yangtze River and Qinghai Lake). We also studied the impact of water-borne micro-organisms on the perpetuation of AIV. Our findings indicated that water is very likely an important route for the epidemic spread of AIV, especially during the autumn and winter seasons. In addition, water-borne micro-organisms might antagonize the persistence of AIV.

  13. Southward autumn migration of waterfowl facilitates cross-continental transmission of the highly pathogenic avian influenza H5N1 virus.

    PubMed

    Xu, Yanjie; Gong, Peng; Wielstra, Ben; Si, Yali

    2016-01-01

    The highly pathogenic avian influenza subtype H5N1 (HPAI H5N1) is a worldwide zoonotic infectious disease, threatening humans, poultry and wild birds. The role of wild birds in the spread of HPAI H5N1 has previously been investigated by comparing disease spread patterns with bird migration routes. However, the different roles that the southward autumn and northward spring migration might play in virus transmission have hardly been explored. Using direction analysis, we analyze HPAI H5N1 transmission directions and angular concentration of currently circulating viral clades, and compare these with waterfowl seasonal migration directions along major waterfowl flyways. Out of 22 HPAI H5N1 transmission directions, 18 had both a southward direction and a relatively high concentration. Differences between disease transmission and waterfowl migration directions were significantly smaller for autumn than for spring migration. The four northward transmission directions were found along Asian flyways, where the initial epicenter of the virus was located. We suggest waterfowl first picked up the virus from East Asia, then brought it to the north via spring migration, and then spread it to other parts of world mainly by autumn migration. We emphasize waterfowl autumn migration plays a relatively important role in HPAI H5N1 transmission compared to spring migration. PMID:27507581

  14. Southward autumn migration of waterfowl facilitates cross-continental transmission of the highly pathogenic avian influenza H5N1 virus

    NASA Astrophysics Data System (ADS)

    Xu, Yanjie; Gong, Peng; Wielstra, Ben; Si, Yali

    2016-08-01

    The highly pathogenic avian influenza subtype H5N1 (HPAI H5N1) is a worldwide zoonotic infectious disease, threatening humans, poultry and wild birds. The role of wild birds in the spread of HPAI H5N1 has previously been investigated by comparing disease spread patterns with bird migration routes. However, the different roles that the southward autumn and northward spring migration might play in virus transmission have hardly been explored. Using direction analysis, we analyze HPAI H5N1 transmission directions and angular concentration of currently circulating viral clades, and compare these with waterfowl seasonal migration directions along major waterfowl flyways. Out of 22 HPAI H5N1 transmission directions, 18 had both a southward direction and a relatively high concentration. Differences between disease transmission and waterfowl migration directions were significantly smaller for autumn than for spring migration. The four northward transmission directions were found along Asian flyways, where the initial epicenter of the virus was located. We suggest waterfowl first picked up the virus from East Asia, then brought it to the north via spring migration, and then spread it to other parts of world mainly by autumn migration. We emphasize waterfowl autumn migration plays a relatively important role in HPAI H5N1 transmission compared to spring migration.

  15. Southward autumn migration of waterfowl facilitates cross-continental transmission of the highly pathogenic avian influenza H5N1 virus.

    PubMed

    Xu, Yanjie; Gong, Peng; Wielstra, Ben; Si, Yali

    2016-08-10

    The highly pathogenic avian influenza subtype H5N1 (HPAI H5N1) is a worldwide zoonotic infectious disease, threatening humans, poultry and wild birds. The role of wild birds in the spread of HPAI H5N1 has previously been investigated by comparing disease spread patterns with bird migration routes. However, the different roles that the southward autumn and northward spring migration might play in virus transmission have hardly been explored. Using direction analysis, we analyze HPAI H5N1 transmission directions and angular concentration of currently circulating viral clades, and compare these with waterfowl seasonal migration directions along major waterfowl flyways. Out of 22 HPAI H5N1 transmission directions, 18 had both a southward direction and a relatively high concentration. Differences between disease transmission and waterfowl migration directions were significantly smaller for autumn than for spring migration. The four northward transmission directions were found along Asian flyways, where the initial epicenter of the virus was located. We suggest waterfowl first picked up the virus from East Asia, then brought it to the north via spring migration, and then spread it to other parts of world mainly by autumn migration. We emphasize waterfowl autumn migration plays a relatively important role in HPAI H5N1 transmission compared to spring migration.

  16. Southward autumn migration of waterfowl facilitates cross-continental transmission of the highly pathogenic avian influenza H5N1 virus

    PubMed Central

    Xu, Yanjie; Gong, Peng; Wielstra, Ben; Si, Yali

    2016-01-01

    The highly pathogenic avian influenza subtype H5N1 (HPAI H5N1) is a worldwide zoonotic infectious disease, threatening humans, poultry and wild birds. The role of wild birds in the spread of HPAI H5N1 has previously been investigated by comparing disease spread patterns with bird migration routes. However, the different roles that the southward autumn and northward spring migration might play in virus transmission have hardly been explored. Using direction analysis, we analyze HPAI H5N1 transmission directions and angular concentration of currently circulating viral clades, and compare these with waterfowl seasonal migration directions along major waterfowl flyways. Out of 22 HPAI H5N1 transmission directions, 18 had both a southward direction and a relatively high concentration. Differences between disease transmission and waterfowl migration directions were significantly smaller for autumn than for spring migration. The four northward transmission directions were found along Asian flyways, where the initial epicenter of the virus was located. We suggest waterfowl first picked up the virus from East Asia, then brought it to the north via spring migration, and then spread it to other parts of world mainly by autumn migration. We emphasize waterfowl autumn migration plays a relatively important role in HPAI H5N1 transmission compared to spring migration. PMID:27507581

  17. Identification and characterization of a highly pathogenic H5N1 avian influenza A virus during an outbreak in vaccinated chickens in Egypt.

    PubMed

    Amen, O; Vemula, S V; Zhao, J; Ibrahim, R; Hussein, A; Hewlett, I K; Moussa, S; Mittal, S K

    2015-12-01

    Highly pathogenic avian influenza A (HPAI) H5N1 viruses continue to be a major veterinary and public health problem in Egypt. Continued surveillance of these viruses is necessary to devise strategies to control the spread of the virus and to monitor its evolutionary patterns. This is a report of the identification of a variant strain of HPAI H5N1 virus during an outbreak in 2010 in vaccinated chicken flocks in a poultry farm in Assiut, Egypt. Vaccination of chickens with an oil-emulsified inactivated A/chicken/Mexico/232/94 (H5N2) vaccine induced high levels of hemagglutination inhibition (HI) antibody titers reaching up to 9 log2. However, all flocks irrespective of the number of vaccine doses and the resultant HI titer levels came down with severe influenza infections. The qRT-PCR and rapid antigen test confirmed the influenza virus to be from H5N1 subtype. Sequencing of the hemagglutinin (HA) gene fragment from ten independent samples demonstrated that a single H5N1 strain was involved. This strain belonged to clade 2.2.1 and had several mutations in the receptor-binding site of the HA protein, thereby producing a variant strain of HPAI H5N1 virus which was antigenically different from the parent clade 2.2.1 virus circulating in Egypt at that time. In order to define the variability in HPAI H5N1 viruses over time in Egypt, we sequenced another H5N1 virus that was causing infections in chickens in 2014. Phylogenetic analysis revealed that both viruses had further distanced from the parent virus circulating during 2010. This study highlights that the antigenic mutations in HPAI H5N1 viruses represent a definitive challenge for the development of an effective vaccine for poultry. Overall, the results emphasize the need for continued surveillance of H5N1 outbreaks and extensive characterization of virus isolates from vaccinated and non-vaccinated poultry populations to better understand genetic changes and their implications.

  18. Migration of Whooper Swans and Outbreaks of Highly Pathogenic Avian Influenza H5N1 Virus in Eastern Asia

    PubMed Central

    Newman, Scott H.; Iverson, Samuel A.; Takekawa, John Y.; Gilbert, Martin; Prosser, Diann J.; Batbayar, Nyambyar; Natsagdorj, Tseveenmyadag; Douglas, David C.

    2009-01-01

    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. PMID:19479053

  19. Migration of whooper swans and outbreaks of highly pathogenic avian influenza H5N1 virus in Eastern Asia

    USGS Publications Warehouse

    Newman, Scott H.; Iverson, Samuel A.; Takekawa, John Y.; Gilbert, Martin; Prosser, Diann J.; Batbayar, Nyambyar; Natsagdorj, Tseveenmyadag; Douglas, David C.

    2009-01-01

    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.

  20. Tropism and Induction of Cytokines in Human Embryonic-Stem Cells-Derived Neural Progenitors upon Inoculation with Highly- Pathogenic Avian H5N1 Influenza Virus

    PubMed Central

    Pringproa, Kidsadagon; Rungsiwiwut, Ruttachuk; Tantilertcharoen, Rachod; Praphet, Reunkeaw; Pruksananonda, Kamthorn; Baumgärtner, Wolfgang; Thanawongnuwech, Roongroje

    2015-01-01

    Central nervous system (CNS) dysfunction caused by neurovirulent influenza viruses is a dreaded complication of infection, and may play a role in some neurodegenerative conditions, such as Parkinson-like diseases and encephalitis lethargica. Although CNS infection by highly pathogenic H5N1 virus has been demonstrated, it is unknown whether H5N1 infects neural progenitor cells, nor whether such infection plays a role in the neuroinflammation and neurodegeneration. To pursue this question, we infected human neural progenitor cells (hNPCs) differentiated from human embryonic stem cells in vitro with H5N1 virus, and studied the resulting cytopathology, cytokine expression, and genes involved in the differentiation. Human embryonic stem cells (BG01) were maintained and differentiated into the neural progenitors, and then infected by H5N1 virus (A/Chicken/Thailand/CUK2/04) at a multiplicity of infection of 1. At 6, 24, 48, and 72 hours post-infection (hpi), cytopathic effects were observed. Then cells were characterized by immunofluorescence and electron microscopy, supernatants quantified for virus titers, and sampled cells studied for candidate genes.The hNPCs were susceptible to H5N1 virus infection as determined by morphological observation and immunofluorescence. The infection was characterized by a significant up-regulation of TNF-α gene expression, while expressions of IFN-α2, IFN-β1, IFN-γ and IL-6 remained unchanged compared to mock-infected controls. Moreover, H5N1 infection did not appear to alter expression of neuronal and astrocytic markers of hNPCs, such as β-III tubulin and GFAP, respectively. The results indicate that hNPCs support H5N1 virus infection and may play a role in the neuroinflammation during acute viral encephalitis. PMID:26274828

  1. A mouse model for the evaluation of pathogenesis and immunity to influenza A (H5N1) viruses isolated from humans.

    PubMed

    Lu, X; Tumpey, T M; Morken, T; Zaki, S R; Cox, N J; Katz, J M

    1999-07-01

    During 1997 in Hong Kong, 18 human cases of respiratory illness, including 6 fatalities, were caused by highly pathogenic avian influenza A (H5N1) viruses. Since H5 viruses had previously been isolated only from avian species, the outbreak raised questions about the ability of these viruses to cause severe disease and death in humans. To better understand the pathogenesis and immunity to these viruses, we have used the BALB/c mouse model. Four H5N1 viruses replicated equally well in the lungs of mice without prior adaptation but differed in lethality for mice. H5N1 viruses that were highly lethal for mice were detected in multiple organs, including the brain. This is the first demonstration of an influenza A virus that replicates systemically in a mammalian species and is neurotropic without prior adaptation. The mouse model was also used to evaluate a strategy of vaccination against the highly pathogenic avian H5N1 viruses, using an inactivated vaccine prepared from nonpathogenic A/Duck/Singapore-Q/F119-3/97 (H5N3) virus that was antigenically related to the human H5N1 viruses. Mice administered vaccine intramuscularly, with or without alum, were completely protected from lethal challenge with H5N1 virus. Protection from infection was also observed in 70% of animals administered vaccine alone and 100% of mice administered vaccine with alum. The protective effect of vaccination correlated with the level of virus-specific serum antibody. These results suggests a strategy of vaccine preparedness for rapid intervention in future influenza pandemics that uses antigenically related nonpathogenic viruses as vaccine candidates. PMID:10364342

  2. Parasite-Mediated Upregulation of NK Cell-Derived Gamma Interferon Protects against Severe Highly Pathogenic H5N1 Influenza Virus Infection▿

    PubMed Central

    O'Brien, Kevin B.; Schultz-Cherry, Stacey; Knoll, Laura J.

    2011-01-01

    Outbreaks of influenza A viruses are associated with significant human morbidity worldwide. Given the increasing resistance to the available influenza drugs, new therapies for the treatment of influenza virus infection are needed. An alternative approach is to identify products that enhance a protective immune response. In these studies, we demonstrate that infecting mice with the Th1-inducing parasite Toxoplasma gondii prior to highly pathogenic avian H5N1 influenza virus infection led to decreased lung viral titers and enhanced survival. A noninfectious fraction of T. gondii soluble antigens (STAg) elicited an immune response similar to that elicited by live parasites, and administration of STAg 2 days after H5N1 influenza virus infection enhanced survival, lowered viral titers, and reduced clinical disease. STAg administration protected H5N1 virus-infected mice lacking lymphocytes, suggesting that while the adaptive immune response was not required for enhanced survival, it was necessary for STAg-mediated viral clearance. Mechanistically, we found that administration of STAg led to increased production of gamma interferon (IFN-γ) from natural killer (NK) cells, which were both necessary and sufficient for survival. Further, administration of exogenous IFN-γ alone enhanced survival from H5N1 influenza virus infection, although not to the same level as STAg treatment. These studies demonstrate that a noninfectious T. gondii extract enhances the protective immune response against severe H5N1 influenza virus infections even when a single dose is administered 2 days postinfection. PMID:21734055

  3. Phylogenetic and pathogenic analyses of three H5N1 avian influenza viruses (clade 2.3.2.1) isolated from wild birds in Northeast China.

    PubMed

    Fan, Zhaobin; Ci, Yanpeng; Liu, Liling; Ma, Yixin; Jia, Ying; Wang, Deli; Guan, Yuntao; Tian, Guobin; Ma, Jianzhang; Li, Yanbing; Chen, Hualan

    2015-01-01

    From April to September 2012, periodic surveillance of avian influenza H5N1 viruses from different wild bird species was conducted in Northeast China. Three highly pathogenic avian influenza (HPAI) H5N1 viruses were isolated from a yellow-browed warbler, common shoveler, and mallard. To trace the genetic lineage of the isolates, nucleotide sequences of all eight gene segments were determined and phylogenetically analyzed. The data indicated that three viruses belonged to the same antigenic virus group: clade 2.3.2.1. To investigate the pathogenicity of these three viruses in different hosts, chickens, ducks, and mice were inoculated. The results showed that chickens were susceptible to each of the three HPAI H5N1 viruses, resulting in 100% mortality within 2-6 days after infection, whereas the three isolates exhibited distinctly different virulence in ducks and mice. The results of this study demonstrated that HPAI H5N1 viruses of clade 2.3.2.1 are still circulating in wild birds through overlapping migratory flyways. Therefore, continuous monitoring of H5N1 in both domestic and wild birds is necessary to prevent a potentially wider outbreak.

  4. Characterization of low-pathogenic H5 subtype influenza viruses from Eurasia: implications for the origin of highly pathogenic H5N1 viruses.

    PubMed

    Duan, L; Campitelli, L; Fan, X H; Leung, Y H C; Vijaykrishna, D; Zhang, J X; Donatelli, I; Delogu, M; Li, K S; Foni, E; Chiapponi, C; Wu, W L; Kai, H; Webster, R G; Shortridge, K F; Peiris, J S M; Smith, Gavin J D; Chen, H; Guan, Y

    2007-07-01

    Highly pathogenic avian influenza (HPAI) H5N1 viruses are now endemic in many Asian countries, resulting in repeated outbreaks in poultry and increased cases of human infection. The immediate precursor of these HPAI viruses is believed to be A/goose/Guangdong/1/96 (Gs/GD)-like H5N1 HPAI viruses first detected in Guangdong, China, in 1996. From 2000 onwards, many novel reassortant H5N1 influenza viruses or genotypes have emerged in southern China. However, precursors of the Gs/GD-like viruses and their subsequent reassortants have not been fully determined. Here we characterize low-pathogenic avian influenza (LPAI) H5 subtype viruses isolated from poultry and migratory birds in southern China and Europe from the 1970s to the 2000s. Phylogenetic analyses revealed that Gs/GD-like virus was likely derived from an LPAI H5 virus in migratory birds. However, its variants arose from multiple reassortments between Gs/GD-like virus and viruses from migratory birds or with those Eurasian viruses isolated in the 1970s. It is of note that unlike HPAI H5N1 viruses, those recent LPAI H5 viruses have not become established in aquatic or terrestrial poultry. Phylogenetic analyses revealed the dynamic nature of the influenza virus gene pool in Eurasia with repeated transmissions between the eastern and western extremities of the continent. The data also show reassortment between influenza viruses from domestic and migratory birds in this region that has contributed to the expanded diversity of the influenza virus gene pool among poultry in Eurasia.

  5. The Continuing Evolution of H5N1 and H9N2 Influenza Viruses in Bangladesh Between 2013 and 2014.

    PubMed

    Marinova-Petkova, Atanaska; Shanmuganatham, Karthik; Feeroz, Mohammed M; Jones-Engel, Lisa; Hasan, M Kamrul; Akhtar, Sharmin; Turner, Jasmine; Walker, David; Seiler, Patrick; Franks, John; McKenzie, Pamela; Krauss, Scott; Webby, Richard J; Webster, Robert G

    2016-05-01

    In 2011, avian influenza surveillance at the Bangladesh live bird markets (LBMs) showed complete replacement of the highly pathogenic avian influenza (HPAI) H5N1 virus of clade 2.2.2 (Qinghai-like H5N1 lineage) by the HPAI H5N1 clade 2.3.2.1. This clade, which continues to circulate in Bangladesh and neighboring countries, is an intra-and interclade reassortant; its HA, polymerase basic 1 (PB1), polymerase (PA), and nonstructural (NS) genes come from subclade 2.3.2.1a; the polymerase basic 2 (PB2) comes from subclade 2.3.2.1c; and the NA, nucleocapsid protein (NP), and matrix (M) gene from clade 2.3.4.2. The H9N2 influenza viruses cocirculating in the Bangladesh LBMs are also reassortants, possessing five genes (NS, M, NP, PA, and PB1) from an HPAI H7N3 virus previously isolated in Pakistan. Despite frequent coinfection of chickens and ducks, reassortment between these H5N1 and H9N2 viruses has been rare. However, all such reassortants detected in 2011 through 2013 have carried seven genes from the local HPAI H5N1 lineage and the PB1 gene from the Bangladeshi H9N2 clade G1 Mideast, itself derived from HPAI H7N3 virus. Although the live birds we sampled in Bangladesh showed no clinical signs of morbidity, the emergence of this reassortant HPAI H5N1 lineage further complicates endemic circulation of H5N1 viruses in Bangladesh, posing a threat to both poultry and humans. PMID:27309046

  6. Living with avian FLU--Persistence of the H5N1 highly pathogenic avian influenza virus in Egypt.

    PubMed

    Njabo, Kevin Yana; Zanontian, Linda; Sheta, Basma N; Samy, Ahmed; Galal, Shereen; Schoenberg, Frederic Paik; Smith, Thomas B

    2016-05-01

    H5N1 highly pathogenic avian influenza virus (HPAIV) continues to cause mortality in poultry and threaten human health at a panzootic scale in Egypt since it was reported in 2006. While the early focus has been in Asia, recent evidence suggests that Egypt is an emerging epicenter for the disease. Despite control measures, epizootic transmission of the disease continues. Here, we investigate the persistence of HPAIV across wild passerine birds and domestic poultry between 2009 and 2012 and the potential risk for continuous viral transmission in Egypt. We use a new weighted cross J-function to investigate the degree and spatial temporal nature of the clustering between sightings of infected birds of different types, and the risk of infection associated with direct contact with infected birds. While we found no infection in wild birds, outbreaks occurred year round between 2009 and 2012, with a positive interaction between chickens and ducks. The disease was more present in the years 2010 and 2011 coinciding with the political unrest in the country. Egypt thus continues to experience endemic outbreaks of avian influenza HPAIV in poultry and an increased potential risk of infection to other species including humans. With the current trends, the elimination of the HPAIV infection is highly unlikely without a complete revamp of current policies. The application of spatial statistics techniques to these types of data may help us to understand the characteristics of the disease and may subsequently allow practitioners to explore possible preventive solutions. PMID:27066713

  7. Living with avian FLU--Persistence of the H5N1 highly pathogenic avian influenza virus in Egypt.

    PubMed

    Njabo, Kevin Yana; Zanontian, Linda; Sheta, Basma N; Samy, Ahmed; Galal, Shereen; Schoenberg, Frederic Paik; Smith, Thomas B

    2016-05-01

    H5N1 highly pathogenic avian influenza virus (HPAIV) continues to cause mortality in poultry and threaten human health at a panzootic scale in Egypt since it was reported in 2006. While the early focus has been in Asia, recent evidence suggests that Egypt is an emerging epicenter for the disease. Despite control measures, epizootic transmission of the disease continues. Here, we investigate the persistence of HPAIV across wild passerine birds and domestic poultry between 2009 and 2012 and the potential risk for continuous viral transmission in Egypt. We use a new weighted cross J-function to investigate the degree and spatial temporal nature of the clustering between sightings of infected birds of different types, and the risk of infection associated with direct contact with infected birds. While we found no infection in wild birds, outbreaks occurred year round between 2009 and 2012, with a positive interaction between chickens and ducks. The disease was more present in the years 2010 and 2011 coinciding with the political unrest in the country. Egypt thus continues to experience endemic outbreaks of avian influenza HPAIV in poultry and an increased potential risk of infection to other species including humans. With the current trends, the elimination of the HPAIV infection is highly unlikely without a complete revamp of current policies. The application of spatial statistics techniques to these types of data may help us to understand the characteristics of the disease and may subsequently allow practitioners to explore possible preventive solutions.

  8. Introduction and enzootic of A/H5N1 in Egypt: Virus evolution, pathogenicity and vaccine efficacy ten years on.

    PubMed

    Abdelwhab, E M; Hassan, M K; Abdel-Moneim, A S; Naguib, M M; Mostafa, A; Hussein, I T M; Arafa, A; Erfan, A M; Kilany, W H; Agour, M G; El-Kanawati, Z; Hussein, H A; Selim, A A; Kholousy, S; El-Naggar, H; El-Zoghby, E F; Samy, A; Iqbal, M; Eid, A; Ibraheem, E M; Pleschka, S; Veits, J; Nasef, S A; Beer, M; Mettenleiter, T C; Grund, C; Ali, M M; Harder, T C; Hafez, H M

    2016-06-01

    It is almost a decade since the highly pathogenic H5N1 avian influenza virus (A/H5N1) of clade 2.2.1 was introduced to Egypt in 2005, most likely, via wild birds; marking the longest endemic status of influenza viruses in poultry outside Asia. The endemic A/H5N1 in Egypt still compromises the poultry industry, poses serious hazards to public health and threatens to become potentially pandemic. The control strategies adopted for A/H5N1 in Egyptian poultry using diverse vaccines in commercialized poultry neither eliminated the virus nor did they decrease its evolutionary rate. Several virus clades have evolved, a few of them disappeared and others prevailed. Disparate evolutionary traits in both birds and humans were manifested by accumulation of clade-specific mutations across viral genomes driven by a variety of selection pressures. Viruses in vaccinated poultry populations displayed higher mutation rates at the immunogenic epitopes, promoting viral escape and reducing vaccine efficiency. On the other hand, viruses isolated from humans displayed changes in the receptor binding domain, which increased the viral affinity to bind to human-type glycan receptors. Moreover, viral pathogenicity exhibited several patterns in different hosts. This review aims to provide an overview of the viral evolution, pathogenicity and vaccine efficacy of A/H5N1 in Egypt during the last ten years. PMID:26917362

  9. Viral replication rate regulates clinical outcome and CD8 T cell responses during highly pathogenic H5N1 influenza virus infection in mice.

    PubMed

    Hatta, Yasuko; Hershberger, Karen; Shinya, Kyoko; Proll, Sean C; Dubielzig, Richard R; Hatta, Masato; Katze, Michael G; Kawaoka, Yoshihiro; Suresh, M

    2010-01-01

    Since the first recorded infection of humans with H5N1 viruses of avian origin in 1997, sporadic human infections continue to occur with a staggering mortality rate of >60%. Although sustained human-to-human transmission has not occurred yet, there is a growing concern that these H5N1 viruses might acquire this trait and raise the specter of a pandemic. Despite progress in deciphering viral determinants of pathogenicity, we still lack crucial information on virus/immune system interactions pertaining to severe disease and high mortality associated with human H5N1 influenza virus infections. Using two human isolates of H5N1 viruses that differ in their pathogenicity in mice, we have defined mechanistic links among the rate of viral replication, mortality, CD8 T cell responses, and immunopathology. The extreme pathogenicity of H5N1 viruses was directly linked to the ability of the virus to replicate rapidly, and swiftly attain high steady-state titers in the lungs within 48 hours after infection. The remarkably high replication rate of the highly pathogenic H5N1 virus did not prevent the induction of IFN-β or activation of CD8 T cells, but the CD8 T cell response was ineffective in controlling viral replication in the lungs and CD8 T cell deficiency did not affect viral titers or mortality. Additionally, BIM deficiency ameliorated lung pathology and inhibited T cell apoptosis without affecting survival of mice. Therefore, rapidly replicating, highly lethal H5N1 viruses could simply outpace and overwhelm the adaptive immune responses, and kill the host by direct cytopathic effects. However, therapeutic suppression of early viral replication and the associated enhancement of CD8 T cell responses improved the survival of mice following a lethal H5N1 infection. These findings suggest that suppression of early H5N1 virus replication is key to the programming of an effective host response, which has implications in treatment of this infection in humans. PMID:20949022

  10. An overview of the epidemic of highly pathogenic H5N1 avian influenza virus in Egypt: epidemiology and control challenges.

    PubMed

    Abdelwhab, E M; Hafez, H M

    2011-05-01

    Emergence of the highly pathogenic avian influenza (HPAI) H5N1 virus in Egypt in mid-February 2006 caused significant losses for the poultry industry and constituted a potential threat to public health. Since late 2007, there has been increasing evidence that stable lineages of H5N1 viruses are being established in chickens and humans in Egypt. The virus has been detected in wild, feral and zoo birds and recently was found in donkeys and pigs. Most of the outbreaks in poultry and humans occurred in the highly populated Nile delta. The temporal pattern of the virus has changed since 2009 with outbreaks now occurring in the warmer months of the year. Challenges to control of endemic disease in Egypt are discussed. For the foreseeable future, unless a global collaboration exists, HPAI H5N1 virus in Egypt will continue to compromise the poultry industry, endanger public health and pose a serious pandemic threat. PMID:21281550

  11. An immunoassay-based reverse-transcription loop-mediated isothermal amplification assay for the rapid detection of avian influenza H5N1 virus viremia.

    PubMed

    Tang, Yi; Yu, Xu; Chen, Hao; Diao, Youxiang

    2016-12-15

    Avian influenza virus (AIV) subtype H5N1 attracts particular consideration because it is a continuous threat to animals and public health systems. The viremia caused by AIV H5N1 infection may increase the risk of blood-borne transmission between humans. Therefore, there is a need to rapidly evaluate and implement screening measures for AIV H5N1 viremia that allows for rapid response to this potentially pandemic threat. The present report describes an immunoassay-based reverse-transcription loop-mediated isothermal amplification (immuno-RT-LAMP) assay for the rapid detection of AIV H5N1 in whole blood samples. Using PCR tubes coated with an H5 subtype monoclonal antibody, AIV H5N1 virions were specifically captured from blood samples. After a thermal lysis step, the released viral N1 gene was exponentially amplified using RT-LAMP on either a real-time PCR instrument for quantitative analysis, or in a water bath system for endpoint analysis. The detection limit of the newly developed immuno-RT-LAMP assay was as low as 1.62×10(1) 50% embryo infectious dose/mL of virus in both regular samples and simulated viremia samples. There were no cross-reactions with non-H5N1 influenza viruses or other avian viruses. The reproducibility of the assay was confirmed using intra- and inter-assay tests with variability ranging from 1.05% to 3.37%. Our results indicate that immuno-RT-LAMP is a novel, effective point-of-care virus identification solution for the rapid diagnosis and monitoring of AIV H5N1 in blood samples.

  12. An immunoassay-based reverse-transcription loop-mediated isothermal amplification assay for the rapid detection of avian influenza H5N1 virus viremia.

    PubMed

    Tang, Yi; Yu, Xu; Chen, Hao; Diao, Youxiang

    2016-12-15

    Avian influenza virus (AIV) subtype H5N1 attracts particular consideration because it is a continuous threat to animals and public health systems. The viremia caused by AIV H5N1 infection may increase the risk of blood-borne transmission between humans. Therefore, there is a need to rapidly evaluate and implement screening measures for AIV H5N1 viremia that allows for rapid response to this potentially pandemic threat. The present report describes an immunoassay-based reverse-transcription loop-mediated isothermal amplification (immuno-RT-LAMP) assay for the rapid detection of AIV H5N1 in whole blood samples. Using PCR tubes coated with an H5 subtype monoclonal antibody, AIV H5N1 virions were specifically captured from blood samples. After a thermal lysis step, the released viral N1 gene was exponentially amplified using RT-LAMP on either a real-time PCR instrument for quantitative analysis, or in a water bath system for endpoint analysis. The detection limit of the newly developed immuno-RT-LAMP assay was as low as 1.62×10(1) 50% embryo infectious dose/mL of virus in both regular samples and simulated viremia samples. There were no cross-reactions with non-H5N1 influenza viruses or other avian viruses. The reproducibility of the assay was confirmed using intra- and inter-assay tests with variability ranging from 1.05% to 3.37%. Our results indicate that immuno-RT-LAMP is a novel, effective point-of-care virus identification solution for the rapid diagnosis and monitoring of AIV H5N1 in blood samples. PMID:27376196

  13. A duck enteritis virus-vectored bivalent live vaccine provides fast and complete protection against H5N1 avian influenza virus infection in ducks.

    PubMed

    Liu, Jinxiong; Chen, Pucheng; Jiang, Yongping; Wu, Li; Zeng, Xianying; Tian, Guobin; Ge, Jinying; Kawaoka, Yoshihiro; Bu, Zhigao; Chen, Hualan

    2011-11-01

    Ducks play an important role in the maintenance of highly pathogenic H5N1 avian influenza viruses (AIVs) in nature, and the successful control of AIVs in ducks has important implications for the eradication of the disease in poultry and its prevention in humans. The inactivated influenza vaccine is expensive, labor-intensive, and usually needs 2 to 3 weeks to induce protective immunity in ducks. Live attenuated duck enteritis virus (DEV; a herpesvirus) vaccine is used routinely to control lethal DEV infections in many duck-producing areas. Here, we first established a system to generate the DEV vaccine strain by using the transfection of overlapping fosmid DNAs. Using this system, we constructed two recombinant viruses, rDEV-ul41HA and rDEV-us78HA, in which the hemagglutinin (HA) gene of the H5N1 virus A/duck/Anhui/1/06 was inserted and stably maintained within the ul41 gene or between the us7 and us8 genes of the DEV genome. Duck studies indicated that rDEV-us78HA had protective efficacy similar to that of the live DEV vaccine against lethal DEV challenge; importantly, a single dose of 10(6) PFU of rDEV-us78HA induced complete protection against a lethal H5N1 virus challenge in as little as 3 days postvaccination. The protective efficacy against both lethal DEV and H5N1 challenge provided by rDEV-ul41HA inoculation in ducks was slightly weaker than that provided by rDEV-us78HA. These results demonstrate, for the first time, that recombinant DEV is suitable for use as a bivalent live attenuated vaccine, providing rapid protection against both DEV and H5N1 virus infection in ducks.

  14. Evolutionary trajectories and diagnostic challenges of potentially zoonotic avian influenza viruses H5N1 and H9N2 co-circulating in Egypt.

    PubMed

    Naguib, Mahmoud M; Arafa, Abdel-Satar A; El-Kady, Magdy F; Selim, Abdullah A; Gunalan, Vithiagaran; Maurer-Stroh, Sebastian; Goller, Katja V; Hassan, Mohamed K; Beer, Martin; Abdelwhab, E M; Harder, Timm C

    2015-08-01

    In Egypt, since 2006, descendants of the highly pathogenic avian influenza virus (HP AIV) H5N1 of clade 2.2 continue to cause sharp losses in poultry production and seriously threaten public health. Potentially zoonotic H9N2 viruses established an endemic status in poultry in Egypt as well and co-circulate with HP AIV H5N1 rising concerns of reassortments between H9N2 and H5N1 viruses along with an increase of mixed infections of poultry. Nucleotide sequences of whole genomes of 15 different isolates (H5N1: 7; H9N2: 8), and of the hemagglutinin (HA) and neuraminidase (NA) encoding segments of nine further clinical samples (H5N1: 2; H9N2: 7) from 2013 and 2014 were generated and analysed. The HA of H5N1 viruses clustered with clade 2.2.1 while the H9 HA formed three distinguishable subgroups within cluster B viruses. BEAST analysis revealed that H9N2 viruses are likely present in Egypt since 2009. Several previously undescribed substituting mutations putatively associated with host tropism and virulence modulation were detected in different proteins of the analysed H9N2 and H5N1 viruses. Reassortment between HP AIV H5N1 and H9N2 is anticipated in Egypt, and timely detection of such events is of public health concern. As a rapid tool for detection of such reassortants discriminative SYBR-Green reverse transcription real-time PCR assays (SG-RT-qPCR), targeting the internal genes of the Egyptian H5N1 and H9N2 viruses were developed for the rapid screening of viral RNAs from both virus isolates and clinical samples. However, in accordance to Sanger sequencing, no reassortants were found by SG-RT-qPCR. Nevertheless, the complex epidemiology of avian influenza in poultry in Egypt will require sustained close observation. Further development and continuing adaptation of rapid and cost-effective screening assays such as the SG-RT-qPCR protocol developed here are at the basis of efforts for improvement the currently critical situation.

  15. Glycine at Position 622 in PB1 Contributes to the Virulence of H5N1 Avian Influenza Virus in Mice

    PubMed Central

    Feng, Xiaoxiao; Wang, Zeng; Shi, Jianzhong; Deng, Guohua; Kong, Huihui; Tao, Shiyu; Li, Changyao; Liu, Liling; Guan, Yuntao

    2015-01-01

    ABSTRACT We isolated two H5N1 viruses, A/duck/Hunan/S4020/2008 (DK/08) and A/chicken/Guangxi/S2039/2009 (CK/09), from live-bird markets during routine surveillance and found that these two viruses are genetically similar but differ in their replication and virulence in mice. The CK/09 virus is lethal for mice with a 50% mouse lethal dose (MLD50) of 1.6 log10 50% egg infectious doses (EID50), whereas the DK/08 virus is nonpathogenic for mice with an MLD50 value of 6.2 log10 EID50. We explored the genetic basis of the virulence difference of these two viruses by generating a series of reassortant viruses and mutants in the lethal virus CK/09 background and evaluating their virulence in mice. We found that the PB1 gene of the DK/08 virus dramatically attenuated the virulence of the CK/09 virus and that the amino acid at position 622 in PB1 made an important contribution. We further demonstrated that the mutation of glycine (G) to aspartic acid (D) at position 622 in PB1 partially impaired the binding of PB1 to viral RNA, thereby dramatically decreasing the polymerase activity and attenuating H5N1 virus virulence in mice. Our results identify a novel virulence-related marker of H5N1 influenza viruses and provide a new target for live attenuated vaccine development. IMPORTANCE H5N1 avian influenza viruses have caused the deaths of nearly 60% of the humans that they have infected since 1997 and clearly represent a threat to public health. A thorough understanding of the genetic basis of virulence determinants will provide important insights for antiviral drug and live attenuated vaccine development. Several virulence-related markers in the PB2, PA, M1, and NS1 proteins of H5N1 viruses have been identified. In this study, we isolated two H5N1 avian influenza viruses that are genetically similar but differ in their virulence in mice, and we identified a new virulence-related marker in the PB1 gene. We found that the mutation of glycine (G) to aspartic acid (D) at position

  16. Assessment of viral replication in eggs and HA protein yield of pre-pandemic H5N1 candidate vaccine viruses.

    PubMed

    Jing, Xianghong; Soto, Jackeline; Gao, Yamei; Phy, Kathryn; Ye, Zhiping

    2013-08-28

    H5N1 infection and the potential for spread from human to human continue to pose a severe public health concern. Since vaccination remains the most effective way to prevent a potential H5N1 pandemic, the World Health Organization (WHO) Collaborating Centers (CCs) and Essential Regulatory Laboratories (ERLs) engineered and developed a panel of H5N1 pre-pandemic vaccine viruses for pandemic vaccine preparedness as well as production of antigen potency testing reagents (reference antigen and reference anti-serum) for vaccine standardization. To develop a strategy utilizing a number of biochemical methods for the characterization of the viral growth properties and protein yield in eggs, we have selected eight H5N1 pre-pandemic viruses and determined the viral Egg Infectious Dose 50 (EID50), total protein yield, hemagglutinin (HA) to nucleoprotein (NP) ratios (HA:NP), and HA1 content of each virus. Our results showed that all the tested H5N1 vaccine viruses grew to high titers in eggs. The total viral protein yield varies within a narrow range, whereas there were greater differences in the HA:NP protein ratios among the eight viruses. The RP-HPLC based HA1 content analysis demonstrated that the viruses A/Anhui/1/2010, A/Hubei/1/2005, and A/goose/Guiyang/337/2006 contained higher HA contents than other five viruses including A/Vietnam/1203/2003. Our approach for analyzing virus growth and protein yield will allow us identify optimal vaccine virus in a timely manner. In addition, we successfully purified the HA proteins of H5N1 vaccine viruses by optimizing bromelain cleavage conditions. Our studies on the HA protein purification may improve the quality control of the production of influenza vaccine test reagent.

  17. Different routes of inoculation impact infectivity and pathogenesis of H5N1 high pathogenicity avian influenza virus infection in chickens and domestic ducks

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The H5N1 type A influenza viruses classified as Qinghai-like virus (clade 2.2) are a unique lineage of type A influenza viruses with the capacity to produce significant disease and mortality in gallinaceous birds and water fowl including ducks. The objective of this study was to determine the suscep...

  18. Mouse lung-adapted mutation of E190G in hemagglutinin from H5N1 influenza virus contributes to attenuation in mice.

    PubMed

    Han, Pengfei; Hu, Yi; Sun, Wei; Zhang, Sen; Li, Yuchang; Wu, Xiaoyan; Yang, Yinhui; Zhu, Qingyu; Jiang, Tao; Li, Jing; Qin, Chengfeng

    2015-11-01

    The highly pathogenic H5N1 avian influenza virus is one of the greatest influenza pandemic threats since 2003. The association of the receptor binding domain (RBD) with the virulence of influenza virus is rarely addressed, particularly of H5N1 influenza viruses. In this study, BALB/c mice were intranasally infected with A/Vietnam/1194/2004 (VN1194, H5N1). The mouse lung-adapted variants were isolated and the mutation of E190G (H3 numbering) in the RBD was recognized. The recombinant virus, rVN-E190G carrying E190G in hemagglutinin (HA) was designed and rescued using reverse genetics techniques. The receptor binding activity, growth curve and pathogenicity in mice of the rVN-E190G were investigated. Results demonstrated that rVN-E190G virus increased the binding avidity to α2,6 SA (sialic acid) and reduced the affinity to α2,3 SA, meanwhile weakened the viral replication in vitro. Moreover, the virulence assessment demonstrated that rVN-E190G was attenuated in mice. These results indicated that the mutation E190G in HA decreases H5N1 viral replication in vitro and significantly attenuates virulence in vivo. These findings identify one of the determinants in RBD which can be associated with H5N1 virulence in mice.

  19. Isolation and identification of highly pathogenic avian influenza H5N1 virus from Houbara bustards (Chlamydotis undulata macqueenii) and contact falcons.

    PubMed

    Khan, Owais Ahmed; Shuaib, Mohammad Adam; Rhman, Salah Shaban Abdel; Ismail, Mahmoud Moussa; Hammad, Yousef Al; Baky, Mansour Hashim Abdel; Fusaro, Alice; Salviato, Annalisa; Cattoli, Giovanni

    2009-02-01

    Highly pathogenic influenza virus (HPAIV) H5N1 has caused mortality and morbidity in many species of domestic and wild bird. The Houbara bustard (Chlamydotis undulata macqueenii) is a solitary bird that inhabits semi-desert regions. It is known to be susceptible to avianpox, avian paramyxovirus type 1, and low-pathogenicity avian influenza H9N2. We report an outbreak of H5N1 HPAIV in Houbara bustards, which were introduced into the Kingdom of Saudi Arabia for falconry purposes. Ninety-three per cent mortality (38 out of 41 birds) in the infected Houbara bustard flock and about 62.5% mortality (10 out of 16 birds) in falcons that came in contact with these birds were observed. Pooled cloacal and tracheal swabs from Houbara bustards as well as visceral organ homogenates collected in Houbara bustards and falcons were tested by real-time reverse transcriptase-polymerase chain reaction, and virus isolation was attempted in specific pathogen free hens' eggs. The viruses isolated were characterized as HPAIV H5N1. Phylogenetic analysis of the haemagglutinating and Neuraminidase (NA) genes revealed that the viruses isolated from Houbara bustards and falcons were closely related to each other and to Kuwaiti H5N1 strains isolated in 2007. Interestingly, they were genetically distinguishable from the co-circulating A/H5N1 viruses in Kingdom of Saudi Arabia causing outbreaks in domestic birds. This case emphasizes the need for surveillance of this endangered species in its natural habitat.

  20. Protection against H5N1 Influenza Virus Induced by Matrix-M Adjuvanted Seasonal Virosomal Vaccine in Mice Requires Both Antibodies and T Cells

    PubMed Central

    Cox, Freek; Baart, Matthijs; Huizingh, Jeroen; Tolboom, Jeroen; Dekking, Liesbeth; Goudsmit, Jaap; Saeland, Eirikur; Radošević, Katarina

    2015-01-01

    Background It remains important to develop the next generation of influenza vaccines that can provide protection against vaccine mismatched strains and to be prepared for potential pandemic outbreaks. To achieve this, the understanding of the immunological parameters that mediate such broad protection is crucial. Method In the current study we assessed the contribution of humoral and cellular immune responses to heterosubtypic protection against H5N1 induced by a Matrix-M (MM) adjuvanted seasonal influenza vaccine by serum transfer and T-cell depletion studies. Results We demonstrate that the heterosubtypic protection against H5N1 induced by MM adjuvanted vaccine is partially mediated by antibodies. The serum contained both H5N1 cross-reactive hemagglutinin (HA)- and neuraminidase (NA)-specific antibodies but with limited virus neutralizing and no hemagglutination inhibiting activity. The cross-reactive antibodies induced antibody-dependent cellular cytotoxicity (ADCC) in vitro, suggesting a role for the Fc part of the antibodies in protection against H5N1. Besides H5N1 specific antibody responses, cross-reactive HA- and NA-specific T-cell responses were induced by the adjuvanted vaccine. T-cell depletion experiments demonstrated that both CD4+ and CD8+ T cells contribute to protection. Conclusion Our study demonstrates that cross-protection against H5N1 induced by MM adjuvanted seasonal virosomal influenza vaccine requires both the humoral and cellular arm of the immune system. PMID:26696245

  1. Differences in the Epidemiology of Human Cases of Avian Influenza A(H7N9) and A(H5N1) Viruses Infection

    PubMed Central

    Qin, Ying; Horby, Peter W.; Tsang, Tim K.; Chen, Enfu; Gao, Lidong; Ou, Jianming; Nguyen, Tran Hien; Duong, Tran Nhu; Gasimov, Viktor; Feng, Luzhao; Wu, Peng; Jiang, Hui; Ren, Xiang; Peng, Zhibin; Li, Sa; Li, Ming; Zheng, Jiandong; Liu, Shelan; Hu, Shixiong; Hong, Rongtao; Farrar, Jeremy J.; Leung, Gabriel M.; Gao, George F.; Cowling, Benjamin J.; Yu, Hongjie

    2015-01-01

    Background. The pandemic potential of avian influenza viruses A(H5N1) and A(H7N9) remains an unresolved but critically important question. Methods. We compared the characteristics of sporadic and clustered cases of human H5N1 and H7N9 infection, estimated the relative risk of infection in blood-related contacts, and the reproduction number (R). Results. We assembled and analyzed data on 720 H5N1 cases and 460 H7N9 cases up to 2 November 2014. The severity and average age of sporadic/index cases of H7N9 was greater than secondary cases (71% requiring intensive care unit admission vs 33%, P = .007; median age 59 years vs 31, P < .001). We observed no significant differences in the age and severity between sporadic/index and secondary H5N1 cases. The upper limit of the 95% confidence interval (CI) for R was 0.12 for H5N1 and 0.27 for H7N9. A higher proportion of H5N1 infections occurred in clusters (20%) compared to H7N9 (8%). The relative risk of infection in blood-related contacts of cases compared to unrelated contacts was 8.96 for H5N1 (95% CI, 1.30, 61.86) and 0.80 for H7N9 (95% CI, .32, 1.97). Conclusions. The results are consistent with an ascertainment bias towards severe and older cases for sporadic H7N9 but not for H5N1. The lack of evidence for ascertainment bias in sporadic H5N1 cases, the more pronounced clustering of cases, and the higher risk of infection in blood-related contacts, support the hypothesis that susceptibility to H5N1 may be limited and familial. This analysis suggests the potential pandemic risk may be greater for H7N9 than H5N1. PMID:25940354

  2. Highly Pathogenic Avian Influenza Virus Subtype H5N1 in Africa: A Comprehensive Phylogenetic Analysis and Molecular Characterization of Isolates

    PubMed Central

    Cattoli, Giovanni; Monne, Isabella; Fusaro, Alice; Joannis, Tony M.; Lombin, Lami H.; Aly, Mona M.; Arafa, Abdel S.; Sturm-Ramirez, Katharine M.; Couacy-Hymann, Emmanuel; Awuni, Joseph A.; Batawui, Komla B.; Awoume, Kodzo A.; Aplogan, Gilbert L.; Sow, Adama; Ngangnou, Andrè C.; El Nasri Hamza, Iman M.; Gamatié, Djibo; Dauphin, Gwenaelle; Domenech, Joseph M.; Capua, Ilaria

    2009-01-01

    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. PMID:19290041

  3. PB1-F2 Attenuates Virulence of Highly Pathogenic Avian H5N1 Influenza Virus in Chickens

    PubMed Central

    Leymarie, Olivier; Embury-Hyatt, Carissa; Chevalier, Christophe; Jouneau, Luc; Moroldo, Marco; Da Costa, Bruno; Berhane, Yohannes; Delmas, Bernard

    2014-01-01

    Highly pathogenic avian influenza virus (HPAIV) is a permanent threat due to its capacity to cross species barriers and generate severe infections and high mortality in humans. Recent findings have highlighted the potential role of PB1-F2, a small accessory influenza protein, in the pathogenesis process mediated by HPAIV in mammals. In this study, using a recombinant H5N1 HPAIV (wt) and its PB1-F2-deleted mutant (ΔF2), we studied the effects of PB1-F2 in a chicken model. Unexpectedly, when using low inoculation dose we observed that the wt-infected chickens had a higher survival rate than the ΔF2-infected chickens, a feature that contrasts with what is usually observed in mammals. High inoculation dose had similar mortality rate for both viruses, and comparison of the bio-distribution of the two viruses indicated that the expression of PB1-F2 allows a better spreading of the virus within chicken embryos. Transcriptomic profiles of lungs and blood cells were characterized at two days post-infection in chickens inoculated with the wild type (wt) or the ΔF2 mutant viruses. In lungs, the expression of PB1-F2 during the infection induced pathways related to calcium signaling and repressed a large panel of immunological functions. In blood cells, PB1-F2 was associated with a gene signature specific for mitochondrial dysfunction and down-modulated leucocytes activation. Finally we compared the effect of PB1-F2 in lungs of chickens and mice. We identified that gene signature associated to tissue damages is a PB1-F2 feature shared by the two species; by contrast, the early inhibition of immune response mediated by PB1-F2 observed in chickens is not seen in mice. In summary, our data suggest that PB1-F2 expression deeply affect the immune response in chickens in a way that may attenuate pathogenicity at low infection dose, a feature differing from what was previously observed in mammal species. PMID:24959667

  4. Modified H5 real-time reverse transcriptase-PCR oligonucleotides for detection of divergent avian influenza H5N1 viruses in Egypt.

    PubMed

    Abdelwhab, E M; Abdelwhab, El-Sayed M; Arafa, Abdel-Satar; Erfan, Ahmed M; Aly, Mona M; Hafez, Hafez M

    2010-12-01

    The efforts exerted to prevent circulation of highly pathogenic avian influenza (HPAI) H5N1 virus in birds are the best way to prevent the emergence of a new virus subtype with pandemic potential. Despite the blanket vaccination strategy against HPAI H5N1 in Egypt, continuous circulation of the virus in poultry has increased since late 2007 as a result of the presence of genetic and antigenic distinct variant strains that have escaped during the immune response of vaccinated birds. Although the suspected poultry flocks have had signs and lesions commonly seen in HPAI H5N1-infected birds, escape of variant strains from detection by real-time reverse transcriptase-PCR (RRT-PCR) was observed. Sequence analysis of these variants revealed multiple single nucleotide substitutions in the primers and probe target sequences of the H5 gene by real-time RT-PCR. This study describes the results of RRT-PCR, modified from an existing protocol with regard to the detection of the partial H5 gene segment of the Egyptian H5N1 divergent viruses and applied to nationwide surveillance. The modified RRT-PCR assay was more sensitive than the original one in the detection of Egyptian isolates, with 104% amplification efficiency. Sixty-one field samples were found to be positive in our assay, but only 51 samples tested positive by the original protocol and were more sensitive than matrix gene RRT-PCR detection assay. A detection limit of 10 mean embryo infective dose (EID50) with the updated oligonucleotides primers and probe set was found. For the foreseeable future, mutation of H5N1 viruses and the endemic situation in developing countries require continuous improvement of current diagnostics to aid in the containment of the H5N1 virus in poultry sectors and to lower the threat of influenza virus spread. PMID:21313854

  5. Experimentally Infected Domestic Ducks Show Efficient Transmission of Indonesian H5N1 Highly Pathogenic Avian Influenza Virus, but Lack Persistent Viral Shedding

    PubMed Central

    Wibawa, Hendra; Bingham, John; Nuradji, Harimurti; Lowther, Sue; Payne, Jean; Harper, Jenni; Junaidi, Akhmad; Middleton, Deborah; Meers, Joanne

    2014-01-01

    Ducks are important maintenance hosts for avian influenza, including H5N1 highly pathogenic avian influenza viruses. A previous study indicated that persistence of H5N1 viruses in ducks after the development of humoral immunity may drive viral evolution following immune selection. As H5N1 HPAI is endemic in Indonesia, this mechanism may be important in understanding H5N1 evolution in that region. To determine the capability of domestic ducks to maintain prolonged shedding of Indonesian clade 2.1 H5N1 virus, two groups of Pekin ducks were inoculated through the eyes, nostrils and oropharynx and viral shedding and transmission investigated. Inoculated ducks (n = 15), which were mostly asymptomatic, shed infectious virus from the oral route from 1 to 8 days post inoculation, and from the cloacal route from 2–8 dpi. Viral ribonucleic acid was detected from 1–15 days post inoculation from the oral route and 1–24 days post inoculation from the cloacal route (cycle threshold <40). Most ducks seroconverted in a range of serological tests by 15 days post inoculation. Virus was efficiently transmitted during acute infection (5 inoculation-infected to all 5 contact ducks). However, no evidence for transmission, as determined by seroconversion and viral shedding, was found between an inoculation-infected group (n = 10) and contact ducks (n = 9) when the two groups only had contact after 10 days post inoculation. Clinical disease was more frequent and more severe in contact-infected (2 of 5) than inoculation-infected ducks (1 of 15). We conclude that Indonesian clade 2.1 H5N1 highly pathogenic avian influenza virus does not persist in individual ducks after acute infection. PMID:24392085

  6. Experimentally infected domestic ducks show efficient transmission of Indonesian H5N1 highly pathogenic avian influenza virus, but lack persistent viral shedding.

    PubMed

    Wibawa, Hendra; Bingham, John; Nuradji, Harimurti; Lowther, Sue; Payne, Jean; Harper, Jenni; Junaidi, Akhmad; Middleton, Deborah; Meers, Joanne

    2014-01-01

    Ducks are important maintenance hosts for avian influenza, including H5N1 highly pathogenic avian influenza viruses. A previous study indicated that persistence of H5N1 viruses in ducks after the development of humoral immunity may drive viral evolution following immune selection. As H5N1 HPAI is endemic in Indonesia, this mechanism may be important in understanding H5N1 evolution in that region. To determine the capability of domestic ducks to maintain prolonged shedding of Indonesian clade 2.1 H5N1 virus, two groups of Pekin ducks were inoculated through the eyes, nostrils and oropharynx and viral shedding and transmission investigated. Inoculated ducks (n = 15), which were mostly asymptomatic, shed infectious virus from the oral route from 1 to 8 days post inoculation, and from the cloacal route from 2-8 dpi. Viral ribonucleic acid was detected from 1-15 days post inoculation from the oral route and 1-24 days post inoculation from the cloacal route (cycle threshold <40). Most ducks seroconverted in a range of serological tests by 15 days post inoculation. Virus was efficiently transmitted during acute infection (5 inoculation-infected to all 5 contact ducks). However, no evidence for transmission, as determined by seroconversion and viral shedding, was found between an inoculation-infected group (n = 10) and contact ducks (n = 9) when the two groups only had contact after 10 days post inoculation. Clinical disease was more frequent and more severe in contact-infected (2 of 5) than inoculation-infected ducks (1 of 15). We conclude that Indonesian clade 2.1 H5N1 highly pathogenic avian influenza virus does not persist in individual ducks after acute infection.

  7. Differences in the Epidemiology of Childhood Infections with Avian Influenza A H7N9 and H5N1 Viruses

    PubMed Central

    Chen, Xiaowen; Zhao, Na; Luo, Mengyun; Dong, Yuanyuan

    2016-01-01

    The difference between childhood infections with avian influenza viruses A(H5N1) and A(H7N9) remains an unresolved but critically important question. We compared the epidemiological characteristics of 244 H5N1 and 41 H7N9 childhood cases (<15 years old), as well as the childhood cluster cases of the two viruses. Our findings revealed a higher proportion of H5N1 than H7N9 childhood infections (31.1% vs. 6.4%, p = 0.000). However, the two groups did not differ significantly in age (median age: 5.0 vs. 5.5 y, p = 0.0651). The proportion of clustered cases was significantly greater among children infected with H5N1 than among children infected with H7N9 [46.7% (71/152) vs. 23.6% (13/55), p = 0.005], and most of the childhood cases were identified as secondary cases [46.4% (45/97) vs. 33.3% (10/30), p = 0.000]. Mild status accounted for 79.49% and 22.66%, severe status for 17.95% and 2.34%, and fatal cases for 2.56% and 75.00% of the H7N9 and H5N1 childhood infection cases (all p<0.05), respectively. The fatality rates for the total, index and secondary childhood cluster cases were 52.86% (37/70), 88.5% (23/26) and 33.33% (15/45), respectively, in the H5N1 group, whereas no fatal H7N9 childhood cluster cases were identified. In conclusion, lower severity and greater transmission were found in the H7N9 childhood cases than in the H5N1 childhood cases. PMID:27695069

  8. Optimal attenuation of a PR8-derived mouse pathogenic H5N1 recombinant virus for testing antigenicity and protective efficacy in mice.

    PubMed

    Kim, Il-Hwan; Kwon, Hyuk-Joon; Park, Jae-Keun; Song, Chang-Seon; Kim, Jae-Hong

    2015-11-17

    The PR8-based reverse genetics vector system is widely used to generate commercial vaccine strains, but the pathogenicity of PR8-derived recombinant viruses in mice hinders further immunological studies. In the present study, we generated PR8-derived H5N1 recombinant viruses, in which haemagglutinin (HA) and neuraminidase (NA) originated from a mouse-pathogenic H5N1 low pathogenic avian influenza virus (LPAIV), and the non-structural proteins (NS) and polymerase basic protein 2 (PB2) originated from different H9N2 LPAIVs. In contrast to the control H5N1 recombinant virus, harboring six internal genes from PR8, the NS and PB2 recombinant viruses did not cause body weight loss in mice. However, the NS recombinant virus replicated in the lungs of mice. It was more immunogenic than the PB2 recombinant virus to protect efficiently against a lethal challenge of a H5N1 highly pathogenic AIV with 89 and 88% amino acid identity in HA and NA, respectively. Therefore, the NS gene may be useful for generating nonpathogenic and immunogenic PR8-derived recombinant viruses for studies of antigenicity and protective efficacy in mice.

  9. Variation in protection by seven inactivated H5 vaccine strains against eight H5N1 high pathogenicity avian influenza viruses in chickens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    H5N1 high pathogenicity avian influenza virus (HPAIV) is an important pathogen for poultry. Vaccines have assisted in control for poultry, and for human pandemic preparedness. However the genetic diversity and rapid antigenic drifting of the field viruses have led to inadequate protection. This s...

  10. Immunization with live nonpathogenic H5N3 duck influenza virus protects chickens against highly pathogenic H5N1 virus.

    PubMed

    Gambaryan, A S; Boravleva, E Y; Lomakina, N F; Kropotkina, E A; Gordeychuk, I V; Chvala, I A; Drygin, V V; Klenk, H-D; Matrosovich, M N

    2016-01-01

    Development of an effective, broadly-active and safe vaccine for protection of poultry from H5N1 highly pathogenic avian influenza viruses (HPAIVs) remains an important practical goal. In this study we used a low pathogenic wild aquatic bird virus isolate А/duck/Moscow/4182/2010 (H5N3) (dk/4182) as a live candidate vaccine. We compared this virus with four live 1:7 reassortant anti-H5N1 candidate vaccine viruses with modified hemagglutinin from either A/Vietnam/1203/04 (H5N1) or A/Kurgan/3/05 (H5N1) and the rest of the genes from either H2N2 cold-adapted master strain A/Leningrad/134/17/57 (rVN-Len and rKu-Len) or H6N2 virus A/gull/Moscow/3100/2006 (rVN-gull and rKu-gull). The viruses were tested in parallel for pathogenicity, immunogenicity and protective effectiveness in chickens using aerosol, intranasal and oral routes of immunization. All five viruses showed zero pathogenicity indexes in chickens. Viruses rVN-gull and rKu-gull were immunogenic and protective, but they were insufficiently attenuated and caused significant mortality of 1-day-old chickens. The viruses with cold-adapted backbones (rVN-Len and rKu-Len) were completely nonpathogenic, but they were significantly less immunogenic and provided lower protection against lethal challenge with HPAIV A/Chicken/Kurgan/3/05 (H5N1) as compared with three other vaccine candidates. Unlike other four viruses, dk/4182 was both safe and highly immunogenic in chickens of any age regardless of inoculation route. Single administration of 106 TCID50 of dk/4182 virus via drinking water provided complete protection of 30-days-old chickens from 100 LD50 of the challenge virus. Our results suggest that low pathogenic viruses of wild aquatic birds can be used as safe and effective live poultry vaccines against highly pathogenic avian viruses. PMID:27640442

  11. Growth and Pathogenic Potential of Naturally Selected Reassortants after Coinfection with Pandemic H1N1 and Highly Pathogenic Avian Influenza H5N1 Viruses

    PubMed Central

    Song, Min-Suk; Baek, Yun Hee; Pascua, Philippe Noriel Q.; Kwon, Hyeok-il; Kim, Eun-Ha; Park, Su-Jin; Kim, Se Mi; Kim, Young-Il; Choi, Won-Suk; Kim, Eung-Gook; Kim, Chul-Joong

    2015-01-01

    Coinfection of ferrets with H5N1 and pH1N1 viruses resulted in two predominate genotypes in the lungs containing surface genes of highly pathogenic avian influenza H5N1 virus in the backbone of pandemic H1N1 2009 (pH1N1). Compared to parental strains, these reassortants exhibited increased growth and virulence in vitro and in mice but failed to be transmitted indirectly to naive contact ferrets. Thus, this demonstrates a possible natural reassortment following coinfection as well as the pathogenicity of the potential reassortants. PMID:26491154

  12. Growth and Pathogenic Potential of Naturally Selected Reassortants after Coinfection with Pandemic H1N1 and Highly Pathogenic Avian Influenza H5N1 Viruses.

    PubMed

    Song, Min-Suk; Baek, Yun Hee; Pascua, Philippe Noriel Q; Kwon, Hyeok-Il; Kim, Eun-Ha; Park, Su-Jin; Kim, Se Mi; Kim, Young-Il; Choi, Won-Suk; Kim, Eung-Gook; Kim, Chul-Joong; Choi, Young Ki

    2016-01-01

    Coinfection of ferrets with H5N1 and pH1N1 viruses resulted in two predominate genotypes in the lungs containing surface genes of highly pathogenic avian influenza H5N1 virus in the backbone of pandemic H1N1 2009 (pH1N1). Compared to parental strains, these reassortants exhibited increased growth and virulence in vitro and in mice but failed to be transmitted indirectly to naive contact ferrets. Thus, this demonstrates a possible natural reassortment following coinfection as well as the pathogenicity of the potential reassortants. PMID:26491154

  13. Detection of HPAI H5N1 viruses in ducks sampled from live bird markets in Vietnam.

    PubMed

    Phan, M Q; Henry, W; Bui, C B; DO, D H; Hoang, N V; Thu, N T; Nguyen, T T; LE, T D; Diep, T Q; Inui, K; Weaver, J; Carrique-Mas, J

    2013-03-01

    In Vietnam, highly pathogenic avian influenza (HPAI) H5N1 infections in poultry often occur without concomitant clinical signs and outbreaks are not consistently reported. Live bird markets represent a convenient site for surveillance that does not rely on farmers' notifications. Two H5N1 surveys were conducted at live bird markets/slaughter points in 39 districts (five provinces) in the Red River, Mekong delta, and central Vietnam during January and May 2011. Oropharyngeal and rectal swab samples from 12 480 ducks were tested for H5N1 by reverse transcription-polymerase chain reaction in pools of five. Traders and stallholders were interviewed using standardized questionnaires; 3·3% of pools tested positive. The highest prevalence (6·6%) corresponded to the Mekong delta, and no H5N1 was detected in the two Red River provinces. The surveys identified key risk behaviours of traders and stallholders. It is recommended that market surveys are implemented over time as a tool to evaluate progress in HPAI control in Vietnam. PMID:22651930

  14. Experimental challenge and pathology of highly pathogenic avian influenza virus H5N1 in dunlin (Calidris alpina), an intercontinental migrant shorebird species

    USGS Publications Warehouse

    Hall, J.S.; Franson, J.C.; Gill, R.E.; Meteyer, C.U.; Teslaa, J.L.; Nashold, S.; Dusek, R.J.; Ip, H.S.

    2011-01-01

    Background Shorebirds (Charadriiformes) are considered one of the primary reservoirs of avian influenza. Because these species are highly migratory, there is concern that infected shorebirds may be a mechanism by which highly pathogenic avian influenza virus (HPAIV) H5N1 could be introduced into North America from Asia. Large numbers of dunlin (Calidris alpina) migrate from wintering areas in central and eastern Asia, where HPAIV H5N1 is endemic, across the Bering Sea to breeding areas in Alaska. Low pathogenic avian influenza virus has been previously detected in dunlin, and thus, dunlin represent a potential risk to transport HPAIV to North America. To date no experimental challenge studies have been performed in shorebirds. Methods Wild dunlin were inoculated intranasally and intrachoanally various doses of HPAIV H5N1. The birds were monitored daily for virus excretion, disease signs, morbidity, and mortality. Results The infectious dose of HPAIV H5N1 in dunlin was determined to be 101.7 EID50/100 ??l and that the lethal dose was 101.83 EID50/100 ??l. Clinical signs were consistent with neurotropic disease, and histochemical analyses revealed that infection was systemic with viral antigen and RNA most consistently found in brain tissues. Infected birds excreted relatively large amounts of virus orally (104 EID50) and smaller amounts cloacally. Conclusions Dunlin are highly susceptible to infection with HPAIV H5N1. They become infected after exposure to relatively small doses of the virus and if they become infected, they are most likely to suffer mortality within 3-5days. These results have important implications regarding the risks of transport and transmission of HPAIV H5N1 to North America by this species and raises questions for further investigation. Published 2011. This article is a US Government work and is in the public domain in the USA.

  15. Experimental infection of a North American raptor, American kestrel (Falco sparverius), with highly pathogenic avian influenza virus (H5N1)

    USGS Publications Warehouse

    Hall, J.S.; Ip, H.S.; Franson, J.C.; Meteyer, C.; Nashold, S.; Teslaa, J.L.; French, J.; Redig, P.; Brand, C.

    2009-01-01

    Several species of wild raptors have been found in Eurasia infected with highly pathogenic avian influenza virus (HPAIV) subtype H5N1. Should HPAIV (H5N1) reach North America in migratory birds, species of raptors are at risk not only from environmental exposure, but also from consuming infected birds and carcasses. In this study we used American kestrels as a representative species of a North American raptor to examine the effects of HPAIV (H5N1) infection in terms of dose response, viral shedding, pathology, and survival. Our data showed that kestrels are highly susceptible to HPAIV (H5N1). All birds typically died or were euthanized due to severe neurologic disease within 4-5 days of inoculation and shed significant amounts of virus both orally and cloacally, regardless of dose administered. The most consistent microscopic lesions were necrosis in the brain and pancreas. This is the first experimental study of HPAIV infection in a North American raptor and highlights the potential risks to birds of prey if HPAIV (H5N1) is introduced into North America.

  16. A novel neutralizing antibody against diverse clades of H5N1 influenza virus and its mutants capable of airborne transmission.

    PubMed

    Wu, Ruiping; Li, Xingxing; Leung, Ho-Chuen; Cao, Zhiliang; Qiu, Zonglin; Zhou, Yusen; Zheng, Bo-Jian; He, Yuxian

    2014-06-01

    Highly pathogenic avian influenza A virus H5N1 continues to spread among poultry and has frequently broken the species barrier to humans. Recent studies have shown that a laboratory-mutated or reassortant H5N1 virus bearing hemagglutinin (HA) with as few as four or five mutations was capable of transmitting more efficiently via respiratory droplets between ferrets, posing a serious threat to public health and underscoring the priority of effective vaccines and therapeutics. In this study, we identified a novel monoclonal antibody (mAb) named HAb21, that has a broadly neutralizing activity against all tested strains of H5N1 covering clades 0, 1, 2.2, 2.3.4, and 2.3.2.1. Importantly, HAb21 efficiently neutralized diverse H5N1 variants with single or combination forms of mutations capable of airborne transmission. We demonstrated that HAb21 blocked viral entry during the receptor-binding step by targeting a previously uncharacterized epitope at the tip of the HA head. This novel epitope closely neighbors the receptor-binding site (RBS) and the interface of HA trimer and is highly conserved among divergent H5N1 strains. Our studies provide a new tool for use either for therapeutic purposes or as a basis of vaccine development. PMID:24681124

  17. Evaluation of a conserved HA274-288 epitope to detect antibodies to highly pathogenic avian influenza virus H5N1 in Indonesian commercial poultry.

    PubMed

    Wawegama, Nadeeka K; Tarigan, Simson; Indriani, Risa; Selleck, Paul; Adjid, Rm Abdul; Syafriati, Tati; Hardiman; Durr, Peter A; Ignjatovic, Jagoda

    2016-08-01

    A peptide enzyme linked immunosorbent assay (ELISA) based on an epitope in the haemagglutinin (HA) of avian influenza virus H5N1, amino acid positions 274-288 (HA274-288) was evaluated for detection of H5N1-specific antibodies. An optimized ELISA based on the tetrameric form of the HA274-288 epitope designated MP15 gave low background with non-immune chicken sera and detected vaccinated and infected birds. The HA274-288 epitope was highly conserved in Indonesian H5N1 strains and antibody responses were detected in the majority of the vaccinated chickens regardless of the H5N1 strain used for vaccination. The HA274-288 epitope was also conserved in the majority of H5N1 strains from the neighbouring Asian region, and other H5 subtypes potentially allowing for a wider use of the MP15 ELISA in H5N1 vaccinated and infected flocks. The MP15 ELISA results correlated significantly with haemagglutination inhibition (HI) test results and test sensitivity and specificity were 87% and 92%, respectively. The MP15 ELISA titres were significantly higher than the HI titres in all immune sera allowing for sera to be tested at a single dilution of 1:400 which is of advantage in routine surveillance. The study indicated that the MP15 ELISA is potentially useful for serological detection of H5N1 vaccinated or infected poultry and to have some advantages over the standard HI test for routine monitoring of flocks' immunity after vaccination.

  18. Pathogenicity of an H5N1 avian influenza virus isolated in Vietnam in 2012 and reliability of conjunctival samples for diagnosis of infection

    PubMed Central

    Bui, Vuong N.; Dao, Tung D.; Nguyen, Tham T. H.; Nguyen, Lien T.; Bui, Anh N.; Trinh, Dai Q.; Pham, Nga T.; Inui, Kenjiro; Runstadler, Jonathan; Ogawa, Haruko; Nguyen, Khong V.; Imai, Kunitoshi

    2013-01-01

    The continued spread of highly pathogenic avian influenza virus (HPAIV) subtype H5N1 among poultry in Vietnam poses a potential threat to animals and public health. To evaluate the pathogenicity of a 2012 H5N1 HPAIV isolate and to assess the utility of conjunctival swabs for viral detection and isolation in surveillance, an experimental infection with HPAIV subtype H5N1 was carried out in domestic ducks. Ducks were infected with 107.2 TCID50 of A/duck/Vietnam/QB1207/2012 (H5N1), which was isolated from a moribund domestic duck. In the infected ducks, clinical signs of disease, including neurological disorder, were observed. Ducks started to die at 3 days-post-infection (dpi), and the study mortality reached 67%. Viruses were recovered from oropharyngeal and conjunctival swabs until 7 dpi and from cloacal swabs until 4 dpi. In the ducks that died or were sacrificed on 3, 5, or 6 dpi, viruses were recovered from lung, brain, heart, pancreas and intestine, among which the highest virus titers were in the lung, brain or heart. Results of virus titration were confirmed by real-time RT-PCR. Genetic and phylogenetic analysis of the HA gene revealed that the isolate belongs to clade 2.3.2.1 similarly to the H5N1 viruses isolated in Vietnam in 2012. The present study demonstrated that this recent HPAI H5N1 virus of clade 2.3.2.1 could replicate efficiently in the systemic organs, including the brain, and cause severe disease with neurological symptoms in domestic ducks. Therefore, this HPAI H5N1 virus seems to retain the neurotrophic feature and has further developed properties of shedding virus from the oropharynx and conjunctiva in addition to the cloaca, potentially posing a higher risk of virus spread through cross-contact and/or environmental transmission. Continued surveillance and diagnostic programs using conjuntcival swabs in the field would further verify the apparent reliability of conjunctival samples for the detection of AIV. PMID:24211664

  19. Pathogenicity of an H5N1 avian influenza virus isolated in Vietnam in 2012 and reliability of conjunctival samples for diagnosis of infection.

    PubMed

    Bui, Vuong N; Dao, Tung D; Nguyen, Tham T H; Nguyen, Lien T; Bui, Anh N; Trinh, Dai Q; Pham, Nga T; Inui, Kenjiro; Runstadler, Jonathan; Ogawa, Haruko; Nguyen, Khong V; Imai, Kunitoshi

    2014-01-22

    The continued spread of highly pathogenic avian influenza virus (HPAIV) subtype H5N1 among poultry in Vietnam poses a potential threat to animals and public health. To evaluate the pathogenicity of a 2012 H5N1 HPAIV isolate and to assess the utility of conjunctival swabs for viral detection and isolation in surveillance, an experimental infection with HPAIV subtype H5N1 was carried out in domestic ducks. Ducks were infected with 10(7.2) TCID50 of A/duck/Vietnam/QB1207/2012 (H5N1), which was isolated from a moribund domestic duck. In the infected ducks, clinical signs of disease, including neurological disorder, were observed. Ducks started to die at 3 days-post-infection (dpi), and the study mortality reached 67%. Viruses were recovered from oropharyngeal and conjunctival swabs until 7 dpi and from cloacal swabs until 4 dpi. In the ducks that died or were sacrificed on 3, 5, or 6 dpi, viruses were recovered from lung, brain, heart, pancreas and intestine, among which the highest virus titers were in the lung, brain or heart. Results of virus titration were confirmed by real-time RT-PCR. Genetic and phylogenetic analysis of the HA gene revealed that the isolate belongs to clade 2.3.2.1 similarly to the H5N1 viruses isolated in Vietnam in 2012. The present study demonstrated that this recent HPAI H5N1 virus of clade 2.3.2.1 could replicate efficiently in the systemic organs, including the brain, and cause severe disease with neurological symptoms in domestic ducks. Therefore, this HPAI H5N1 virus seems to retain the neurotrophic feature and has further developed properties of shedding virus from the oropharynx and conjunctiva in addition to the cloaca, potentially posing a higher risk of virus spread through cross-contact and/or environmental transmission. Continued surveillance and diagnostic programs using conjunctival swabs in the field would further verify the apparent reliability of conjunctival samples for the detection of AIV.

  20. Pathogenesis of 1918 Pandemic and H5N1 Influenza Virus Infections in a Guinea Pig Model: Antiviral Potential of Exogenous Alpha Interferon To Reduce Virus Shedding▿

    PubMed Central

    Van Hoeven, Neal; Belser, Jessica A.; Szretter, Kristy J.; Zeng, Hui; Staeheli, Peter; Swayne, David E.; Katz, Jacqueline M.; Tumpey, Terrence M.

    2009-01-01

    Although highly pathogenic avian influenza H5N1 viruses have yet to acquire the ability to transmit efficiently among humans, the increasing genetic diversity among these viruses and continued outbreaks in avian species underscore the need for more effective measures for the control and prevention of human H5N1 virus infection. Additional small animal models with which therapeutic approaches against virulent influenza viruses can be evaluated are needed. In this study, we used the guinea pig model to evaluate the relative virulence of selected avian and human influenza A viruses. We demonstrate that guinea pigs can be infected with avian and human influenza viruses, resulting in high titers of virus shedding in nasal washes for up to 5 days postinoculation (p.i.) and in lung tissue of inoculated animals. However, other physiologic indicators typically associated with virulent influenza virus strains were absent in this species. We evaluated the ability of intranasal treatment with human alpha interferon (α-IFN) to reduce lung and nasal wash titers in guinea pigs challenged with the reconstructed 1918 pandemic H1N1 virus or a contemporary H5N1 virus. IFN treatment initiated 1 day prior to challenge significantly reduced or prevented infection of guinea pigs by both viruses, as measured by virus titer determination and seroconversion. The expression of the antiviral Mx protein in lung tissue correlated with the reduction of virus titers. We propose that the guinea pig may serve as a useful small animal model for testing the efficacy of antiviral compounds and that α-IFN treatment may be a useful antiviral strategy against highly virulent strains with pandemic potential. PMID:19144714

  1. Avian influenza virus A/HK/483/97(H5N1) NS1 protein induces apoptosis in human airway epithelial cells.

    PubMed

    Lam, W Y; Tang, Julian W; Yeung, Apple C M; Chiu, Lawrence C M; Sung, Joseph J Y; Chan, Paul K S

    2008-03-01

    Avian H5N1 influenza virus causes a remarkably severe disease in humans, with an overall case fatality rate of greater than 50%. Human influenza A viruses induce apoptosis in infected cells, which can lead to organ dysfunction. To verify the role of H5N1-encoded NS1 in inducing apoptosis, the NS1 gene was cloned and expressed in human airway epithelial cells (NCI-H292 cells). The apoptotic events posttransfection were examined by a terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick-end-labeling assay, flow cytometric measurement of propidium iodide, annexin V staining, and Western blot analyses with antibodies specific for proapoptotic and antiapoptotic proteins. We demonstrated that the expression of H5N1 NS1 protein in NCI-H292 cells was sufficient to induce apoptotic cell death. Western blot analyses also showed that there was prominent cleavage of poly(ADP-ribose) polymerase and activation of caspase-3, caspase-7, and caspase-8 during the NS1-induced apoptosis. The results of caspase inhibitor assays further confirmed the involvement of caspase-dependent pathways in the NS1-induced apoptosis. Interestingly, the ability of H5N1 NS1 protein to induce apoptosis was much enhanced in cells pretreated with Fas ligand (the time posttransfection required to reach >30% apoptosis was reduced from 24 to 6 h). Furthermore, 24 h posttransfection, an increase in Fas ligand mRNA expression of about 5.6-fold was detected in cells transfected with H5N1 NS1. In conclusion, we demonstrated that the NS1 protein encoded by avian influenza A virus H5N1 induced apoptosis in human lung epithelial cells, mainly via the caspase-dependent pathway, which encourages further investigation into the potential for the NS1 protein to be a novel therapeutic target.

  2. Lessons from emergence of A/goose/Guangdong/1996-like H5N1 highly pathogenic avian influenza viruses and recent influenza surveillance efforts in southern China.

    PubMed

    Wan, X F

    2012-09-01

    Southern China is proposed as an influenza epicentre. At least two of the three pandemics in the last century, including 1957 and 1968 influenza pandemics, originated from this area. In 1996, A/goose/Guangdong/1/1996 (H5N1), the precursor of currently circulating highly pathogenic H5N1 avian influenza viruses (HPAIVs) was identified in farmed geese in southern China. These H5N1 HPAIVs have been spread across Asia, Europe and Africa and poses a continuous threat to both animal and human health. However, how and where this H5N1 HPAIV emerged are not fully understood. In the past decade, many influenza surveillance efforts have been carried out in southern China, and our understanding of the genetic diversity of non-human influenza A viruses in this area has been much better than ever. Here, the historical and first-hand experimental data on A/goose/Guangdong/1/1996(H5N1)-like HPAIVs are reviewed within the context of the findings from recent surveillance efforts on H5N1 HPAIVs and other non-human influenza A viruses. Such a retrospective recapitulation suggests that long-term and systematic surveillance programmes should continue to be implemented in southern China that the wet markets on the animal-human interface shall be the priority area and that the surveillance on the animal species bridging the interface between wildlife and domestic animal populations and the interface between the aquatics and territories shall be the strengthened. PMID:22958248

  3. A human antibody recognizing a conserved epitope of H5 hemagglutinin broadly neutralizes highly pathogenic avian influenza H5N1 viruses.

    PubMed

    Hu, Hongxing; Voss, Jarrod; Zhang, Guoliang; Buchy, Philippi; Zuo, Teng; Wang, Lulan; Wang, Feng; Zhou, Fan; Wang, Guiqing; Tsai, Cheguo; Calder, Lesley; Gamblin, Steve J; Zhang, Linqi; Deubel, Vincent; Zhou, Boping; Skehel, John J; Zhou, Paul

    2012-03-01

    Influenza A virus infection is a persistent threat to public health worldwide due to its ability to evade immune surveillance through rapid genetic drift and shift. Current vaccines against influenza A virus provide immunity to viral isolates that are similar to vaccine strains. High-affinity neutralizing antibodies against conserved epitopes could provide immunity to diverse influenza virus strains and protection against future pandemic viruses. In this study, by using a highly sensitive H5N1 pseudotype-based neutralization assay to screen human monoclonal antibodies produced by memory B cells from an H5N1-infected individual and molecular cloning techniques, we developed three fully human monoclonal antibodies. Among them, antibody 65C6 exhibited potent neutralization activity against all H5 clades and subclades except for subclade 7.2 and prophylactic and therapeutic efficacy against highly pathogenic avian influenza H5N1 viruses in mice. Studies on hemagglutinin (HA)-antibody complexes by electron microscopy and epitope mapping indicate that antibody 65C6 binds to a conformational epitope comprising amino acid residues at positions 118, 121, 161, 164, and 167 (according to mature H5 numbering) on the tip of the membrane-distal globular domain of HA. Thus, we conclude that antibody 65C6 recognizes a neutralization epitope in the globular head of HA that is conserved among almost all divergent H5N1 influenza stains. PMID:22238297

  4. Identification of climate factors related to human infection with avian influenza A H7N9 and H5N1 viruses in China.

    PubMed

    Li, Jing; Rao, Yuhan; Sun, Qinglan; Wu, Xiaoxu; Jin, Jiao; Bi, Yuhai; Chen, Jin; Lei, Fumin; Liu, Qiyong; Duan, Ziyuan; Ma, Juncai; Gao, George F; Liu, Di; Liu, Wenjun

    2015-01-01

    Human influenza infections display a strongly seasonal pattern. However, whether H7N9 and H5N1 infections correlate with climate factors has not been examined. Here, we analyzed 350 cases of H7N9 infection and 47 cases of H5N1 infection. The spatial characteristics of these cases revealed that H5N1 infections mainly occurred in the South, Middle, and Northwest of China, while the occurrence of H7N9 was concentrated in coastal areas of East and South of China. Aside from spatial-temporal characteristics, the most adaptive meteorological conditions for the occurrence of human infections by these two viral subtypes were different. We found that H7N9 infections correlate with climate factors, especially temperature (TEM) and relative humidity (RHU), while H5N1 infections correlate with TEM and atmospheric pressure (PRS). Hence, we propose a risky window (TEM 4-14 °C and RHU 65-95%) for H7N9 infection and (TEM 2-22 °C and PRS 980-1025 kPa) for H5N1 infection. Our results represent the first step in determining the effects of climate factors on two different virus infections in China and provide warning guidelines for the future when provinces fall into the risky windows. These findings revealed integrated predictive meteorological factors rooted in statistic data that enable the establishment of preventive actions and precautionary measures against future outbreaks. PMID:26656876

  5. Identification of climate factors related to human infection with avian influenza A H7N9 and H5N1 viruses in China

    PubMed Central

    Li, Jing; Rao, Yuhan; Sun, Qinglan; Wu, Xiaoxu; Jin, Jiao; Bi, Yuhai; Chen, Jin; Lei, Fumin; Liu, Qiyong; Duan, Ziyuan; Ma, Juncai; Gao, George F.; Liu, Di; Liu, Wenjun

    2015-01-01

    Human influenza infections display a strongly seasonal pattern. However, whether H7N9 and H5N1 infections correlate with climate factors has not been examined. Here, we analyzed 350 cases of H7N9 infection and 47 cases of H5N1 infection. The spatial characteristics of these cases revealed that H5N1 infections mainly occurred in the South, Middle, and Northwest of China, while the occurrence of H7N9 was concentrated in coastal areas of East and South of China. Aside from spatial-temporal characteristics, the most adaptive meteorological conditions for the occurrence of human infections by these two viral subtypes were different. We found that H7N9 infections correlate with climate factors, especially temperature (TEM) and relative humidity (RHU), while H5N1 infections correlate with TEM and atmospheric pressure (PRS). Hence, we propose a risky window (TEM 4–14 °C and RHU 65–95%) for H7N9 infection and (TEM 2–22 °C and PRS 980-1025 kPa) for H5N1 infection. Our results represent the first step in determining the effects of climate factors on two different virus infections in China and provide warning guidelines for the future when provinces fall into the risky windows. These findings revealed integrated predictive meteorological factors rooted in statistic data that enable the establishment of preventive actions and precautionary measures against future outbreaks. PMID:26656876

  6. Understanding of Drug-Target Interactions: A case Study in Influenza Virus A Subtype H5N1

    NASA Astrophysics Data System (ADS)

    Rungrotmongkol, Thanyada; Malaisree, Maturos; Decha, Panita; Laohpongspaisan, Chittima; Aruksakunwong, Ornjira; Intharathep, Pathumwadee; Pianwanit, Somsak; Sompornpisut, Pornthep; Parasuk, Vudhichai; Megnassan, Eugene; Frecer, Vladimir; Miertus, Stanislav; Hannongbua, Supot

    2007-12-01

    This study aims at gaining insight into molecular mechanisms of action of three drug targets of the life cycle of influenza virus A subtype H5N1, namely Hemagglutinin (H5), Neuraminidase (N1) and M2 ion channel (M2), using molecular mechanics and molecular dynamics techniques. In hemagglutinin, interest is focused on the high pathogenicity of the H5 due to the -RRRKK- insertion. MD simulations carried out for H5 in both high and low pathogenic forms (HPH5 and LPH5), aimed at understanding why HPH5 was experimentally observed to be 5-fold better cleaved by furin relative to the non-inserted sequence of LPH5. As the results, the cleavage loop of HPH5 was found to fit well and bind strongly into the catalytic site of human furin, serving as a conformation suitable for the proteolytic reaction. The second target, neuraminidase was studied by two different approaches. Firstly with MD simulations, rotation of the -NHAc and—OCHEt2 side chains of oseltamivir (OTV), leading directly to rearrangement of the catalytic cavity, was found to be a primary source of the lower susceptibility of OTV to neuraminidase subtype N1 than to N2 and N9. In addition, three inhibitiors, OTV, zanamivir (ZNV) and peramivir (PRV), complexed with neuraminidase subtype N1 were studied to understand the drug-target interactions. The structural properties, position and conformation of PRV and its side chains are uniformly preferential, i.e., its conformation fits very well with the N1 active site. At the N1 target, another approach, combinatorial chemistry, was used to design a library of new potent inhibitors, which well fit to the active site and the 150-loop residues of N1. Investigation was also extended to the M2 proton channel. Five different protonation states of the selectivity filter residue (His) where 0H, 1H, 2aH, 2dH and 4H represent the systems with none, mono-protonated, di-protonated at adjacent and opposite positions, and tetra-protonated, respectively, were taken into account both

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

    PubMed

    Adlhoch, C; Gossner, C; Koch, G; Brown, I; Bouwstra, R; 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 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.

  8. Vaccination with Astragalus and Ginseng Polysaccharides Improves Immune Response of Chickens against H5N1 Avian Influenza Virus

    PubMed Central

    Kallon, Sanpha; Yu, Xingang

    2016-01-01

    To determine the effect of astragalus and ginseng polysaccharides (APS, GPS) on immune response and improvement of H5N1 vaccine, 360-day-old broilers were randomly divided into 8 groups of 45 chicks, comprising APS groups (1–3); GPS groups (4–6); vaccine group (7); and blank control (8) (without polysaccharide and vaccine). From day 12 after hatch groups 1–3 were given APS and groups 4–6 with GPS both at 100, 200, and 400 (mg/kg), respectively. At day 15 after hatch, groups 1–7 were vaccinated with 0.3 mL H5N1 vaccine subcutaneously; daily weight gain (DWG) and serum Ig antibody (by HI-test) were measured on 3, 7, 14, and 28 days after vaccination. Serum antibody titers and expression of cytokines (IL-2, IL-10, I FN-γ, and TNF) were determined by ELISA and RT-PCR. Results revealed that all the polysaccharide groups were numerically increased in antibody levels and the expression of cytokines was significant (P < 0.05) in the APS and GPS groups compared to corresponding vaccine group and blank control. DWG was higher (P < 0.05) in 400 mg/kg APS groups than control groups. Thus oral supplements of GPS and APS have shown their potential in the improvement of immune response and could be used as adjuvant in a formulation of H5N1 vaccine. PMID:27597953

  9. Vaccination with Astragalus and Ginseng Polysaccharides Improves Immune Response of Chickens against H5N1 Avian Influenza Virus

    PubMed Central

    Kallon, Sanpha; Yu, Xingang

    2016-01-01

    To determine the effect of astragalus and ginseng polysaccharides (APS, GPS) on immune response and improvement of H5N1 vaccine, 360-day-old broilers were randomly divided into 8 groups of 45 chicks, comprising APS groups (1–3); GPS groups (4–6); vaccine group (7); and blank control (8) (without polysaccharide and vaccine). From day 12 after hatch groups 1–3 were given APS and groups 4–6 with GPS both at 100, 200, and 400 (mg/kg), respectively. At day 15 after hatch, groups 1–7 were vaccinated with 0.3 mL H5N1 vaccine subcutaneously; daily weight gain (DWG) and serum Ig antibody (by HI-test) were measured on 3, 7, 14, and 28 days after vaccination. Serum antibody titers and expression of cytokines (IL-2, IL-10, I FN-γ, and TNF) were determined by ELISA and RT-PCR. Results revealed that all the polysaccharide groups were numerically increased in antibody levels and the expression of cytokines was significant (P < 0.05) in the APS and GPS groups compared to corresponding vaccine group and blank control. DWG was higher (P < 0.05) in 400 mg/kg APS groups than control groups. Thus oral supplements of GPS and APS have shown their potential in the improvement of immune response and could be used as adjuvant in a formulation of H5N1 vaccine.

  10. Vaccination with Astragalus and Ginseng Polysaccharides Improves Immune Response of Chickens against H5N1 Avian Influenza Virus.

    PubMed

    Abdullahi, Auwalu Yusuf; Kallon, Sanpha; Yu, Xingang; Zhang, Yongliang; Li, Guoqing

    2016-01-01

    To determine the effect of astragalus and ginseng polysaccharides (APS, GPS) on immune response and improvement of H5N1 vaccine, 360-day-old broilers were randomly divided into 8 groups of 45 chicks, comprising APS groups (1-3); GPS groups (4-6); vaccine group (7); and blank control (8) (without polysaccharide and vaccine). From day 12 after hatch groups 1-3 were given APS and groups 4-6 with GPS both at 100, 200, and 400 (mg/kg), respectively. At day 15 after hatch, groups 1-7 were vaccinated with 0.3 mL H5N1 vaccine subcutaneously; daily weight gain (DWG) and serum Ig antibody (by HI-test) were measured on 3, 7, 14, and 28 days after vaccination. Serum antibody titers and expression of cytokines (IL-2, IL-10, I FN-γ, and TNF) were determined by ELISA and RT-PCR. Results revealed that all the polysaccharide groups were numerically increased in antibody levels and the expression of cytokines was significant (P < 0.05) in the APS and GPS groups compared to corresponding vaccine group and blank control. DWG was higher (P < 0.05) in 400 mg/kg APS groups than control groups. Thus oral supplements of GPS and APS have shown their potential in the improvement of immune response and could be used as adjuvant in a formulation of H5N1 vaccine. PMID:27597953

  11. Highly pathogenic avian influenza H5N1 Clade 2.3.2.1c virus in migratory birds, 2014-2015.

    PubMed

    Bi, Yuhai; Chen, Jianjun; Zhang, Zhenjie; Li, Mingxin; Cai, Tianlong; Sharshov, Kirill; Susloparov, Ivan; Shestopalov, Alexander; Wong, Gary; He, Yubang; Xing, Zhi; Sun, Jianqing; Liu, Di; Liu, Yingxia; Liu, Lei; Liu, Wenjun; Lei, Fumin; Shi, Weifeng; Gao, George F

    2016-08-01

    A novel Clade 2.3.2.1c H5N1 reassortant virus caused several outbreaks in wild birds in some regions of China from late 2014 to 2015. Based on the genetic and phylogenetic analyses, the viruses possess a stable gene constellation with a Clade 2.3.2.1c HA, a H9N2-derived PB2 gene and the other six genes of Asian H5N1-origin. The Clade 2.3.2.1c H5N1 reassortants displayed a high genetic relationship to a human H5N1 strain (A/Alberta/01/2014). Further analysis showed that similar viruses have been circulating in wild birds in China, Russia, Dubai (Western Asia), Bulgaria and Romania (Europe), as well as domestic poultry in some regions of Africa. The affected areas include the Central Asian, East Asian-Australasian, West Asian-East African, and Black Sea/Mediterranean flyways. These results show that the novel Clade 2.3.2.1c reassortant viruses are circulating worldwide and may have gained a selective advantage in migratory birds, thus posing a serious threat to wild birds and potentially humans.

  12. Indications that Live Poultry Markets Are a Major Source of Human H5N1 Influenza Virus Infection in China ▿ †

    PubMed Central

    Wan, Xiu-Feng; Dong, Libo; Lan, Yu; Long, Li-Ping; Xu, Cuiling; Zou, Shumei; Li, Zi; Wen, Leying; Cai, Zhipeng; Wang, Wei; Li, Xiaodan; Yuan, Fan; Sui, Hongtao; Zhang, Ye; Dong, Jie; Sun, Shanhua; Gao, Yan; Wang, Min; Bai, Tian; Yang, Lei; Li, Dexin; Yang, Weizhong; Yu, Hongjie; Wang, Shiwen; Feng, Zijian; Wang, Yu; Guo, Yuanji; Webby, Richard J.; Shu, Yuelong

    2011-01-01

    Human infections of H5N1 highly pathogenic avian influenza virus have continued to occur in China without corresponding outbreaks in poultry, and there is little conclusive evidence of the source of these infections. Seeking to identify the source of the human infections, we sequenced 31 H5N1 viruses isolated from humans in China (2005 to 2010). We found a number of viral genotypes, not all of which have similar known avian virus counterparts. Guided by patient questionnaire data, we also obtained environmental samples from live poultry markets and dwellings frequented by six individuals prior to disease onset (2008 and 2009). H5N1 viruses were isolated from 4 of the 6 live poultry markets sampled. In each case, the genetic sequences of the environmental and corresponding human isolates were highly similar, demonstrating a link between human infection and live poultry markets. Therefore, infection control measures in live poultry markets are likely to reduce human H5N1 infection in China. PMID:21976646

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 specific pathogen free (SPF) birds immunized with 0.2 ...

  14. Impact of vaccination on infection with Vietnam H5N1 high pathogenicity avian influenza virus in hens and the eggs they lay

    Technology Transfer Automated Retrieval System (TEKTRAN)

    High pathogenicity avian influenza virus (HPAIV) infections in chickens decrease egg production and eggs that are laid contain HPAIV. Vaccination once or twice was examined as a way to protect chickens from Vietnamese H5N1 HPAIV. Eighty-three percent of hens without vaccination died within 3 days ...

  15. Highly pathogenic avian influenza H5N1 Clade 2.3.2.1c virus in migratory birds, 2014-2015.

    PubMed

    Bi, Yuhai; Chen, Jianjun; Zhang, Zhenjie; Li, Mingxin; Cai, Tianlong; Sharshov, Kirill; Susloparov, Ivan; Shestopalov, Alexander; Wong, Gary; He, Yubang; Xing, Zhi; Sun, Jianqing; Liu, Di; Liu, Yingxia; Liu, Lei; Liu, Wenjun; Lei, Fumin; Shi, Weifeng; Gao, George F

    2016-08-01

    A novel Clade 2.3.2.1c H5N1 reassortant virus caused several outbreaks in wild birds in some regions of China from late 2014 to 2015. Based on the genetic and phylogenetic analyses, the viruses possess a stable gene constellation with a Clade 2.3.2.1c HA, a H9N2-derived PB2 gene and the other six genes of Asian H5N1-origin. The Clade 2.3.2.1c H5N1 reassortants displayed a high genetic relationship to a human H5N1 strain (A/Alberta/01/2014). Further analysis showed that similar viruses have been circulating in wild birds in China, Russia, Dubai (Western Asia), Bulgaria and Romania (Europe), as well as domestic poultry in some regions of Africa. The affected areas include the Central Asian, East Asian-Australasian, West Asian-East African, and Black Sea/Mediterranean flyways. These results show that the novel Clade 2.3.2.1c reassortant viruses are circulating worldwide and may have gained a selective advantage in migratory birds, thus posing a serious threat to wild birds and potentially humans. PMID:27405930

  16. The PA-gene-mediated lethal dissemination and excessive innate immune response contribute to the high virulence of H5N1 avian influenza virus in mice.

    PubMed

    Hu, Jiao; Hu, Zenglei; Song, Qingqing; Gu, Min; Liu, Xiaowen; Wang, Xiaoquan; Hu, Shunlin; Chen, Chaoyang; Liu, Huimou; Liu, Wenbo; Chen, Sujuan; Peng, Daxin; Liu, Xiufan

    2013-03-01

    Highly pathogenic H5N1 influenza A virus remains a substantial threat to public health. To understand the molecular basis and host mechanism for the high virulence of H5N1 viruses in mammals, we compared two H5N1 isolates which have similar genetic backgrounds but greatly differ in their virulence in mice. A/Chicken/Jiangsu/k0402/2010 (CK10) is highly pathogenic, whereas A/Goose/Jiangsu/k0403/2010 (GS10) is nonpathogenic. We first showed that CK10 elicited a more potent innate immune response than did GS10 in mouse lungs by increasing the number and expression levels of activated genes. We then generated a series of reassortants between the two viruses and evaluated their virulence in mice. Inclusion of the CK10 PA gene in the GS10 background resulted in a dramatic increase in virulence. Conversely, expression of the GS10 PA gene in the CK10 background significantly attenuated the virulence. These results demonstrated that the PA gene mainly determines the pathogenicity discrepancy between CK10 and GS10 in mice. We further determined that arginine (R) at position 353 of the PA gene contributes to the high virulence of CK10 in mice. The reciprocal substitution at position 353 in PA or the exchange of the entire PA gene largely caused the transfer of viral phenotypes, including virus replication, polymerase activity, and manipulation of the innate response, between CK10 and GS10. We therefore defined a novel molecular marker associated with the high virulence of H5N1 influenza viruses, providing further insights into the pathogenesis of H5N1 viruses in mammals.

  17. Pathogenesis in Eurasian tree sparrows inoculated with H5N1 highly pathogenic avian influenza virus and experimental virus transmission from tree sparrows to chickens.

    PubMed

    Yamamoto, Yu; Nakamura, Kikuyasu; Yamada, Manabu; Mase, Masaji

    2013-06-01

    Small wild birds that routinely enter poultry farms may be possible vectors of Asian lineage H5N1 highly pathogenic avian influenza virus. In this study, we conducted experimental infections using wild-caught Eurasian tree sparrows (Passer montanus) to evaluate their possible epidemiological involvement in virus transmission. When tree sparrows were intranasally inoculated with the virus at a low or high dose, all sparrows excluding euthanatized birds died within 11 days after inoculation. Viruses were frequently isolated from the drinking water, oral swabs, and visceral organs of the sparrows. Immunohistochemical analysis revealed that the virus replicated strongly in the central nervous system, heart, and adrenal gland following primary infection in the upper respiratory tract and a probable subsequent viremic stage. In the contact infection study using virus-inoculated sparrows and untreated contact chickens, more than half of all chickens died from viral infection. In the virus transmission study in which chickens were given drinking water collected from virus-inoculated sparrows, mortality due to viral infection was observed in chickens. Our data suggest that Eurasian tree sparrows could be biological vectors of the H5N1 highly pathogenic avian influenza virus. In addition to frequent virus detection in the drinking water of sparrows, the results of the virus transmission study suggest that waterborne pathways could be important for viral transmission from tree sparrows to poultry.

  18. Prophylactic and Therapeutic Efficacy of Avian Antibodies Against Influenza Virus H5N1 and H1N1 in Mice

    PubMed Central

    Nguyen, Huan H.; Tumpey, Terrence M.; Park, Hae-Jung; Byun, Young-Ho; Tran, Linh D.; Nguyen, Van D.; Kilgore, Paul E.; Czerkinsky, Cecil; Katz, Jacqueline M.; Seong, Baik Lin; Song, Jae Min; Kim, Young Bong; Do, Hoa T.; Nguyen, Tung; Nguyen, Cam V.

    2010-01-01

    Background Pandemic influenza poses a serious threat to global health and the world economy. While vaccines are currently under development, passive immunization could offer an alternative strategy to prevent and treat influenza virus infection. Attempts to develop monoclonal antibodies (mAbs) have been made. However, passive immunization based on mAbs may require a cocktail of mAbs with broader specificity in order to provide full protection since mAbs are generally specific for single epitopes. Chicken immunoglobulins (IgY) found in egg yolk have been used mainly for treatment of infectious diseases of the gastrointestinal tract. Because the recent epidemic of highly pathogenic avian influenza virus (HPAIV) strain H5N1 has resulted in serious economic losses to the poultry industry, many countries including Vietnam have introduced mass vaccination of poultry with H5N1 virus vaccines. We reasoned that IgY from consumable eggs available in supermarkets in Vietnam could provide protection against infections with HPAIV H5N1. Methods and Findings We found that H5N1-specific IgY that are prepared from eggs available in supermarkets in Vietnam by a rapid and simple water dilution method cross-protect against infections with HPAIV H5N1 and related H5N2 strains in mice. When administered intranasally before or after lethal infection, the IgY prevent the infection or significantly reduce viral replication resulting in complete recovery from the disease, respectively. We further generated H1N1 virus-specific IgY by immunization of hens with inactivated H1N1 A/PR/8/34 as a model virus for the current pandemic H1N1/09 and found that such H1N1-specific IgY protect mice from lethal influenza virus infection. Conclusions The findings suggest that readily available H5N1-specific IgY offer an enormous source of valuable biological material to combat a potential H5N1 pandemic. In addition, our study provides a proof-of-concept for the approach using virus-specific IgY as affordable

  19. Experimental challenge and pathology of highly pathogenic avian influenza virus H5N1 in dunlin (Calidris alpina), an intercontinental migrant shorebird species

    PubMed Central

    Hall, Jeffrey S.; Franson, J. Christian; Gill, Robert E.; Meteyer, Carol U.; TeSlaa, Joshua L.; Nashold, Sean; Dusek, Robert J.; Ip, Hon S.

    2011-01-01

    Please cite this paper as: Hall et al. (2011). Experimental challenge and pathology of highly pathogenic avian influenza virus H5N1 in dunlin (Calidris alpina), an intercontinental migrant shorebird species. Influenza and Other Respiratory Viruses 5(5), 365–372. Background  Shorebirds (Charadriiformes) are considered one of the primary reservoirs of avian influenza. Because these species are highly migratory, there is concern that infected shorebirds may be a mechanism by which highly pathogenic avian influenza virus (HPAIV) H5N1 could be introduced into North America from Asia. Large numbers of dunlin (Calidris alpina) migrate from wintering areas in central and eastern Asia, where HPAIV H5N1 is endemic, across the Bering Sea to breeding areas in Alaska. Low pathogenic avian influenza virus has been previously detected in dunlin, and thus, dunlin represent a potential risk to transport HPAIV to North America. To date no experimental challenge studies have been performed in shorebirds. Methods  Wild dunlin were inoculated intranasally and intrachoanally various doses of HPAIV H5N1. The birds were monitored daily for virus excretion, disease signs, morbidity, and mortality. Results  The infectious dose of HPAIV H5N1 in dunlin was determined to be 101.7 EID50/100 μl and that the lethal dose was 101.83 EID50/100 μl. Clinical signs were consistent with neurotropic disease, and histochemical analyses revealed that infection was systemic with viral antigen and RNA most consistently found in brain tissues. Infected birds excreted relatively large amounts of virus orally (104 EID50) and smaller amounts cloacally. Conclusions  Dunlin are highly susceptible to infection with HPAIV H5N1. They become infected after exposure to relatively small doses of the virus and if they become infected, they are most likely to suffer mortality within 3–5 days. These results have important implications regarding the risks of transport and transmission of HPAIV H5N1 to North

  20. Increased Acid Stability of the Hemagglutinin Protein Enhances H5N1 Influenza Virus Growth in the Upper Respiratory Tract but Is Insufficient for Transmission in Ferrets

    PubMed Central

    Zaraket, Hassan; Bridges, Olga A.; Duan, Susu; Baranovich, Tatiana; Yoon, Sun-Woo; Reed, Mark L.; Salomon, Rachelle; Webby, Richard J.; Webster, Robert G.

    2013-01-01

    Influenza virus entry is mediated by the acidic-pH-induced activation of hemagglutinin (HA) protein. Here, we investigated how a decrease in the HA activation pH (an increase in acid stability) influences the properties of highly pathogenic H5N1 influenza virus in mammalian hosts. We generated isogenic A/Vietnam/1203/2004 (H5N1) (VN1203) viruses containing either wild-type HA protein (activation pH 6.0) or an HA2-K58I point mutation (K to I at position 58) (activation pH 5.5). The VN1203-HA2-K58I virus had replication kinetics similar to those of wild-type VN1203 in MDCK and normal human bronchial epithelial cells and yet had reduced growth in human alveolar A549 cells, which were found to have a higher endosomal pH than MDCK cells. Wild-type and HA2-K58I viruses promoted similar levels of morbidity and mortality in C57BL/6J mice and ferrets, and neither virus transmitted efficiently to naive contact cage-mate ferrets. The acid-stabilizing HA2-K58I mutation, which diminishes H5N1 replication and transmission in ducks, increased the virus load in the ferret nasal cavity early during infection while simultaneously reducing the virus load in the lungs. Overall, a single, acid-stabilizing mutation was found to enhance the growth of an H5N1 influenza virus in the mammalian upper respiratory tract, and yet it was insufficient to enable contact transmission in ferrets in the absence of additional mutations that confer α(2,6) receptor binding specificity and remove a critical N-linked glycosylation site. The information provided here on the contribution of HA acid stability to H5N1 influenza virus fitness and transmissibility in mammals in the background of a non-laboratory-adapted virus provides essential information for the surveillance and assessment of the pandemic potential of currently circulating H5N1 viruses. PMID:23824818

  1. Isolation and identification of highly pathogenic avian influenza H5N1 virus from Houbara bustards (Chlamydotis undulata macqueenii) and contact falcons.

    PubMed

    Khan, Owais Ahmed; Shuaib, Mohammad Adam; Rhman, Salah Shaban Abdel; Ismail, Mahmoud Moussa; Hammad, Yousef Al; Baky, Mansour Hashim Abdel; Fusaro, Alice; Salviato, Annalisa; Cattoli, Giovanni

    2009-02-01

    Highly pathogenic influenza virus (HPAIV) H5N1 has caused mortality and morbidity in many species of domestic and wild bird. The Houbara bustard (Chlamydotis undulata macqueenii) is a solitary bird that inhabits semi-desert regions. It is known to be susceptible to avianpox, avian paramyxovirus type 1, and low-pathogenicity avian influenza H9N2. We report an outbreak of H5N1 HPAIV in Houbara bustards, which were introduced into the Kingdom of Saudi Arabia for falconry purposes. Ninety-three per cent mortality (38 out of 41 birds) in the infected Houbara bustard flock and about 62.5% mortality (10 out of 16 birds) in falcons that came in contact with these birds were observed. Pooled cloacal and tracheal swabs from Houbara bustards as well as visceral organ homogenates collected in Houbara bustards and falcons were tested by real-time reverse transcriptase-polymerase chain reaction, and virus isolation was attempted in specific pathogen free hens' eggs. The viruses isolated were characterized as HPAIV H5N1. Phylogenetic analysis of the haemagglutinating and Neuraminidase (NA) genes revealed that the viruses isolated from Houbara bustards and falcons were closely related to each other and to Kuwaiti H5N1 strains isolated in 2007. Interestingly, they were genetically distinguishable from the co-circulating A/H5N1 viruses in Kingdom of Saudi Arabia causing outbreaks in domestic birds. This case emphasizes the need for surveillance of this endangered species in its natural habitat. PMID:19130352

  2. Transmission of H5N1 high pathogenicity avian influenza virus to Herring gulls (Larus argentatus) through intranasal inoculation of virus and ingestion of virus-infected chicken meat

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In order to evaluate the susceptibility of herring gulls (Larus argentatus) to H5N1 highly pathogenic avian influenza (HPAI) virus under natural routes of infection, we exposed gulls to two Asian lineage H5N1 HPAI viruses (A/whooper swan/Mongolia/244/05 and A/duck meat/Anyang/AVL-1/01) via intranasa...

  3. The N-linked glycosylation site at position 158 on the head of hemagglutinin and the virulence of H5N1 avian influenza virus in mice.

    PubMed

    Suptawiwat, Ornpreya; Boonarkart, Chompunuch; Chakritbudsabong, Warunya; Uiprasertkul, Mongkol; Puthavathana, Pilaipan; Wiriyarat, Witthawat; Auewarakul, Prasert

    2015-02-01

    N-linked glycosylation of the influenza virus hemagglutinin (HA) protein plays crucial roles in HA structure and function, evasion of neutralizing antibodies, and susceptibility to innate soluble antiviral factors. The N-linked glycosylation site at position 158 of highly pathogenic H5N1 virus was previously shown to affect viral receptor-binding preference. H5N1 viruses show heterogeneity with respect to the presence of this glycosylation site. Clade 1 viruses that caused outbreaks in Southeast Asia in 2004 contained this glycosylation site, while the site is absent in the more recent clade 2 viruses. Here, we show that elimination of this glycosylation site increases viral virulence in mice. The mutant lacking the glycosylation site at position 158 showed unaltered growth kinetics in vitro and a comparable level of sensitivity to a major antiviral protein found in respiratory secretions, surfactant protein D (SP-D).

  4. Live Bird Markets of Bangladesh: H9N2 Viruses and the Near Absence of Highly Pathogenic H5N1 Influenza

    PubMed Central

    Negovetich, Nicholas J.; Feeroz, Mohammed M.; Jones-Engel, Lisa; Walker, David; Alam, S. M. Rabiul; Hasan, Kamrul; Seiler, Patrick; Ferguson, Angie; Friedman, Kim; Barman, Subrata; Franks, John; Turner, Jasmine; Krauss, Scott; Webby, Richard J.; Webster, Robert G.

    2011-01-01

    Avian influenza surveillance in Bangladesh has been passive, relying on poultry farmers to report suspected outbreaks of highly pathogenic H5N1 influenza. Here, the results of an active surveillance effort focusing on the live-bird markets are presented. Prevalence of influenza infection in the birds of the live bird markets is 23.0%, which is similar to that in poultry markets in other countries. Nearly all of the isolates (94%) were of the non-pathogenic H9N2 subtype, but viruses of the H1N2, H1N3, H3N6, H4N2, H5N1, and H10N7 subtypes were also observed. The highly pathogenic H5N1-subtype virus was observed at extremely low prevalence in the surveillance samples (0.08%), and we suggest that the current risk of infection for humans in the retail poultry markets in Bangladesh is negligible. However, the high prevalence of the H9 subtype and its potential for interaction with the highly pathogenic H5N1-subtype, i.e., reassortment and attenuation of host morbidity, highlight the importance of active surveillance of the poultry markets. PMID:21541296

  5. Evidence for differing evolutionary dynamics of A/H5N1 viruses among countries applying or not applying avian influenza vaccination in poultry.

    PubMed

    Cattoli, Giovanni; Fusaro, Alice; Monne, Isabella; Coven, Fethiye; Joannis, Tony; El-Hamid, Hatem S Abd; Hussein, Aly Ahmed; Cornelius, Claire; Amarin, Nadim Mukhles; Mancin, Marzia; Holmes, Edward C; Capua, Ilaria

    2011-11-21

    Highly pathogenic avian influenza (HPAI) H5N1 (clade 2.2) was introduced into Egypt in early 2006. Despite the control measures taken, including mass vaccination of poultry, the virus rapidly spread among commercial and backyard flocks. Since the initial outbreaks, the virus in Egypt has evolved into a third order clade (clade 2.2.1) and diverged into antigenically and genetically distinct subclades. To better understand the dynamics of HPAI H5N1 evolution in countries that differ in vaccination policy, we undertook an in-depth analysis of those virus strains circulating in Egypt between 2006 and 2010, and compared countries where vaccination was adopted (Egypt and Indonesia) to those where it was not (Nigeria, Turkey and Thailand). This study incorporated 751 sequences (Egypt n=309, Indonesia n=149, Nigeria n=106, Turkey n=87, Thailand n=100) of the complete haemagglutinin (HA) open reading frame, the major antigenic determinant of influenza A virus. Our analysis revealed that two main Egyptian subclades (termed A and B) have co-circulated in domestic poultry since late 2007 and exhibit different profiles of positively selected codons and rates of nucleotide substitution. The mean evolutionary rate of subclade A H5N1 viruses was 4.07×10(-3) nucleotide substitutions per site, per year (HPD 95%, 3.23-4.91), whereas subclade B possessed a markedly higher substitution rate (8.87×10(-3); 95% HPD 7.0-10.72×10(-3)) and a stronger signature of positive selection. Although the direct association between H5N1 vaccination and virus evolution is difficult to establish, we found evidence for a difference in the evolutionary dynamics of H5N1 viruses among countries where vaccination was or was not adopted. In particular, both evolutionary rates and the number of positively selected sites were higher in virus populations circulating in countries applying avian influenza vaccination for H5N1, compared to viruses circulating in countries which had never used vaccination. We

  6. Evidence for differing evolutionary dynamics of A/H5N1 viruses among countries applying or not applying avian influenza vaccination in poultry.

    PubMed

    Cattoli, Giovanni; Fusaro, Alice; Monne, Isabella; Coven, Fethiye; Joannis, Tony; El-Hamid, Hatem S Abd; Hussein, Aly Ahmed; Cornelius, Claire; Amarin, Nadim Mukhles; Mancin, Marzia; Holmes, Edward C; Capua, Ilaria

    2011-11-21

    Highly pathogenic avian influenza (HPAI) H5N1 (clade 2.2) was introduced into Egypt in early 2006. Despite the control measures taken, including mass vaccination of poultry, the virus rapidly spread among commercial and backyard flocks. Since the initial outbreaks, the virus in Egypt has evolved into a third order clade (clade 2.2.1) and diverged into antigenically and genetically distinct subclades. To better understand the dynamics of HPAI H5N1 evolution in countries that differ in vaccination policy, we undertook an in-depth analysis of those virus strains circulating in Egypt between 2006 and 2010, and compared countries where vaccination was adopted (Egypt and Indonesia) to those where it was not (Nigeria, Turkey and Thailand). This study incorporated 751 sequences (Egypt n=309, Indonesia n=149, Nigeria n=106, Turkey n=87, Thailand n=100) of the complete haemagglutinin (HA) open reading frame, the major antigenic determinant of influenza A virus. Our analysis revealed that two main Egyptian subclades (termed A and B) have co-circulated in domestic poultry since late 2007 and exhibit different profiles of positively selected codons and rates of nucleotide substitution. The mean evolutionary rate of subclade A H5N1 viruses was 4.07×10(-3) nucleotide substitutions per site, per year (HPD 95%, 3.23-4.91), whereas subclade B possessed a markedly higher substitution rate (8.87×10(-3); 95% HPD 7.0-10.72×10(-3)) and a stronger signature of positive selection. Although the direct association between H5N1 vaccination and virus evolution is difficult to establish, we found evidence for a difference in the evolutionary dynamics of H5N1 viruses among countries where vaccination was or was not adopted. In particular, both evolutionary rates and the number of positively selected sites were higher in virus populations circulating in countries applying avian influenza vaccination for H5N1, compared to viruses circulating in countries which had never used vaccination. We

  7. Effects of a nutrient mixture on infectious properties of the highly pathogenic strain of avian influenza virus A/H5N1.

    PubMed

    Deryabin, Petr G; Lvov, Dmitry K; Botikov, Andrey G; Ivanov, Vadim; Kalinovsky, Tatiana; Niedzwiecki, Aleksandra; Rath, Matthias

    2008-01-01

    Numerous outbreaks of avian influenza virus infection (A/H5N1) have occurred recently, infecting domestic birds, chicken and ducks. The possibility of the emergence of a new strain of influenza virus capable of causing a pandemic in humans is high and no vaccine effective against such a strain currently exists. A unique nutrient mixture (NM), containing lysine, proline, ascorbic acid, green tea extract, N-acetyl cysteine, selenium among other micro nutrients, has been shown to exert a wide range of biochemical and pharmacological effects, including an inhibitory effect on replication of influenza virus and HIV. This prompted us to investigate the potential anti-viral activity of a nutrient mixture (NM) and its components on avian influenza virus A/H5N1at viral dosages of 1.0, 0.1 and 0.01 TCID(50). Antiviral activity was studied in cultured cell lines PK, BHK-21, and Vero-E6. Virus lysing activity was determined by co-incubation of virus A/H5N1 with NM for 0-60 min, followed residual virulence titration in cultured SPEV or BHK-21 cells. NM demonstrated high antiviral activity evident even at prolonged periods after infection. NM antiviral properties were comparable to those of conventional drugs (amantadine and oseltamivir); however, NM had the advantage of affecting viral replication at the late stages of the infection process.

  8. The virulence of 1997 H5N1 influenza viruses in the mouse model is increased by correcting a defect in their NS1 proteins.

    PubMed

    Spesock, April; Malur, Meghana; Hossain, M Jaber; Chen, Li-Mei; Njaa, Bradley L; Davis, Charles T; Lipatov, Aleksandr S; York, Ian A; Krug, Robert M; Donis, Ruben O

    2011-07-01

    The NS1 protein of human influenza A viruses binds the 30-kDa subunit of the cleavage and polyadenylation specificity factor (CPSF30), a protein required for 3' end processing of cellular pre-mRNAs, thereby inhibiting production of beta interferon (IFN-β) mRNA. The NS1 proteins of pathogenic 1997 H5N1 viruses contain the CPSF30-binding site but lack the consensus amino acids at positions 103 and 106, F and M, respectively, that are required for the stabilization of CPSF30 binding, resulting in nonoptimal CPSF30 binding in infected cells. Here we have demonstrated that strengthening CPSF30 binding, by changing positions 103 and 106 in the 1997 H5N1 NS1 protein to the consensus amino acids, results in a remarkable 300-fold increase in the lethality of the virus in mice. Unexpectedly, this increase in virulence is not associated with increased lung pathology but rather is characterized by faster systemic spread of the virus, particularly to the brain, where increased replication and severe pathology occur. This increased spread is associated with increased cytokine and chemokine levels in extrapulmonary tissues. We conclude that strengthening CPSF30 binding by the NS1 protein of 1997 H5N1 viruses enhances virulence in mice by increasing the systemic spread of the virus from the lungs, particularly to the brain.

  9. The Virulence of 1997 H5N1 Influenza Viruses in the Mouse Model Is Increased by Correcting a Defect in Their NS1 Proteins ▿

    PubMed Central

    Spesock, April; Malur, Meghana; Hossain, M. Jaber; Chen, Li-Mei; Njaa, Bradley L.; Davis, Charles T.; Lipatov, Aleksandr S.; York, Ian A.; Krug, Robert M.; Donis, Ruben O.

    2011-01-01

    The NS1 protein of human influenza A viruses binds the 30-kDa subunit of the cleavage and polyadenylation specificity factor (CPSF30), a protein required for 3′ end processing of cellular pre-mRNAs, thereby inhibiting production of beta interferon (IFN-β) mRNA. The NS1 proteins of pathogenic 1997 H5N1 viruses contain the CPSF30-binding site but lack the consensus amino acids at positions 103 and 106, F and M, respectively, that are required for the stabilization of CPSF30 binding, resulting in nonoptimal CPSF30 binding in infected cells. Here we have demonstrated that strengthening CPSF30 binding, by changing positions 103 and 106 in the 1997 H5N1 NS1 protein to the consensus amino acids, results in a remarkable 300-fold increase in the lethality of the virus in mice. Unexpectedly, this increase in virulence is not associated with increased lung pathology but rather is characterized by faster systemic spread of the virus, particularly to the brain, where increased replication and severe pathology occur. This increased spread is associated with increased cytokine and chemokine levels in extrapulmonary tissues. We conclude that strengthening CPSF30 binding by the NS1 protein of 1997 H5N1 viruses enhances virulence in mice by increasing the systemic spread of the virus from the lungs, particularly to the brain. PMID:21593152

  10. The antigenic drift molecular basis of the H5N1 influenza viruses in a novel branch of clade 2.3.4.

    PubMed

    Zhong, Lei; Zhao, Qingqing; Zhao, Kunkun; Wang, Xiaoquan; Zhao, Guo; Li, Qunhui; Gu, Min; Peng, Daxin; Liu, Xiufan

    2014-06-25

    H5N1 subtype influenza A virus has evolved into many HA clades since late 1990 s. Six circulating H5N1 influenza viruses clustered to a novel branch in clade 2.3.4 and could escape vaccine protection, indicating their antigenic drift. Eleven amino acids substitutions in three antigenic sites of the hemagglutinin of these isolates were found when compared with the hemagglutinin of the primary viruses in clade 2.3.4. On the backbone of the novel isolates A/chicken/Northern China/k0602/2010, we generated a panel of recombinant viruses with HA mutations of restoring the primary vaccine strain Re-5's amino acid and homologous antisera to determine the role of these substitutions. The results of cross-HI assay, micro-neutralization assay and the antigen map of the mutated recombinant viruses showed that three substitutions in antigenic site B, especially D205K, are the major contributors to the antigenic drift of the novel branch of clade 2.3.4. Our study highlights the importance of surveillance of antigenic drift of H5N1 viruses for the control and preparedness of pandemic threats.

  11. Post-exposure treatment with whole inactivated H5N1 avian influenza virus protects against lethal homologous virus infection in mice

    PubMed Central

    Hagan, Mable; Ranadheera, Charlene; Audet, Jonathan; Morin, Jocelyn; Leung, Anders; Kobasa, Darwyn

    2016-01-01

    Concerns with H5N1 influenza viruses include their prevalence in wild and domestic poultry, high mortality rate (~60%) in humans with some strains, lack of pre-existing immunity in humans, and the possibility that these viruses acquire mutations that enable efficient transmission between humans. H5 subtype viruses of Eurasian origin have recently appeared in wild and domestic bird populations in North America, and have led to the generation of new virus strains that are highly pathogenic in poultry. These new H5 HA containing viruses with their ability to evolve rapidly represent an unknown threat to humans in contact with infected poultry, and vaccination with an off-the-shelf vaccine may be impractical to provide protection to at-risk individuals. Instead, we have evaluated the efficacy of a formalin-inactivated vaccine, which could be derived directly from a circulating virus, to provide post-exposure protection. This strategy was evaluated using a prototypic highly pathogenic avian H5N1 strain, A/Vietnam/1203/2004, and demonstrated rapid induction of adaptive immune responses providing protection in a mammalian model of lethal infection. Additionally, this post-exposure vaccine was highly efficacious when administered 24 hours after exposure. This study offers a platform for developing effective post-exposure vaccines for treatment of highly virulent influenza infections. PMID:27405487

  12. Recombinant baculovirus vaccine containing multiple M2e and adjuvant LTB induces T cell dependent, cross-clade protection against H5N1 influenza virus in mice.

    PubMed

    Zhang, Jie; Fan, Hui-Ying; Zhang, Zhen; Zhang, Juan; Zhang, Jiao; Huang, Jian-Ni; Ye, Yu; Liao, Ming

    2016-01-27

    H5N1, highly pathogenic avian influenza poses, a threat to animal and human health. Rapid changes in H5N1 viruses require periodic reformulation of the conventional strain-matched vaccines, thus emphasizing the need for a broadly protective influenza vaccine. Here, we constructed BV-Dual-3M2e-LTB, a recombinant baculovirus based on baculovirus display and BacMam technology. BV-Dual-3M2e-LTB harbors a gene cassette expressing three tandem copies of the highly conserved extracellular domain of influenza M2 protein (M2e) and the mucosal adjuvant, LTB. We showed that BV-Dual-3M2e-LTB displayed the target protein (M2e/LTB) on the baculoviral surface and expressed it in transduced mammalian cells. BV-Dual-3M2e-LTB, when delivered nasally in mice, was highly immunogenic and induced superior levels of anti-M2e IgA than the non-adjuvanted baculovirus (BV-Dual-3M2e). Importantly, after challenge with different H5N1 clades (clade 0, 2.3.2.1, 2.3.4 and 4), mice inoculated with BV-Dual-3M2e-LTB displayed improved survival and decreased lung virus shedding compared with mice inoculated with BV-Dual-3M2e. The enhanced protection from BV-Dual-3M2e-LTB is mediated by T cell immunity and is primarily based on CD8(+) T cells, while mucosal antibodies alone were insufficient for protection from lethal H5N1 challenge. These results suggest that BV-Dual-3M2e-LTB has potential to protect against a broad range of H5N1 strains thereby providing a novel direction for developing broadly protective vaccines based on cellular immunity. PMID:26724200

  13. Transmissibility of the highly pathogenic avian influenza virus, subtype H5N1 in domestic poultry: a spatio-temporal estimation at the global scale.

    PubMed

    Zhang, Zhijie; Chen, Dongmei; Ward, Michael P; Jiang, Qingwu

    2012-11-01

    The highly pathogenic avian influenza virus (HPAIV), subtype H5N1 poses a serious threat not only to the poultry industry and wild birds but also to humans. Despite a large number of studies conducted on various aspects of this virus, its transmissibility is still poorly understood. This study quantifies the basic reproductive number (R0) of the global HPAIV H5N1 spread within domestic poultry during December 2003 to December 2009. Three different approaches were applied to estimate R0 for HPAIV H5N1: (i) epidemic doubling time; (ii) spatial distance-based nearest neighbour; and (iii) spatio-temporal distance-based nearest neighbour. These three approaches represent temporal (tR0), spatial (sR0) and spatio-temporal transmissibility (stR0), respectively. The joint application of these three approaches provides a more complete profile by characterising the transmissibility traits of infectious diseases from different perspectives. Estimates of tR0 gradually decreased over the six sequential epidemic waves (EWs) examined, suggesting that the implemented control measures were effective in reducing the number of outbreaks. However, sR0 and stR0 increased from EW1, peaked in EW3 and then gradually decreased during EW4-EW6, reflecting different aspects of disease transmissibility compared to tR0. The application of all three methods in the final EW6 showed R0 >1, suggesting that the control measures implemented did not completely interrupt the transmission cycle, and hence were insufficient to eliminate HPAIV H5N1. Close monitoring of HPAIV H5N1 outbreaks and enhanced control policies is advised. PMID:23242687

  14. Global epidemiology of avian influenza A H5N1 virus infection in humans, 1997-2015: a systematic review of individual case data.

    PubMed

    Lai, Shengjie; Qin, Ying; Cowling, Benjamin J; Ren, Xiang; Wardrop, Nicola A; Gilbert, Marius; Tsang, Tim K; Wu, Peng; Feng, Luzhao; Jiang, Hui; Peng, Zhibin; Zheng, Jiandong; Liao, Qiaohong; Li, Sa; Horby, Peter W; Farrar, Jeremy J; Gao, George F; Tatem, Andrew J; Yu, Hongjie

    2016-07-01

    Avian influenza A H5N1 viruses have caused many, typically severe, human infections since the first human case was reported in 1997. However, no comprehensive epidemiological analysis of global human cases of H5N1 from 1997 to 2015 exists. Moreover, few studies have examined in detail the changing epidemiology of human H5N1 cases in Egypt, especially given the outbreaks since November, 2014, which have the highest number of cases ever reported worldwide in a similar period. Data on individual patients were collated from different sources using a systematic approach to describe the global epidemiology of 907 human H5N1 cases between May, 1997, and April, 2015. The number of affected countries rose between 2003 and 2008, with expansion from east and southeast Asia, then to west Asia and Africa. Most cases (67·2%) occurred from December to March, and the overall case-fatality risk was 483 (53·5%) of 903 cases which varied across geographical regions. Although the incidence in Egypt has increased dramatically since November, 2014, compared with the cases beforehand, there were no significant differences in the fatality risk, history of exposure to poultry, history of patient contact, and time from onset to hospital admission in the recent cases. PMID:27211899

  15. An avian influenza H5N1 virus vaccine candidate based on the extracellular domain produced in yeast system as subviral particles protects chickens from lethal challenge.

    PubMed

    Pietrzak, Maria; Macioła, Agnieszka; Zdanowski, Konrad; Protas-Klukowska, Anna Maria; Olszewska, Monika; Śmietanka, Krzysztof; Minta, Zenon; Szewczyk, Bogusław; Kopera, Edyta

    2016-09-01

    Highly pathogenic avian influenza is an on-going problem in poultry and a potential human pandemic threat. Pandemics occur suddenly and vaccine production must be fast and effective to be of value in controlling the spread of the virus. In this study we evaluated the potential of a recombinant protein from the extracellular domain of an H5 hemagglutinin protein produced in a yeast expression system to act as an effective vaccine. Protein production was efficient, with up to 200 mg purified from 1 L of culture medium. We showed that the deletion of the multibasic cleavage site from the protein improves oligomerization and, consequentially, its immunogenicity. We also showed that immunization with this deleted protein protected chickens from challenge with a highly pathogenic avian influenza H5N1 virus. Our results suggest that this recombinant protein produced in yeast may be an effective vaccine against H5N1 virus in poultry. PMID:27498036

  16. Cross-protective efficacies of highly-pathogenic avian influenza H5N1 vaccines against a recent H5N8 virus.

    PubMed

    Park, Su-Jin; Si, Young-Jae; Kim, Jihye; Song, Min-Suk; Kim, Se-Mi; Kim, Eun-Ha; Kwon, Hyeok-Il; Kim, Young-Il; Lee, Ok-Jun; Shin, Ok Sarah; Kim, Chul-Joong; Shin, Eui-Cheol; Choi, Young Ki

    2016-11-01

    To investigate cross-protective vaccine efficacy of highly-pathogenic avian influenza H5N1 viruses against a recent HPAI H5N8 virus, we immunized C57BL/6 mice and ferrets with three alum-adjuvanted inactivated whole H5N1 vaccines developed through reverse-genetics (Rg): [Vietnam/1194/04xPR8 (clade 1), Korea/W149/06xPR8 (clade 2.2), and Korea/ES223N/03xPR8 (clade 2.5)]. Although relatively low cross-reactivities (10-40 HI titer) were observed against heterologous H5N8 virus, immunized animals were 100% protected from challenge with the 20 mLD50 of H5N8 virus, with the exception of mice vaccinated with 3.5μg of Rg Vietnam/1194/04xPR8. Of note, the Rg Korea/ES223N/03xPR8 vaccine provided not only effective protection, but also markedly inhibited viral replication in the lungs and nasal swabs of vaccine recipients within five days of HPAI H5N8 virus challenge. Further, we demonstrated that antibody-dependent cell-mediated cytotoxicity (ADCC) of an antibody-coated target cell by cytotoxic effector cells also plays a role in the heterologous protection of H5N1 vaccines against H5N8 challenge. PMID:27543757

  17. The contribution of PA-X to the virulence of pandemic 2009 H1N1 and highly pathogenic H5N1 avian influenza viruses.

    PubMed

    Gao, Huijie; Sun, Yipeng; Hu, Jiao; Qi, Lu; Wang, Jinliang; Xiong, Xin; Wang, Yu; He, Qiming; Lin, Yang; Kong, Weili; Seng, Lai-Giea; Sun, Honglei; Pu, Juan; Chang, Kin-Chow; Liu, Xiufan; Liu, Jinhua

    2015-02-05

    PA-X is a novel protein encoded by PA mRNA and is found to decrease the pathogenicity of pandemic 1918 H1N1 virus in mice. However, the importance of PA-X proteins in current epidemiologically important influenza A virus strains is not known. In this study, we report on the pathogenicity and pathological effects of PA-X deficient 2009 pandemic H1N1 (pH1N1) and highly pathogenic avian influenza H5N1 viruses. We found that loss of PA-X expression in pH1N1 and H5N1 viruses increased viral replication and apoptosis in A549 cells and increased virulence and host inflammatory response in mice. In addition, PA-X deficient pH1N1 and H5N1 viruses up-regulated PA mRNA and protein synthesis and increased viral polymerase activity. Loss of PA-X was also accompanied by accelerated nuclear accumulation of PA protein and reduced suppression of PA on non-viral protein expression. Our study highlights the effects of PA-X on the moderation of viral pathogenesis and pathogenicity.

  18. Encephalitis in a stone marten (Martes foina) after natural infection with highly pathogenic avian influenza virus subtype H5N1.

    PubMed

    Klopfleisch, R; Wolf, P U; Wolf, C; Harder, T; Starick, E; Niebuhr, M; Mettenleiter, T C; Teifke, J P

    2007-01-01

    Recent outbreaks of disease in different avian species, caused by the highly pathogenic avian influenza virus (HPAIV), have involved infection by subtype H5N1 of the virus. This virus has also crossed species barriers and infected felines and humans. Here, we report the natural infection of a stone marten (Martes foina) from an area with numerous confirmed cases of H5N1 HPAIV infection in wild birds. Histopathological examination of tissues from this animal revealed a diffuse nonsuppurative panencephalitis with perivascular cuffing, multifocal gliosis and neuronal necrosis. Additionally, focal necrosis of pancreatic acinar cells was observed. Immunohistochemically, lesions in these organs were associated with avian influenza virus antigen in neurons, glial cells and pancreatic acinar cells. Thus, the microscopical lesions and viral antigen distribution in this stone marten differs from that recently described for cats naturally and experimentally infected with the same virus subtype. This is the first report of natural infection of a mustelid with HPAIV H5N1.

  19. Evaluation of vaccines for H5N1 influenza virus in ferrets reveals the potential for protective single-shot immunization.

    PubMed

    Middleton, Deborah; Rockman, Steven; Pearse, Martin; Barr, Ian; Lowther, Sue; Klippel, Jessica; Ryan, David; Brown, Lorena

    2009-08-01

    As part of influenza pandemic preparedness, policy decisions need to be made about how best to utilize vaccines once they are manufactured. Since H5N1 avian influenza virus has the potential to initiate the next human pandemic, isolates of this subtype have been used for the production and testing of prepandemic vaccines. Clinical trials of such vaccines indicate that two injections of preparations containing adjuvant will be required to induce protective immunity. However, this is a working assumption based on classical serological measures only. Examined here are the dose of viral hemagglutinin (HA) and the number of inoculations required for two different H5N1 vaccines to achieve protection in ferrets after lethal H5N1 challenge. Ferrets inoculated twice with 30 microg of A/Vietnam/1194/2004 HA vaccine with AlPO4, or with doses as low as 3.8 microg of HA with Iscomatrix (ISCOMATRIX, referred to as Iscomatrix herein, is a registered trademark of CSL Limited) adjuvant, were completely protected against death and disease after H5N1 challenge, and the protection lasted at least 15 months. Cross-clade protection was also observed with both vaccines. Significantly, complete protection against death could be achieved with only a single inoculation of H5N1 vaccine containing as little as 15 microg of HA with AlPO4 or 3.8 microg of HA with Iscomatrix adjuvant. Ferrets vaccinated with the single-injection Iscomatrix vaccines showed fewer clinical manifestations of infection than those given AlPO4 vaccines and remained highly active. Our data provide the first indication that in the event of a future influenza pandemic, effective mass vaccination may be achievable with a low-dose "single-shot" vaccine and provide not only increased survival but also significant reduction in disease severity.

  20. Characterization and phylogenetic analysis of a highly pathogenic avian influenza H5N1 virus isolated from diseased ostriches (Struthio camelus) in the Kingdom of Saudi Arabia.

    PubMed

    Ismail, Mahmoud Moussa; El-Sabagh, I M; Al-Ankari, Abdul-Rahman

    2014-06-01

    During 2007, two outbreaks of avian influenza virus (AIV) in backyard and commercial ostrich flocks were first reported in the Kingdom of Saudi Arabia (KSA). The infected ostriches suffered from depression, anorexia, and diarrhea and some exhibited sudden death. A rapid AIV-group antigen detection and real-time reverse-transcription PCR (rtRT-PCR) were initially performed on cloacal and tracheal swabs collected from diseased birds. Pools from positive-tested swabs for each flock were utilized for virus isolation in specific-pathogen-free embryonating chicken eggs. H5N1 AIV was identified in the harvested allantoic fluids by hemagglutination followed by hemagglutination inhibition and rtRT-PCR. The viruses responsible for these two outbreaks were sequenced and characterized as HPAIV H5N1 (A/ostrich/Saudi Arabia/6732-3/2007 and A/ostrich/Saudi Arabia/3489-73VIR08/ 2007) from backyard and commercial flocks, respectively. Phylogenetic analysis of both isolates revealed that the two viruses belong to clade 2.2 sublineage II and cluster with the HPAIV H5N1 isolated from falcons and turkeys during 2007 in KSA. PMID:25055639

  1. Novel H5 clade 2.3.4.4 reassortant (H5N1) virus from a green-winged teal in Washington, USA.

    USGS Publications Warehouse

    Torchetti, Mia Kim; Killian, Mary-Lea; Dusek, Robert J.; Pedersen, Janice C.; Hines, Nichole; Bodenstein, Barbara L.; White, C. LeAnn; Ip, Hon S.

    2015-01-01

    Eurasian (EA)-origin H5N8 clade 2.3.4.4 avian influenza viruses were first detected in North America during December 2014. Subsequent reassortment with North American (AM) low-pathogenic wild-bird-origin avian influenza has generated at least two reassortants, including an EA/AM H5N1 from an apparently healthy wild green-winged teal, suggesting continued ongoing reassortment.

  2. Reintroduction of H5N1 highly pathogenic avian influenza virus by migratory water birds, causing poultry outbreaks in the 2010-2011 winter season in Japan.

    PubMed

    Sakoda, Yoshihiro; Ito, Hiroshi; Uchida, Yuko; Okamatsu, Masatoshi; Yamamoto, Naoki; Soda, Kosuke; Nomura, Naoki; Kuribayashi, Saya; Shichinohe, Shintaro; Sunden, Yuji; Umemura, Takashi; Usui, Tatsufumi; Ozaki, Hiroichi; Yamaguchi, Tsuyoshi; Murase, Toshiyuki; Ito, Toshihiro; Saito, Takehiko; Takada, Ayato; Kida, Hiroshi

    2012-03-01

    H5N1 highly pathogenic avian influenza virus (HPAIV) was reintroduced and caused outbreaks in chickens in the 2010-2011 winter season in Japan, which had been free from highly pathogenic avian influenza (HPAI) since 2007 when HPAI outbreaks occurred and were controlled. On 14 October 2010 at Lake Ohnuma, Wakkanai, the northernmost part of Hokkaido, Japan, H5N1 HPAIVs were isolated from faecal samples of ducks flying from their nesting lakes in Siberia. Since then, in Japan, H5N1 HPAIVs have been isolated from 63 wild birds in 17 prefectures and caused HPAI outbreaks in 24 chicken farms in nine prefectures by the end of March in 2011. Each of these isolates was genetically closely related to the HPAIV isolates at Lake Ohnuma, and those in China, Mongolia, Russia and Korea, belonging to genetic clade 2.3.2.1. In addition, these isolates were genetically classified into three groups, suggesting that the viruses were transmitted by migratory water birds through at least three different routes from their northern territory to Japan. These isolates were antigenic variants, which is consistent with selection in poultry under the immunological pressure induced by vaccination. To prevent the perpetuation of viruses in the lakes where water birds nest in summer in Siberia, prompt eradication of HPAIVs in poultry is urgently needed in Asian countries where HPAI has not been controlled.

  3. Molecular analysis of hemagglutinin-1 fragment of avian influenza H5N1 viruses isolated from chicken farms in Indonesia from 2008 to 2010.

    PubMed

    Mahardika, Gusti N; Jonas, Melina; Murwijati, Theresia; Fitria, Nur; Suartha, I Nyoman; Suartini, I Gusti A A; Wibawan, I Wayan Teguh

    2016-04-15

    Highly pathogenic avian influenza virus of subtype H5N1 (AIV-H5N1) has been circulating in Indonesia since 2003. To understand the genetic diversity of these viruses, and to predict vaccine efficacy, the hemaglutinin-1 (HA-1) fragment of viruses isolated from chicken farms in Indonesia from 2008 to 2010 was sequenced and analyzed. The effects of these molecular changes were investigated in challenge experiments and HI assays of homologous and heterologous strains. Molecular analysis showed that these AIV-H5N1 isolates had evolved into three distinct sub-lineages from an ancestor circulating since 2003. Although no significant positive selection of residues was detected, 12 negatively selected sites were identified (p<0.05). Moreover, four sites showed evidence of significant episodic diversifying selection. The findings indicated complete protectivity and high HI titers with homologous strains, compared with protectivity ranging from 40 to 100% and lower HI titers with heterologous strains resulting from polymorphisms at antigenic sites. Our findings provide valuable insight into the molecular evolution of AIV and have important implications for vaccine efficacy and future vaccination strategies.

  4. The Effect of the PB2 Mutation 627K on Highly Pathogenic H5N1 Avian Influenza Virus Is Dependent on the Virus Lineage

    PubMed Central

    Howard, Wendy A.; Núñez, Alejandro; Moncorgé, Olivier; Lycett, Samantha; Banks, Jill

    2013-01-01

    Clade 2.2 Eurasian-lineage H5N1 highly pathogenic avian influenza viruses (HPAIVs) were first detected in Qinghai Lake, China, in 2005 and subsequently spread through Asia, Europe, and Africa. Importantly, these viruses carried a lysine at amino acid position 627 of the PB2 protein (PB2 627K), a known mammalian adaptation motif. Previous avian influenza virus isolates have carried glutamic acid in this position (PB2 627E), commonly described to restrict virus polymerase function in the mammalian host. We sought to examine the effect of PB2 627K on viral maintenance in the avian reservoir. Viruses constructed by reverse genetics were engineered to contain converse PB2 627K/E mutations in a Eurasian H5N1 virus (A/turkey/Turkey/5/2005 [Ty/05]) and, for comparison, a historical pre-Asian H5N1 HPAIV that naturally bears PB2 627E (A/turkey/England/50-92/1991 [50-92]). The 50-92 PB2 627K was genetically unstable during virus propagation, resulting in reversion to PB2 627E or the accumulation of the additional mutation PB2 628R and/or a synonymous mutation from an A to a G nucleotide at nucleotide position 1869 (PB2 A1869G). Intriguingly, PB2 628R and/or A1869G appeared to improve the genetic stability of 50-92 PB2 627K. However, the replication of 50-92 PB2 627K in conjunction with these stabilizing mutations was significantly restricted in experimentally infected chickens, where reversion to PB2 627E occurred. In contrast, no significant effects on viral fitness were observed for Ty/05 PB2 627E or 627K in in vitro or in vivo experiments. Our observations suggest that PB2 627K is supported in Eurasian-lineage viruses; in contrast, PB2 627K carries a significant fitness cost in the historical pre-Asian 50-92 virus. PMID:23843645

  5. Different routes of inoculation impact infectivity and pathogenesis of H5N1 high pathogenicity avian influenza virus infection in chickens and domestic ducks.

    PubMed

    Kwon, Y K; Swayne, D E

    2010-12-01

    The H5N1 type A influenza viruses classified as Qinghai-like virus (clade 2.2) are a unique lineage of type A influenza viruses with the capacity to produce significant disease and mortality in gallinaceous and anseriform birds, including domestic and wild ducks. The objective of this study was to determine the susceptibility and pathogenesis of chickens and domestic ducks to A/Whooper Swan/Mongolia/224/05 (H5N1) high pathogenicity avian influenza (HPAI) virus when administered through respiratory or alimentary routes of exposure. The chickens and ducks were more susceptible to the H5N1 HPAI virus, as evidenced by low infectious and lethal viral doses, when exposed by intranasal as compared to alimentary routes of inoculation (intragastric or oral-fed infected chicken meat). In the alimentary exposure pathogenesis study, pathologic changes included hemorrhage, necrosis, and inflammation in association with virus detection. These changes were generally observed in most of the visceral organs of chickens, between 2 and 4 days postinoculation (DPI), and are similar to lesions and virus localization seen in birds in natural cases or in experimental studies using the intranasal route. Alimentary exposure to the virus caused systemic infection in the ducks, characterized by moderate lymphocytic encephalitis, necrotized hepatitis, and pancreatitis with a corresponding demonstration of virus within the lesions. In both chickens and ducks with alimentary exposure, lesions, virus, or both were first demonstrated in the upper alimentary tract on 1 DPI, suggesting that the alimentary tract was the initial site affected upon consumption of infected meat or on gavage of virus in liquid medium. However, as demonstrated in the infectivity study in chickens, alimentary infection required higher exposure doses to produce infection as compared to intranasal exposure in chickens. These data suggest that upper respiratory exposure to H5N1 HPAI virus in birds is more likely to result in

  6. A Single Electroporation Delivery of a DNA Vaccine Containing the Hemagglutinin Gene of Asian H5N1 Avian Influenza Virus Generated a Protective Antibody Response in Chickens against a North American Virus Strain

    PubMed Central

    Pasick, John; Kobinger, Gary P.; Hannaman, Drew; Berhane, Yohannes; Clavijo, Alfonso; van Drunen Littel-van den Hurk, Sylvia

    2013-01-01

    Protection against the avian influenza (AI) H5N1 virus is suspected to be mainly conferred by the presence of antibodies directed against the hemagglutinin (HA) protein of the virus. A single electroporation delivery of 100 or 250 μg of a DNA vaccine construct, pCAG-HA, carrying the HA gene of strain A/Hanoi/30408/2005 (H5N1), in chickens led to the development of anti-HA antibody response in 16 of 17 immunized birds, as measured by a hemagglutination inhibition (HI) test, competitive enzyme-linked immunosorbent assay (cELISA), and an indirect ELISA. Birds vaccinated by electroporation (n = 11) were protected from experimental AI challenge with strain A/chicken/Pennsylvania/1370/1/1983 (H5N2) as judged by low viral load, absence of clinical symptoms, and absence of mortality (n = 11). In contrast, only two out of 10 birds vaccinated with the same vaccine dose (100 or 250 μg) but without electroporation developed antibodies. These birds showed high viral loads and significant morbidity and mortality after the challenge. Seroconversion was reduced in birds electroporated with a low vaccine dose (10 μg), but the antibody-positive birds were protected against virus challenge. Nonelectroporation delivery of a low-dose vaccine did not result in seroconversion, and the birds were as susceptible as those in the control groups that received the control pCAG vector. Electroporation delivery of the DNA vaccine led to enhanced antibody responses and to protection against the AI virus challenge. The HI test, cELISA, or indirect ELISA for anti-H5 antibodies might serve as a good predictor of the potency and efficacy of a DNA immunization strategy against AI in chickens. PMID:23365205

  7. Comparison of efficacies of RWJ-270201, zanamivir, and oseltamivir against H5N1, H9N2, and other avian influenza viruses.

    PubMed

    Govorkova, E A; Leneva, I A; Goloubeva, O G; Bush, K; Webster, R G

    2001-10-01

    The orally administered neuraminidase (NA) inhibitor RWJ-270201 was tested in parallel with zanamivir and oseltamivir against a panel of avian influenza viruses for inhibition of NA activity and replication in tissue culture. The agents were then tested for protection of mice against lethal H5N1 and H9N2 virus infection. In vitro, RWJ-270201 was highly effective against all nine NA subtypes. NA inhibition by RWJ-270201 (50% inhibitory concentration, 0.9 to 4.3 nM) was superior to that by zanamivir and oseltamivir carboxylate. RWJ-270201 inhibited the replication of avian influenza viruses of both Eurasian and American lineages in MDCK cells (50% effective concentration, 0.5 to 11.8 microM). Mice given 10 mg of RWJ-270201 per kg of body weight per day were completely protected against lethal challenge with influenza A/Hong Kong/156/97 (H5N1) and A/quail/Hong Kong/G1/97 (H9N2) viruses. Both RWJ-270201 and oseltamivir significantly reduced virus titers in mouse lungs at daily dosages of 1.0 and 10 mg/kg and prevented the spread of virus to the brain. When treatment began 48 h after exposure to H5N1 virus, 10 mg of RWJ-270201/kg/day protected 50% of mice from death. These results suggest that RWJ-270201 is at least as effective as either zanamivir or oseltamivir against avian influenza viruses and may be of potential clinical use for treatment of emerging influenza viruses that may be transmitted from birds to humans.

  8. Monoclonal Antibodies against the Fusion Peptide of Hemagglutinin Protect Mice from Lethal Influenza A Virus H5N1 Infection ▿

    PubMed Central

    Prabhu, Nayana; Prabakaran, Mookkan; Ho, Hui-Ting; Velumani, Sumathy; Qiang, Jia; Goutama, Michael; Kwang, Jimmy

    2009-01-01

    The HA2 glycopolypeptide (gp) is highly conserved in all influenza A virus strains, and it is known to play a major role in the fusion of the virus with the endosomal membrane in host cells during the course of viral infection. Vaccines and therapeutics targeting this HA2 gp could induce efficient broad-spectrum immunity against influenza A virus infections. So far, there have been no studies on the possible therapeutic effects of monoclonal antibodies (MAbs), specifically against the fusion peptide of hemagglutinin (HA), upon lethal infections with highly pathogenic avian influenza (HPAI) H5N1 virus. We have identified MAb 1C9, which binds to GLFGAIAGF, a part of the fusion peptide of the HA2 gp. We evaluated the efficacy of MAb 1C9 as a therapy for influenza A virus infections. This MAb, which inhibited cell fusion in vitro when administered passively, protected 100% of mice from challenge with five 50% mouse lethal doses of HPAI H5N1 influenza A viruses from two different clades. Furthermore, it caused earlier clearance of the virus from the lung. The influenza virus load was assessed in lung samples from mice challenged after pretreatment with MAb 1C9 (24 h prior to challenge) and from mice receiving early treatment (24 h after challenge). The study shows that MAb 1C9, which is specific to the antigenically conserved fusion peptide of HA2, can contribute to the cross-clade protection of mice infected with H5N1 virus and mediate more effective recovery from infection. PMID:19109379

  9. Susceptibility to and transmission of H5N1 and H7N1 highly pathogenic avian influenza viruses in bank voles (Myodes glareolus).

    PubMed

    Romero Tejeda, Aurora; Aiello, Roberta; Salomoni, Angela; Berton, Valeria; Vascellari, Marta; Cattoli, Giovanni

    2015-01-01

    The study of influenza type A (IA) infections in wild mammals populations is a critical gap in our knowledge of how IA viruses evolve in novel hosts that could be in close contact with avian reservoir species and other wild animals. The aim of this study was to evaluate the susceptibility to infection, the nasal shedding and the transmissibility of the H7N1 and H5N1 highly pathogenic avian influenza (HPAI) viruses in the bank vole (Myodes glareolus), a wild rodent common throughout Europe and Asia. Two out of 24 H5N1-infected voles displayed evident respiratory distress, while H7N1-infected voles remained asymptomatic. Viable virus was isolated from nasal washes collected from animals infected with both HPAI viruses, and extra-pulmonary infection was confirmed in both experimental groups. Histopathological lesions were evident in the respiratory tract of infected animals, although immunohistochemistry positivity was only detected in lungs and trachea of two H7N1-infected voles. Both HPAI viruses were transmitted by direct contact, and seroconversion was confirmed in 50% and 12.5% of the asymptomatic sentinels in the H7N1 and H5N1 groups, respectively. Interestingly, viable virus was isolated from lungs and nasal washes collected from contact sentinels of both groups. The present study demonstrated that two non-rodent adapted HPAI viruses caused asymptomatic infection in bank voles, which shed high amounts of the viruses and were able to infect contact voles. Further investigations are needed to determine whether bank voles could be involved as silent hosts in the transmission of HPAI viruses to other mammals and domestic poultry. PMID:25963535

  10. Identifying live bird markets with the potential to act as reservoirs of avian influenza A (H5N1) virus: a survey in northern Viet Nam and Cambodia.

    PubMed

    Fournié, Guillaume; Guitian, Javier; Desvaux, Stéphanie; Mangtani, Punam; Ly, Sowath; Cong, Vu Chi; San, Sorn; Dung, Do Huu; Holl, Davun; Pfeiffer, Dirk U; Vong, Sirenda; Ghani, Azra C

    2012-01-01

    Wet markets are common in many parts of the world and may promote the emergence, spread and maintenance of livestock pathogens, including zoonoses. A survey was conducted in order to assess the potential of Vietnamese and Cambodian live bird markets (LBMs) to sustain circulation of highly pathogenic avian influenza virus subtype H5N1 (HPAIV H5N1). Thirty Vietnamese and 8 Cambodian LBMs were visited, and structured interviews were conducted with the market managers and 561 Vietnamese and 84 Cambodian traders. Multivariate and cluster analysis were used to construct a typology of traders based on their poultry management practices. As a result of those practices and large poultry surplus (unsold poultry reoffered for sale the following day), some poultry traders were shown to promote conditions favorable for perpetuating HPAIV H5N1 in LBMs. More than 80% of these traders operated in LBMs located in the most densely populated areas, Ha Noi and Phnom Penh. The profiles of sellers operating at a given LBM could be reliably predicted using basic information about the location and type of market. Consequently, LBMs with the largest combination of risk factors for becoming virus reservoirs could be easily identified, potentially allowing control strategies to be appropriately targeted. These findings are of particular relevance to resource-scarce settings with extensively developed LBM systems, commonly found in South-East Asia. PMID:22675502

  11. Antiviral susceptibility of highly pathogenic avian influenza A(H5N1) viruses isolated from poultry, Vietnam, 2009-2011.

    PubMed

    Nguyen, Ha T; Nguyen, Tung; Mishin, Vasiliy P; Sleeman, Katrina; Balish, Amanda; Jones, Joyce; Creanga, Adrian; Marjuki, Henju; Uyeki, Timothy M; Nguyen, Dang H; Nguyen, Diep T; Do, Hoa T; Klimov, Alexander I; Davis, Charles T; Gubareva, Larisa V

    2013-12-01

    We assessed drug susceptibilities of 125 avian influenza A(H5N1) viruses isolated from poultry in Vietnam during 2009-2011. Of 25 clade 1.1 viruses, all possessed a marker of resistance to M2 blockers amantadine and rimantadine; 24 were inhibited by neuraminidase inhibitors. One clade 1.1 virus contained the R430W neuraminidase gene and reduced inhibition by oseltamivir, zanamivir, and laninamivir 12-, 73-, and 29-fold, respectively. Three of 30 clade 2.3.4 viruses contained a I223T mutation and showed 7-fold reduced inhibition by oseltamivir. One of 70 clade 2.3.2.1 viruses had the H275Y marker of oseltamivir resistance and exhibited highly reduced inhibition by oseltamivir and peramivir; antiviral agents DAS181 and favipiravir inhibited H275Y mutant virus replication in MDCK-SIAT1 cells. Replicative fitness of the H275Y mutant virus was comparable to that of wildtype virus. These findings highlight the role of drug susceptibility monitoring of H5N1 subtype viruses circulating among birds to inform antiviral stockpiling decisions for pandemic preparedness.

  12. Assessment of the removal and inactivation of influenza viruses H5N1 and H1N1 by drinking water treatment.

    PubMed

    Lénès, Dorothée; Deboosere, Nathalie; Ménard-Szczebara, Florence; Jossent, Jérôme; Alexandre, Virginie; Machinal, Claire; Vialette, Michèle

    2010-04-01

    Since 2003, there has been significant concern about the possibility of an outbreak of avian influenza virus subtype H5N1. Moreover, in the last few months, a pandemic of a novel swine-origin influenza A virus, namely A(H1N1), has already caused hundreds of thousands of human cases of illness and thousands of deaths. As those viruses could possibly contaminate water resources through wild birds excreta or through sewage, the aim of our work was to find out whether the treatment processes in use in the drinking water industry are suitable for eradicating them. The effectiveness of physical treatments (coagulation-flocculation-settling, membrane ultrafiltration and ultraviolet) was assessed on H5N1, and that of disinfectants (monochloramine, chlorine dioxide, chlorine, and ozone) was established for both the H5N1 and H1N1 viruses. Natural water samples were spiked with human H5N1/H1N1 viruses. For the coagulation-settling experiments, raw surface water was treated in jar-test pilots with 3 different coagulating agents (aluminum sulfate, ferric chloride, aluminum polychorosulfate). Membrane performance was quantified using a hollow-fiber ultrafiltration system. Ultraviolet irradiation experiments were conducted with a collimated beam that made it possible to assess the effectiveness of various UV doses (25-60 mJ/cm2). In the case of ozone, 0.5 mg/L and 1 mg/L residual concentrations were tested with a contact time of 10 min. Finally, for chlorine, chlorine dioxide and monochloramine treatments, several residual oxidant target levels were tested (from 0.3 to 3 mg/L) with contact times of 5-120 min. The infectivity of the H5N1 and H1N1 viruses in water samples was quantified in cell culture using a microtiter endpoint titration. The impact of coagulation-settling on the H5N1 subtype was quite low and variable. In contrast, ultrafiltration achieved more than a 3-log reduction (and more than a 4-log removal in most cases), and UV treatment was readily effective on its

  13. Assessment of the removal and inactivation of influenza viruses H5N1 and H1N1 by drinking water treatment.

    PubMed

    Lénès, Dorothée; Deboosere, Nathalie; Ménard-Szczebara, Florence; Jossent, Jérôme; Alexandre, Virginie; Machinal, Claire; Vialette, Michèle

    2010-04-01

    Since 2003, there has been significant concern about the possibility of an outbreak of avian influenza virus subtype H5N1. Moreover, in the last few months, a pandemic of a novel swine-origin influenza A virus, namely A(H1N1), has already caused hundreds of thousands of human cases of illness and thousands of deaths. As those viruses could possibly contaminate water resources through wild birds excreta or through sewage, the aim of our work was to find out whether the treatment processes in use in the drinking water industry are suitable for eradicating them. The effectiveness of physical treatments (coagulation-flocculation-settling, membrane ultrafiltration and ultraviolet) was assessed on H5N1, and that of disinfectants (monochloramine, chlorine dioxide, chlorine, and ozone) was established for both the H5N1 and H1N1 viruses. Natural water samples were spiked with human H5N1/H1N1 viruses. For the coagulation-settling experiments, raw surface water was treated in jar-test pilots with 3 different coagulating agents (aluminum sulfate, ferric chloride, aluminum polychorosulfate). Membrane performance was quantified using a hollow-fiber ultrafiltration system. Ultraviolet irradiation experiments were conducted with a collimated beam that made it possible to assess the effectiveness of various UV doses (25-60 mJ/cm2). In the case of ozone, 0.5 mg/L and 1 mg/L residual concentrations were tested with a contact time of 10 min. Finally, for chlorine, chlorine dioxide and monochloramine treatments, several residual oxidant target levels were tested (from 0.3 to 3 mg/L) with contact times of 5-120 min. The infectivity of the H5N1 and H1N1 viruses in water samples was quantified in cell culture using a microtiter endpoint titration. The impact of coagulation-settling on the H5N1 subtype was quite low and variable. In contrast, ultrafiltration achieved more than a 3-log reduction (and more than a 4-log removal in most cases), and UV treatment was readily effective on its

  14. Impact of vaccination on infection with Vietnam H5N1 high pathogenicity avian influenza virus in hens and the eggs they lay.

    PubMed

    Bertran, Kateri; Moresco, Kira; Swayne, David E

    2015-03-10

    High pathogenicity avian influenza virus (HPAIV) infections in chickens negatively impact egg production and cause egg contamination. Previously, vaccination maintained egg production and reduced egg contamination when challenged with a North American H5N2 HPAIV. However, Asian H5N1 HPAIV infection has some characteristics of increased pathogenicity compared to other H5 HPAIV such as more rapid drop and complete cessation in egg production. Sham (vaccinated at 25 and 28 weeks of age), inactivated H5N1 Once (1X-H5-Vax; vaccinated at 28 weeks of age only) and inactivated H5N1 Twice (2X-H5-Vax; vaccinated at 25 and 28 weeks of age) vaccinated adult White Leghorn hens were challenged intranasally at 31 weeks of age with 6.1 log10 mean embryo infectious doses (EID50) of clade 2.3.2.1a H5N1 HPAIV (A/chicken/Vietnam/NCVD-675/2011) which was homologous to the inactivated vaccine. Sham-vaccinated layers experienced 100% mortality within 3 days post-challenge; laid soft and thin-shelled eggs; had recovery of virus from oral swabs and in 53% of the eggs from eggshell surface (35%), yolk (24%), and albumin (41%); and had very high titers of virus (average 7.91 log10 EID50/g) in all segments of the oviduct and ovary. By comparison, 1X- and 2X-H5-Vax challenged hens survived infection, laid similar number of eggs pre- and post-challenge, all eggs had normal egg shell quality, and had significantly fewer contaminated eggs with reduced virus quantity. The 2X-H5-Vax hens had significantly higher HI titers by the day of challenge (304 GMT) and at termination (512 GMT) than 1X-H5-Vax hens (45 GMT and 128 GMT). The current study demonstrated that AIV infections caused by clade 2.3.2.1a H5N1 variants can be effectively controlled by either double or single homologous vaccination. PMID:25657093

  15. Predicting Disease Severity and Viral Spread of H5N1 Influenza Virus in Ferrets in the Context of Natural Exposure Routes

    PubMed Central

    Edenborough, Kathryn M.; Lowther, Suzanne; Laurie, Karen; Yamada, Manabu; Long, Fenella; Bingham, John; Payne, Jean; Harper, Jennifer; Haining, Jessica; Arkinstall, Rachel; Gilbertson, Brad; Middleton, Deborah

    2015-01-01

    ABSTRACT Although avian H5N1 influenza virus has yet to develop the capacity for human-to-human spread, the severity of the rare cases of human infection has warranted intensive follow-up of potentially exposed individuals that may require antiviral prophylaxis. For countries where antiviral drugs are limited, the World Health Organization (WHO) has developed a risk categorization for different levels of exposure to environmental, poultry, or human sources of infection. While these take into account the infection source, they do not account for the likely mode of virus entry that the individual may have experienced from that source and how this could affect the disease outcome. Knowledge of the kinetics and spread of virus after natural routes of exposure may further inform the risk of infection, as well as the likely disease severity. Using the ferret model of H5N1 infection, we compared the commonly used but artificial inoculation method that saturates the total respiratory tract (TRT) with virus to upper respiratory tract (URT) and oral routes of delivery, those likely to be encountered by humans in nature. We show that there was no statistically significant difference in survival rate with the different routes of infection, but the disease characteristics were somewhat different. Following URT infection, viral spread to systemic organs was comparatively delayed and more focal than after TRT infection. By both routes, severe disease was associated with early viremia and central nervous system infection. After oral exposure to the virus, mild infections were common suggesting consumption of virus-contaminated liquids may be associated with seroconversion in the absence of severe disease. IMPORTANCE Risks for human H5N1 infection include direct contact with infected birds and frequenting contaminated environments. We used H5N1 ferret infection models to show that breathing in the virus was more likely to produce clinical infection than swallowing contaminated

  16. Human microRNA-24 modulates highly pathogenic avian-origin H5N1 influenza A virus infection in A549 cells by targeting secretory pathway furin.

    PubMed

    Loveday, Emma-Kate; Diederich, Sandra; Pasick, John; Jean, François

    2015-01-01

    A common critical cellular event that many human enveloped viruses share is the requirement for proteolytic cleavage of the viral glycoprotein by furin in the host secretory pathway. For example, the furin-dependent proteolytic activation of highly pathogenic (HP) influenza A (infA) H5 and H7 haemagglutinin precursor (HA0) subtypes is critical for yielding fusion-competent infectious virions. In this study, we hypothesized that viral hijacking of the furin pathway by HP infA viruses to permit cleavage of HA0 could represent a novel molecular mechanism controlling the dynamic production of fusion-competent infectious virus particles during the viral life cycle. We explored the biological role of a newly identified furin-directed human microRNA, miR-24, in this process as a potential post-transcriptional regulator of the furin-mediated activation of HA0 and production of fusion-competent virions in the host secretory pathway. We report that miR-24 and furin are differentially expressed in human A549 cells infected with HP avian-origin infA H5N1. Using miR-24 mimics, we demonstrated a robust decrease in both furin mRNA levels and intracellular furin activity in A549 cells. Importantly, pretreatment of A549 cells with miR-24 mimicked these results: a robust decrease of H5N1 infectious virions and a complete block of H5N1 virus spread that was not observed in A549 cells infected with low-pathogenicity swine-origin infA H1N1 virus. Our results suggest that viral-specific downregulation of furin-directed microRNAs such as miR-24 during the life cycle of HP infA viruses may represent a novel regulatory mechanism that governs furin-mediated proteolytic activation of HA0 glycoproteins and production of infectious virions.

  17. Surveillance plan for the early detection of H5N1 highly pathogenic avian influenza virus in migratory birds in the United States: surveillance year 2009

    USGS Publications Warehouse

    Brand, Christopher J.

    2009-01-01

    Executive Summary: This Surveillance Plan (Plan) describes plans for conducting surveillance of wild birds in the United States and its Territories and Freely-Associated States to provide for early detection of the introduction of the H5N1 Highly Pathogenic Avian Influenza (HPAI) subtype of the influenza A virus by migratory birds during the 2009 surveillance year, spanning the period of April 1, 2009 - March 31, 2010. The Plan represents a continuation of surveillance efforts begun in 2006 under the Interagency Strategic Plan for the Early Detection of H5N1 Highly Pathogenic Avian Influenza in Wild Migratory Birds (U.S. Department of Agriculture and U.S. Department of the Interior, 2006). The Plan sets forth sampling plans by: region, target species or species groups to be sampled, locations of sampling, sample sizes, and sampling approaches and methods. This Plan will be reviewed annually and modified as appropriate for subsequent surveillance years based on evaluation of information from previous years of surveillance, changing patterns and threats of H5N1 HPAI, and changes in funding availability for avian influenza surveillance. Specific sampling strategies will be developed accordingly within each of six regions, defined here as Alaska, Hawaiian/Pacific Islands, Lower Pacific Flyway (Washington, Oregon, California, Idaho, Nevada, Arizona), Central Flyway, Mississippi Flyway, and Atlantic Flyway.

  18. Effect of statin treatments on highly pathogenic avian influenza H5N1, seasonal and H1N1pdm09 virus infections in BALB/c mice

    PubMed Central

    Kumaki, Yohichi; Morrey, John D; Barnard, Dale L

    2013-01-01

    Statins are used to control elevated cholesterol or hypercholesterolemia, but have previously been reported to have antiviral properties. Aims To show efficacy of statins in various influenza virus mouse models. Materials & methods BALB/c mice were treated intraperitoneally or orally with several types of statins (simvastatin, lovastatin, mevastatin, pitavastatin, atorvastatin or rosuvastatin) at various concentrations before or after infection with either influenza A/Duck/ MN/1525/81 H5N1 virus, influenza A/Vietnam/1203/2004 H5N1 virus, influenza A/ Victoria/3/75 H3N2 virus, influenza A/NWS/33 H1N1 virus or influenza A/CA/04/09 H1N1pdm09 virus. Results The statins administered intraperitoneally or orally at any dose did not significantly enhance the total survivors relative to untreated controls. In addition, infected mice receiving any concentration of statin were not protected against weight loss due to the infection. None of the statins significantly increased the mean day of death relative to mice in the placebo treatment group. Furthermore, the statins had relatively few ameliorative effects on lung pathology or lung weights at day 3 and 6 after virus exposure, although mice treated with simvastatin did have improved lung function as measured by arterial saturated oxygen levels in one experiment. Conclusion Statins showed relatively little efficacy in any mouse model used by any parameter tested. PMID:23420457

  19. The Influenza Virus H5N1 Infection Can Induce ROS Production for Viral Replication and Host Cell Death in A549 Cells Modulated by Human Cu/Zn Superoxide Dismutase (SOD1) Overexpression

    PubMed Central

    Lin, Xian; Wang, Ruifang; Zou, Wei; Sun, Xin; Liu, Xiaokun; Zhao, Lianzhong; Wang, Shengyu; Jin, Meilin

    2016-01-01

    Highly pathogenic H5N1 infections are often accompanied by excessive pro-inflammatory response, high viral titer, and apoptosis; as such, the efficient control of these infections poses a great challenge. The pathogenesis of influenza virus infection is also related to oxidative stress. However, the role of endogenic genes with antioxidant effect in the control of influenza viruses, especially H5N1 viruses, should be further investigated. In this study, the H5N1 infection in lung epithelial cells decreased Cu/Zn superoxide dismutase (SOD1) expression at mRNA and protein levels. Forced SOD1 expression significantly inhibited the H5N1-induced increase in reactive oxygen species, decreased pro-inflammatory response, prevented p65 and p38 phosphorylation, and impeded viral ribonucleoprotein nuclear export and viral replication. The SOD1 overexpression also rescued H5N1-induced cellular apoptosis and alleviated H5N1-caused mitochondrial dysfunction. Therefore, this study described the role of SOD1 in the replication of H5N1 influenza virus and emphasized the relevance of this enzyme in the control of H5N1 replication in epithelial cells. Pharmacological modulation or targeting SOD1 may open a new way to fight H5N1 influenza virus. PMID:26761025

  20. The Influenza Virus H5N1 Infection Can Induce ROS Production for Viral Replication and Host Cell Death in A549 Cells Modulated by Human Cu/Zn Superoxide Dismutase (SOD1) Overexpression.

    PubMed

    Lin, Xian; Wang, Ruifang; Zou, Wei; Sun, Xin; Liu, Xiaokun; Zhao, Lianzhong; Wang, Shengyu; Jin, Meilin

    2016-01-08

    Highly pathogenic H5N1 infections are often accompanied by excessive pro-inflammatory response, high viral titer, and apoptosis; as such, the efficient control of these infections poses a great challenge. The pathogenesis of influenza virus infection is also related to oxidative stress. However, the role of endogenic genes with antioxidant effect in the control of influenza viruses, especially H5N1 viruses, should be further investigated. In this study, the H5N1 infection in lung epithelial cells decreased Cu/Zn superoxide dismutase (SOD1) expression at mRNA and protein levels. Forced SOD1 expression significantly inhibited the H5N1-induced increase in reactive oxygen species, decreased pro-inflammatory response, prevented p65 and p38 phosphorylation, and impeded viral ribonucleoprotein nuclear export and viral replication. The SOD1 overexpression also rescued H5N1-induced cellular apoptosis and alleviated H5N1-caused mitochondrial dysfunction. Therefore, this study described the role of SOD1 in the replication of H5N1 influenza virus and emphasized the relevance of this enzyme in the control of H5N1 replication in epithelial cells. Pharmacological modulation or targeting SOD1 may open a new way to fight H5N1 influenza virus.

  1. The evolutionary dynamics of highly pathogenic avian influenza H5N1 in south-central Vietnam reveals multiple clades evolving from Chinese and Cambodian viruses.

    PubMed

    Nguyen, Tinh Huu; Than, Van Thai; Thanh, Hien Dang; Nguyen, Van Quang; Nguyen, Kim Hue; Nguyen, Duc Tan; Park, Jong-Hwa; Chung, In Sik; Jeong, Dae Gwin; Chang, Kyu-Tae; Oh, Tae Kwang; Kim, Wonyong

    2015-10-01

    In Vietnam, highly pathogenic avian influenza (HPAI), such as that caused by H5N1 viruses, is the most highly contagious infectious disease that has been affecting domestic poultry in recent years. Vietnam might be an evolutionary hotspot and a potential source of globally pandemic strains. However, few studies have reported viruses circulating in the south-central region of Vietnam. In the present study, 47 H5N1-positive samples were collected from both vaccinated and unvaccinated poultry farms in the South Central Coast region of Vietnam during 2013-2014, and their genetic diversity was analyzed. A common sequence motif for HPAI virus was identified at HA-cleavage sites in all samples: either RERRRKR/G (clades 2.3.2.1c and 2.3.2.1a) or REGRRKKR/G (clade 1.1.2). Phylogenetic analysis of HA genes identified three clades of HPAI H5N1: 1.1.2 (n=1), 2.3.2.1a (n=1), and 2.3.2.1c (n=45). The phylogenetic analysis indicated that these Vietnamese clades may have evolved from Chinese and Cambodian virus clades isolated in 2012-2013 but are less closely related to the clades detected from the Tyva Republic, Bulgaria, Mongolia, Japan, and Korea in 2009-2011. Detection of the coexistence of virus clades 2.3.2.1 and the very virulent 1.1.2 in the south-central regions suggests their local importance and highlights concerns regarding their spread, both northwards and southwards, as well as the potential for reassortment. The obtained data highlight the importance of regular identification of viral evolution and the development and use of region-specific vaccines.

  2. The evolutionary dynamics of highly pathogenic avian influenza H5N1 in south-central Vietnam reveals multiple clades evolving from Chinese and Cambodian viruses.

    PubMed

    Nguyen, Tinh Huu; Than, Van Thai; Thanh, Hien Dang; Nguyen, Van Quang; Nguyen, Kim Hue; Nguyen, Duc Tan; Park, Jong-Hwa; Chung, In Sik; Jeong, Dae Gwin; Chang, Kyu-Tae; Oh, Tae Kwang; Kim, Wonyong

    2015-10-01

    In Vietnam, highly pathogenic avian influenza (HPAI), such as that caused by H5N1 viruses, is the most highly contagious infectious disease that has been affecting domestic poultry in recent years. Vietnam might be an evolutionary hotspot and a potential source of globally pandemic strains. However, few studies have reported viruses circulating in the south-central region of Vietnam. In the present study, 47 H5N1-positive samples were collected from both vaccinated and unvaccinated poultry farms in the South Central Coast region of Vietnam during 2013-2014, and their genetic diversity was analyzed. A common sequence motif for HPAI virus was identified at HA-cleavage sites in all samples: either RERRRKR/G (clades 2.3.2.1c and 2.3.2.1a) or REGRRKKR/G (clade 1.1.2). Phylogenetic analysis of HA genes identified three clades of HPAI H5N1: 1.1.2 (n=1), 2.3.2.1a (n=1), and 2.3.2.1c (n=45). The phylogenetic analysis indicated that these Vietnamese clades may have evolved from Chinese and Cambodian virus clades isolated in 2012-2013 but are less closely related to the clades detected from the Tyva Republic, Bulgaria, Mongolia, Japan, and Korea in 2009-2011. Detection of the coexistence of virus clades 2.3.2.1 and the very virulent 1.1.2 in the south-central regions suggests their local importance and highlights concerns regarding their spread, both northwards and southwards, as well as the potential for reassortment. The obtained data highlight the importance of regular identification of viral evolution and the development and use of region-specific vaccines. PMID:26577194

  3. Surveillance, epidemiological, and virological detection of highly pathogenic H5N1 avian influenza viruses in duck and poultry from Bangladesh.

    PubMed

    Ansari, Wahedul Karim; Parvej, Md Shafiullah; El Zowalaty, Mohamed E; Jackson, Sally; Bustin, Stephen A; Ibrahim, Adel K; El Zowalaty, Ahmed E; Rahman, Md Tanvir; Zhang, Han; Khan, Mohammad Ferdousur Rahman; Ahamed, Md Mostakin; Rahman, Md Fasiur; Rahman, Marzia; Nazir, K H M Nazmul Hussain; Ahmed, Sultan; Hossen, Md Liakot; Kafi, Md Abdul; Yamage, Mat; Debnath, Nitish C; Ahmed, Graba; Ashour, Hossam M; Masudur Rahman, Md; Noreddin, Ayman; Rahman, Md Bahanur

    2016-09-25

    Avian influenza viruses (AIVs) continue to pose a global threat. Waterfowl are the main reservoir and are responsible for the spillover of AIVs to other hosts. This study was conducted as part of routine surveillance activities in Bangladesh and it reports on the serological and molecular detection of H5N1 AIV subtype. A total of 2169 cloacal and 2191 oropharyngeal swabs as well as 1725 sera samples were collected from live birds including duck and chicken in different locations in Bangladesh between the years of 2013 and 2014. Samples were tested using virus isolation, serological tests and molecular methods of RT-PCR. Influenza A viruses were detected using reverse transcription PCR targeting the virus matrix (M) gene in 41/4360 (0.94%) samples including both cloacal and oropharyngeal swab samples, 31 of which were subtyped as H5N1 using subtype-specific primers. Twenty-one live H5N1 virus isolates were recovered from those 31 samples. Screening of 1,868 blood samples collected from the same birds using H5-specific ELISA identified 545/1603 (34%) positive samples. Disconcertingly, an analysis of 221 serum samples collected from vaccinated layer chicken in four districts revealed that only 18 samples (8.1%) were seropositive for anti H5 antibodies, compared to unvaccinated birds (n=105), where 8 samples (7.6%) were seropositive. Our result indicates that the vaccination program as currently implemented should be reviewed and updated. In addition, surveillance programs are crucial for monitoring the efficacy of the current poultry vaccinations programs, and to monitor the circulating AIV strains and emergence of AIV subtypes in Bangladesh. PMID:27599930

  4. [Screening efficient siRNAs in vitro as the candidate genes for chicken anti-avian influenza virus H5N1 breeding].

    PubMed

    Zhang, P; Wang, J G; Wan, J G; Liu, W Q

    2010-01-01

    The frequent disease outbreaks caused by avian influenza virus not only affect the poultry industry but also pose a threat to human safety. To address the problem, RNA interference (RNAi) has recently been widely used as a potential antiviral approach. Transgenesis in combination with RNAi to specifically inhibit avian enza virus gene expression has been proposed to make chickens resistant to the infection. For the transgenic breeding, screening in vitro efficient siRNAs as the candidate genes is one of the most important tasks. Here, we combined an online search tool and a series of bioinformatics programs with a set of rules for designing siRNAs targeted towards different mRNA regions of H5N1 avian influenza virus. Five rational siRNAs were chosen by this method, five U6 promoter-driven shRNA expression plasmids containing the siRNA genes were constructed and used for producing stably transfected MDCK cells. The data obtained by virus titration, IFA, PI-stained flow cytometry, real-time quantitative RT-PCR, and DAS-ELISA analyses showed that all five stably transfected cell lines we re resistant to virusreplication when exposed to 100 CCID50 of avian influenza virus H5N1. Finally, most effective plasmids (pSi-604i and pSi-1597i) as the candidates for making the transgenic chickens were chosen. These findings provide baseline information on use of RNAi technique for breeding transgenic chickens resistant to avian influenza virus.

  5. Highly (H5N1) and Low (H7N2) Pathogenic Avian Influenza Virus Infection in Falcons Via Nasochoanal Route and Ingestion of Experimentally Infected Prey

    PubMed Central

    Bertran, Kateri; Busquets, Núria; Abad, Francesc Xavier; García de la Fuente, Jorge; Solanes, David; Cordón, Iván; Costa, Taiana; Dolz, Roser; Majó, Natàlia

    2012-01-01

    An experimental infection with highly pathogenic avian influenza (HPAI) and low pathogenic avian influenza (LPAI) viruses was carried out on falcons in order to examine the effects of these viruses in terms of pathogenesis, viral distribution in tissues and viral shedding. The distribution pattern of influenza virus receptors was also assessed. Captive-reared gyr-saker (Falco rusticolus x Falco cherrug) hybrid falcons were challenged with a HPAI H5N1 virus (A/Great crested grebe/Basque Country/06.03249/2006) or a LPAI H7N2 virus (A/Anas plathyrhynchos/Spain/1877/2009), both via the nasochoanal route and by ingestion of previously infected specific pathogen free chicks. Infected falcons exhibited similar infection dynamics despite the different routes of exposure, demonstrating the effectiveness of in vivo feeding route. H5N1 infected falcons died, or were euthanized, between 5–7 days post-infection (dpi) after showing acute severe neurological signs. Presence of viral antigen in several tissues was confirmed by immunohistochemistry and real time RT-PCR (RRT-PCR), which were generally associated with significant microscopical lesions, mostly in the brain. Neither clinical signs, nor histopathological findings were observed in any of the H7N2 LPAI infected falcons, although all of them had seroconverted by 11 dpi. Avian receptors were strongly present in the upper respiratory tract of the falcons, in accordance with the consistent oral viral shedding detected by RRT-PCR in both H5N1 HPAI and H7N2 LPAI infected falcons. The present study demonstrates that gyr-saker hybrid falcons are highly susceptible to H5N1 HPAI virus infection, as previously observed, and that they may play a major role in the spreading of both HPAI and LPAI viruses. For the first time in raptors, natural infection by feeding on infected prey was successfully reproduced. The use of avian prey species in falconry husbandry and wildlife rehabilitation facilities could put valuable birds of prey

  6. Highly (H5N1) and low (H7N2) pathogenic avian influenza virus infection in falcons via nasochoanal route and ingestion of experimentally infected prey.

    PubMed

    Bertran, Kateri; Busquets, Núria; Abad, Francesc Xavier; García de la Fuente, Jorge; Solanes, David; Cordón, Iván; Costa, Taiana; Dolz, Roser; Majó, Natàlia

    2012-01-01

    An experimental infection with highly pathogenic avian influenza (HPAI) and low pathogenic avian influenza (LPAI) viruses was carried out on falcons in order to examine the effects of these viruses in terms of pathogenesis, viral distribution in tissues and viral shedding. The distribution pattern of influenza virus receptors was also assessed. Captive-reared gyr-saker (Falco rusticolus x Falco cherrug) hybrid falcons were challenged with a HPAI H5N1 virus (A/Great crested grebe/Basque Country/06.03249/2006) or a LPAI H7N2 virus (A/Anas plathyrhynchos/Spain/1877/2009), both via the nasochoanal route and by ingestion of previously infected specific pathogen free chicks. Infected falcons exhibited similar infection dynamics despite the different routes of exposure, demonstrating the effectiveness of in vivo feeding route. H5N1 infected falcons died, or were euthanized, between 5-7 days post-infection (dpi) after showing acute severe neurological signs. Presence of viral antigen in several tissues was confirmed by immunohistochemistry and real time RT-PCR (RRT-PCR), which were generally associated with significant microscopical lesions, mostly in the brain. Neither clinical signs, nor histopathological findings were observed in any of the H7N2 LPAI infected falcons, although all of them had seroconverted by 11 dpi. Avian receptors were strongly present in the upper respiratory tract of the falcons, in accordance with the consistent oral viral shedding detected by RRT-PCR in both H5N1 HPAI and H7N2 LPAI infected falcons. The present study demonstrates that gyr-saker hybrid falcons are highly susceptible to H5N1 HPAI virus infection, as previously observed, and that they may play a major role in the spreading of both HPAI and LPAI viruses. For the first time in raptors, natural infection by feeding on infected prey was successfully reproduced. The use of avian prey species in falconry husbandry and wildlife rehabilitation facilities could put valuable birds of prey and

  7. A Single Amino Acid in the M1 Protein Responsible for the Different Pathogenic Potentials of H5N1 Highly Pathogenic Avian Influenza Virus Strains

    PubMed Central

    Nao, Naganori; Kajihara, Masahiro; Manzoor, Rashid; Maruyama, Junki; Yoshida, Reiko; Muramatsu, Mieko; Miyamoto, Hiroko; Igarashi, Manabu; Eguchi, Nao; Sato, Masahiro; Kondoh, Tatsunari; Okamatsu, Masatoshi; Sakoda, Yoshihiro; Kida, Hiroshi; Takada, Ayato

    2015-01-01

    Two highly pathogenic avian influenza virus strains, A/duck/Hokkaido/WZ83/2010 (H5N1) (WZ83) and A/duck/Hokkaido/WZ101/2010 (H5N1) (WZ101), which were isolated from wild ducks in Japan, were found to be genetically similar, with only two amino acid differences in their M1 and PB1 proteins at positions 43 and 317, respectively. We found that both WZ83 and WZ101 caused lethal infection in chickens but WZ101 killed them more rapidly than WZ83. Interestingly, ducks experimentally infected with WZ83 showed no or only mild clinical symptoms, whereas WZ101 was highly lethal. We then generated reassortants between these viruses and found that exchange of the M gene segment completely switched the pathogenic phenotype in both chickens and ducks, indicating that the difference in the pathogenicity for these avian species between WZ83 and WZ101 was determined by only a single amino acid in the M1 protein. It was also found that WZ101 showed higher pathogenicity than WZ83 in mice and that WZ83, whose M gene was replaced with that of WZ101, showed higher pathogenicity than wild-type WZ83, although this reassortant virus was not fully pathogenic compared to wild-type WZ101. These results suggest that the amino acid at position 43 of the M1 protein is one of the factors contributing to the pathogenicity of H5N1 highly pathogenic avian influenza viruses in both avian and mammalian hosts. PMID:26368015

  8. Spatial and Temporal Association of Outbreaks of H5N1 Influenza Virus Infection in Wild Birds with the 0°C Isotherm

    PubMed Central

    Reperant, Leslie A.; Fučkar, Neven S.; Osterhaus, Albert D. M. E.; Dobson, Andrew P.; Kuiken, Thijs

    2010-01-01

    Wild bird movements and aggregations following spells of cold weather may have resulted in the spread of highly pathogenic avian influenza virus (HPAIV) H5N1 in Europe during the winter of 2005–2006. Waterbirds are constrained in winter to areas where bodies of water remain unfrozen in order to feed. On the one hand, waterbirds may choose to winter as close as possible to their breeding grounds in order to conserve energy for subsequent reproduction, and may be displaced by cold fronts. On the other hand, waterbirds may choose to winter in regions where adverse weather conditions are rare, and may be slowed by cold fronts upon their journey back to the breeding grounds, which typically starts before the end of winter. Waterbirds will thus tend to aggregate along cold fronts close to the 0°C isotherm during winter, creating conditions that favour HPAIV H5N1 transmission and spread. We determined that the occurrence of outbreaks of HPAIV H5N1 infection in waterbirds in Europe during the winter of 2005–2006 was associated with temperatures close to 0°C. The analysis suggests a significant spatial and temporal association of outbreaks caused by HPAIV H5N1 in wild birds with maximum surface air temperatures of 0°C–2°C on the day of the outbreaks and the two preceding days. At locations where waterbird census data have been collected since 1990, maximum mallard counts occurred when average and maximum surface air temperatures were 0°C and 3°C, respectively. Overall, the abundance of mallards (Anas platyrhynchos) and common pochards (Aythya ferina) was highest when surface air temperatures were lower than the mean temperatures of the region investigated. The analysis implies that waterbird movements associated with cold weather, and congregation of waterbirds along the 0°C isotherm likely contributed to the spread and geographical distribution of outbreaks of HPAIV H5N1 infection in wild birds in Europe during the winter of 2005–2006. PMID:20386716

  9. Spatial modeling of wild bird risk factors to investigate highly pathogenic A(H5N1) avian influenza virus transmission

    USGS Publications Warehouse

    Prosser, Diann J.; Hungerford, Laura L.; Erwin, R. Michael; Ottinger, Mary Ann; Takekawa, John Y.; Newman, Scott H.; Xiao, Xianming; Ellis, Erie C.

    2016-01-01

    One of the longest-persisting avian influenza viruses in history, highly pathogenic avian influenza virus (HPAIV) A(H5N1), continues to evolve after 18 years, advancing the threat of a global pandemic. Wild waterfowl (family Anatidae), are reported as secondary transmitters of HPAIV, and primary reservoirs for low-pathogenic avian influenza viruses, yet spatial inputs for disease risk modeling for this group have been lacking. Using GIS and Monte Carlo simulations, we developed geospatial indices of waterfowl abundance at 1 and 30 km resolutions and for the breeding and wintering seasons for China, the epicenter of H5N1. Two spatial layers were developed: cumulative waterfowl abundance (WAB), a measure of predicted abundance across species, and cumulative abundance weighted by H5N1 prevalence (WPR), whereby abundance for each species was adjusted based on prevalence values then totaled across species. Spatial patterns of the model output differed between seasons, with higher WAB and WPR in the northern and western regions of China for the breeding season and in the southeast for the wintering season. Uncertainty measures indicated highest error in southeastern China for both WAB and WPR. We also explored the effect of resampling waterfowl layers from 1 km to 30 km resolution for multi-scale risk modeling. Results indicated low average difference (less than 0.16 and 0.01 standard deviations for WAB and WPR, respectively), with greatest differences in the north for the breeding season and southeast for the wintering season. This work provides the first geospatial models of waterfowl abundance available for China. The indices provide important inputs for modeling disease transmission risk at the interface of poultry and wild birds. These models are easily adaptable, have broad utility to both disease and conservation needs, and will be available to the scientific community for advanced modeling applications.

  10. Avian Influenza A(H5N1) Virus Outbreak Investigation: Application of the FAO-OIE-WHO Four-way Linking Framework in Indonesia.

    PubMed

    Setiawaty, V; Dharmayanti, N L P I; Misriyah; Pawestri, H A; Azhar, M; Tallis, G; Schoonman, L; Samaan, G

    2015-08-01

    WHO, FAO and OIE developed a 'four-way linking' framework to enhance the cross-sectoral sharing of epidemiological and virological information in responding to zoonotic disease outbreaks. In Indonesia, outbreak response challenges include completeness of data shared between human and animal health authorities. The four-way linking framework (human health laboratory/epidemiology and animal health laboratory/epidemiology) was applied in the investigation of the 193 rd human case of avian influenza A(H5N1) virus infection. As recommended by the framework, outbreak investigation and risk assessment findings were shared. On 18 June 2013, a hospital in West Java Province reported a suspect H5N1 case in a 2-year-old male. The case was laboratory-confirmed that evening, and the information was immediately shared with the Ministry of Agriculture. The human health epidemiology/laboratory team investigated the outbreak and conducted an initial risk assessment on 19 June. The likelihood of secondary cases was deemed low as none of the case contacts were sick. By 3 July, no secondary cases associated with the outbreak were identified. The animal health epidemiology/laboratory investigation was conducted on 19-25 June and found that a live bird market visited by the case was positive for H5N1 virus. Once both human and market virus isolates were sequenced, a second risk assessment was conducted jointly by the human health and animal health epidemiology/laboratory teams. This assessment concluded that the likelihood of additional human cases associated with this outbreak was low but that future sporadic human infections could not be ruled out because of challenges in controlling H5N1 virus contamination in markets. Findings from the outbreak investigation and risk assessments were shared with stakeholders at both Ministries. The four-way linking framework clarified the type of data to be shared. Both human health and animal health teams made ample data available, and there was

  11. 14-Deoxy-11,12-didehydroandrographolide attenuates excessive inflammatory responses and protects mice lethally challenged with highly pathogenic A(H5N1) influenza viruses.

    PubMed

    Cai, Wentao; Chen, Sunrui; Li, Yongtao; Zhang, Anding; Zhou, Hongbo; Chen, Huanchun; Jin, Meilin

    2016-09-01

    Traditional Chinese medicine (TCM) has been an excellent treasury for centuries' accumulation of clinical experiences, which deserves to be tapped for potential drugs and improved using modern scientific methods. 14-Deoxy-11,12-didehydroandrographolide (DAP), a major component of an important TCM named Andrographis paniculata, with non-toxic concentration of 1000 mg/kg/day, effectively reduced the mortality and weight loss of mice lethally challenged with A/chicken/Hubei/327/2004 (H5N1) or A/PR/8/34 (H1N1) influenza A viruses (IAV) when initiated at 4 h before infection, or A/duck/Hubei/XN/2007 (H5N1) when initiated at 4 h or 48 h before infection, or 4 h post-infection (pi). DAP (1000 or 500 mg/kg/day) also significantly diminished lung virus titres of infected mice when initiated at 4 h or 48 h before infection, or 4 h pi. In the infection of A/duck/Hubei/XN/2007 (H5N1), DAP (1000 mg/kg/day) treatment initiated at 48 h before infection gained the best efficacy that virus titres in lungs of mice in log10TCID50/mL reduced from 2.61 ± 0.14 on 3 days post-infection (dpi), 2.98 ± 0.17 on 5 dpi, 3.54 ± 0.19 on 7 dpi to 1.46 ± 0.14 on 3 dpi, 1.86 ± 0.18 on 5 dpi, 2.03 ± 0.21 on 7 dpi. Moreover, DAP obviously alleviated lung histopathology and also strongly inhibited proinflammatory cytokines/chemokines expression. The mRNA levels of TNF-α, IL-1β, IL-6, CCL-2/MCP-1, IFN-α, IFN-β, IFN-γ, MIP-1α, MIP-1β in lungs of A/duck/Hubei/XN/2007 (H5N1)-infected mice and serum protein expression of TNF-α, IL-1β, IL-6, CCL-2/MCP-1 and CXCL-10/IP-10 in mice infected with all the three strains of IAV were all significantly reduced by DAP. Results demonstrated that DAP could restrain both the host intense inflammatory responses and high viral load, which were considered to contribute to the pathogenesis of H5N1 virus and should be controlled together in a clinical setting. Considering the anti-inflammatory and anti-IAV activities of DAP, DAP may

  12. Reverse genetics based rgH5N2 vaccine provides protection against high dose challenge of H5N1 avian influenza virus in chicken.

    PubMed

    Bhatia, S; Khandia, R; Sood, R; Bhat, S; Siddiqui, A; Jahagirdhar, G; Mishra, S; Mishra, A; Pateriya, A K; Kulkarni, D D

    2016-08-01

    An inactivated vaccine was developed using the rgH5N2 virus (6 + 2 reassortant) generated by plasmid based reverse genetics system (RGS) with WSN/33/H1N1 as backbone virus. Following mutation of the basic amino acid cleavage site RRRKKR*GLF to IETR*GLF, the H5-HA (haemagglutinin) gene of the selected donor H5N1 virus (A/chicken/West Bengal/80995/2008) of antigenic clade 2.2 was used along with the N2-NA gene from H9N2 field isolate (A/chicken/Uttar Pradesh/2543/2004) for generation of the rgH5N2 virus. A single dose (0.5 ml/bird) of the inactivated rgH5N2 vaccine protected 100% of the vaccinated chickens (n = 10) on 28(th) dpv (early challenge) and 90% of the vaccinated chickens (n = 10) on 200(th) dpv (late challenge) against high dose challenge with HPAI virus (10(9) EID50/bird). Challenge virus shedding via oropharynx and cloaca of the vaccinated chickens was detectable by realtime RT-PCR during 1-5 dpc and 1-9 days dpc in the early and the late challenge, respectively. The protective level of antibodies (mean HI titre > 128) was maintained without booster vaccination for 200 days. The present study provides the experimental evidence about the extent of protection provided by a reverse genetics based vaccine for clade 2.2 H5N1 viruses against challenge with high dose of field virus at two different time points (28 dpv and 200 dpv). The challenge study is uniquely different from the previous similar experiments on account of 1000 times higher dose of challenge and protection at 200 dpv. The protection and virus shedding data of the study may be useful for countries planning to use H5 vaccine in poultry especially against the clade 2.2 H5N1 viruses. PMID:27296706

  13. Effects of preventive administration of oxidized dextran on liver injury and reparative regeneration in mice infected with influenza A/H5N1 virus.

    PubMed

    Shkurupy, V A; Potapova, O V; Sharkova, T V; Shestopalov, A M; Troitskii, A V

    2015-02-01

    Intranasal infection of outbred male mice with influenza A/H5N1 A/goose/Krasnoozerskoye/627/05 virus led to high (85%) mortality of animals. Morphological studies of liver specimens showed destructive changes in the parenchyma (93.5% hepatocytes), caused by long persistence of the virus in the liver. The virus persistence was conjugated with activation of cellular immunity, manifesting by an increase in the counts of cells with high expression of proinflammatory cytokines (TNF-α) and lysosomal enzymes (lysozyme, cathepsin D). Injections of oxidized dextran 3 and 1 days before infection reduced mortality and 2-fold attenuated destructive changes in the liver, presumably due to prevention of virus penetration into the target cells, modulation of immune reactions, and stimulation of reparative plastic processes.

  14. Effects of preventive administration of oxidized dextran on liver injury and reparative regeneration in mice infected with influenza A/H5N1 virus.

    PubMed

    Shkurupy, V A; Potapova, O V; Sharkova, T V; Shestopalov, A M; Troitskii, A V

    2015-02-01

    Intranasal infection of outbred male mice with influenza A/H5N1 A/goose/Krasnoozerskoye/627/05 virus led to high (85%) mortality of animals. Morphological studies of liver specimens showed destructive changes in the parenchyma (93.5% hepatocytes), caused by long persistence of the virus in the liver. The virus persistence was conjugated with activation of cellular immunity, manifesting by an increase in the counts of cells with high expression of proinflammatory cytokines (TNF-α) and lysosomal enzymes (lysozyme, cathepsin D). Injections of oxidized dextran 3 and 1 days before infection reduced mortality and 2-fold attenuated destructive changes in the liver, presumably due to prevention of virus penetration into the target cells, modulation of immune reactions, and stimulation of reparative plastic processes. PMID:25708331

  15. Pathogenicity in domestic ducks and mice of highly pathogenic H5N1 clade 2.3.2.1 influenza viruses recently circulating in Eastern Asia.

    PubMed

    Kang, Hyun-Mi; Choi, Jun-Gu; Kim, Kwang-Il; Kim, Bang-Sil; Batchuluun, Damdinjav; Erdene-Ochir, Tseren-Ochir; Kim, Min-Chul; Kwon, Jun-Hun; Park, Choi-Kyu; Lee, Youn-Jeong

    2013-12-27

    Influenza virus A (H5N1) clade 2.3.2.1 has recently caused widespread outbreaks of disease in domestic poultry and wild birds in Eastern Asia. In the current study, the antigenicity and pathogenicity of three clade 2.3.2.1 viruses (Ck/Kr/Gimje/08, Ws/Mongolia/1/09, and Ws/Mongolia/7/10) were investigated in domestic ducks and mice. The H5N1 influenza viruses in this study were antigenically similar to each other (r-values of 0.35-1.4). The three viruses replicated systemically in all tissues tested in domestic ducks, indicating high pathogenicity. However, the viruses produced different clinical signs and mortality rates: Ck/Kr/Gimje/08 and Ws/Mongolia/1/09 resulted in 100% mortality with severe neurological signs, whereas Ws/Mongolia/7/10 resulted in 50% mortality with relatively mild neurological signs. In mice, infection with Ck/Kr/Gimje/08 and Ws/Mongolia/7/10 resulted in weight loss that peaked at 4 days post-infection (22.3% and 20.8%, respectively), same MLD50 (2.2 Log10 EID50) and systemic replication. The three viruses had K deletion at the -2 position of the HA1-connecting peptide (PQRERRRK-R), which is associated with increased virulence in domestic ducks and harbored NA stalk deletion, NS1 deletion and mutation of P42S in NS1, and full length (90aa) in PB1-F2, which confer increased virulence in mice. Our study shows that clade 2.3.2.1 viruses from Korea and Mongolia are antigenically similar and highly pathogenic in both domestic ducks and mice. Moreover, we provide molecular determinants of the clade 2.3.2.1 viruses associated with the pathogenicity in domestic ducks and mice, respectively.

  16. ELPylated haemagglutinins produced in tobacco plants induce potentially neutralizing antibodies against H5N1 viruses in mice.

    PubMed

    Phan, Hoang T; Pohl, Julia; Floss, Doreen M; Rabenstein, Frank; Veits, Jutta; Le, Binh T; Chu, Ha H; Hause, Gerd; Mettenleiter, Thomas; Conrad, Udo

    2013-06-01

    Reducing the cost of vaccine production is a key priority for veterinary research, and the possibility of heterologously expressing antigen in plants provides a particularly attractive means of achieving this. Here, we report the expression of the avian influenza virus haemagglutinin (AIV HA) in tobacco, both as a monomer and as a trimer in its native and its ELPylated form. We firstly presented evidence to produce stabilized trimers of soluble HA in plants. ELPylation of these trimers does not influence the trimerization. Strong expression enhancement in planta caused by ELPylation was demonstrated for trimerized H5-ELP. ELPylated trimers could be purified by a membrane-based inverse transition cycling procedure with the potential of successful scale-up. The trimeric form of AIV HA was found to enhance the HA-specific immune response compared with the monomeric form. Plant-derived AIV HA trimers elicited potentially neutralizing antibodies interacting with both homologous virus-like particles from plants and heterologous inactivated AIV. ELPylation did not influence the functionality and the antigenicity of the stabilized H5 trimers. These data allow further developments including scale-up of production, purification and virus challenge experiments with the final goal to achieve suitable technologies for efficient avian flu vaccine production.

  17. Combinations of Oseltamivir and T-705 Extend the Treatment Window for Highly Pathogenic Influenza A(H5N1) Virus Infection in Mice

    PubMed Central

    Marathe, Bindumadhav M.; Wong, Sook-San; Vogel, Peter; Garcia-Alcalde, Fernando; Webster, Robert G.; Webby, Richard J.; Najera, Isabel; Govorkova, Elena A.

    2016-01-01

    Current anti-influenza therapy depends on administering drugs soon after infection, which is often impractical. We assessed whether combinations of oseltamivir (a neuraminidase inhibitor) and T-705 (a nonspecific inhibitor of viral polymerases) could extend the window for treating lethal infection with highly pathogenic A(H5N1) influenza virus in mice. Combination therapy protected 100% of mice, even when delayed until 96 h postinoculation. Compared to animals receiving monotherapy, mice receiving combination therapy had reduced viral loads and restricted viral spread in lung tissues, limited lung damage, and decreased inflammatory cytokine production. Next-generation sequencing showed that virus populations in T-705–treated mice had greater genetic variability, with more frequent transversion events, than did populations in control and oseltamivir-treated mice, but no substitutions associated with resistance to oseltamivir or T-705 were detected. Thus, combination therapy extended the treatment window for A(H5N1) influenza infection in mice and should be considered for evaluation in a clinical setting. PMID:27221530

  18. Combinations of Oseltamivir and T-705 Extend the Treatment Window for Highly Pathogenic Influenza A(H5N1) Virus Infection in Mice.

    PubMed

    Marathe, Bindumadhav M; Wong, Sook-San; Vogel, Peter; Garcia-Alcalde, Fernando; Webster, Robert G; Webby, Richard J; Najera, Isabel; Govorkova, Elena A

    2016-01-01

    Current anti-influenza therapy depends on administering drugs soon after infection, which is often impractical. We assessed whether combinations of oseltamivir (a neuraminidase inhibitor) and T-705 (a nonspecific inhibitor of viral polymerases) could extend the window for treating lethal infection with highly pathogenic A(H5N1) influenza virus in mice. Combination therapy protected 100% of mice, even when delayed until 96 h postinoculation. Compared to animals receiving monotherapy, mice receiving combination therapy had reduced viral loads and restricted viral spread in lung tissues, limited lung damage, and decreased inflammatory cytokine production. Next-generation sequencing showed that virus populations in T-705-treated mice had greater genetic variability, with more frequent transversion events, than did populations in control and oseltamivir-treated mice, but no substitutions associated with resistance to oseltamivir or T-705 were detected. Thus, combination therapy extended the treatment window for A(H5N1) influenza infection in mice and should be considered for evaluation in a clinical setting. PMID:27221530

  19. Delayed antiviral plus immunomodulator treatment still reduces mortality in mice infected by high inoculum of influenza A/H5N1 virus.

    PubMed

    Zheng, Bo-Jian; Chan, Kwok-Wah; Lin, Yong-Ping; Zhao, Guang-Yu; Chan, Chris; Zhang, Hao-Jie; Chen, Hong-Lin; Wong, Samson S Y; Lau, Susanna K P; Woo, Patrick C Y; Chan, Kwok-Hung; Jin, Dong-Yan; Yuen, Kwok-Yung

    2008-06-10

    The mortality of human infection by influenza A/H5N1 virus can exceed 80%. The high mortality and its poor response to the neuraminidase inhibitor oseltamivir have been attributed to uncontrolled virus-induced cytokine storm. We challenged BALB/c mice with 1,000 LD50 of influenza A/Vietnam/1194/04. Survival, body weight, histopathology, inflammatory markers, viral loads, T lymphocyte counts, and neutralizing antibody response were documented in infected mice treated individually or in combination with zanamvir, celecoxib, gemfibrozil, and mesalazine. To imitate the real-life scenario, treatment was initiated at 48 h after viral challenge. There were significant improvements in survival rate (P = 0.02), survival time (P < 0.02), and inflammatory markers (P < 0.01) in the group treated with a triple combination of zanamivir, celecoxib, and mesalazine when compared with zanamivir alone. Zanamivir with or without immunomodulators reduced viral load to a similar extent. Insignificant prolongation of survival was observed when individual agents were used alone. Significantly higher levels of CD4+ and CD8+ T lymphocytes and less pulmonary inflammation were also found in the group receiving triple therapy. Zanamivir alone reduced viral load but not inflammation and mortality. The survival benefits of adding celecoxib and mesalazine to zanamivir could be caused by their synergistic effects in reducing cytokine dysfunction and preventing apoptosis. Combinations of a neuraminidase inhibitor with these immunomodulators should be considered in randomized controlled treatment trials of patients suffering from H5N1 infection.

  20. The amino-terminal region of the neuraminidase protein from avian H5N1 influenza virus is important for its biosynthetic transport to the host cell surface.

    PubMed

    Qian, Guomin; Wang, Song; Chi, Xiaojuan; Li, Hua; Wei, Haitao; Zhu, Xiaomei; Chen, Yuhai; Chen, Ji-Long

    2014-12-01

    Influenza virus neuraminidase (NA) is a major viral envelope glycoprotein, which plays a critical role in viral infection. Although NA functional domains have been determined previously, the precise role of the amino acids located at the N-terminus of avian H5N1 NA for protein expression and intracellular transport to the host plasma membrane is not fully understood. In the present study, a series of N-terminal truncation or deletion mutants of H5N1 NA were generated and their expression and intracellular trafficking were investigated. Protein expression from mutants NAΔ20, NAΔ35, NAΔ40, NAΔ7-20 and NAΔ7-35 was undetectable by immunoblotting and by performing NA activity assays. Mutants NAΔ6, NAΔ11 and NAΔ15-20 showed a marked decreased in protein expression, whereas mutants NAΔ7-15 and NAΔ15 displayed a slight increase in protein expression, compared with that of the native NA protein. These data suggest that amino acid residues 16-20 are vital for NA protein expression, while amino acids 7-15 might suppress NA protein expression. In deletion mutants NAΔ7-15 and NAΔ15 there was an accumulation of NA protein at the juxta-nuclear region, with reduced expression of NA at the cell surface. Although active Cdc42 could promote transport of wild-type NA to the host cell surface, this member of the Rho family of GTPases failed to regulate transport of mutants NAΔ7-15 and NAΔ15. The results of the study reveal that amino acid residues 7-15 of H5N1 NA are critical for its biosynthetic transport to the host cell surface.

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

    PubMed

    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

    2015-07-01

    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

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

    PubMed

    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

    2015-07-01

    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

  3. Serological Evidence for Non-Lethal Exposures of Mongolian Wild Birds to Highly Pathogenic Avian Influenza H5N1 Virus

    PubMed Central

    Gilbert, Martin; Koel, Björn F.; Bestebroer, Theo M.; Lewis, Nicola S.; Smith, Derek J.; Fouchier, Ron A. M.

    2014-01-01

    Surveillance for highly pathogenic avian influenza viruses (HPAIV) in wild birds is logistically demanding due to the very low rates of virus detection. Serological approaches may be more cost effective as they require smaller sample sizes to identify exposed populations. We hypothesized that antigenic differences between classical Eurasian H5 subtype viruses (which have low pathogenicity in chickens) and H5N1 viruses of the Goose/Guangdong/96 H5 lineage (which are HPAIV) may be used to differentiate populations where HPAIVs have been circulating, from those where they have not. To test this we performed hemagglutination inhibition assays to compare the reactivity of serum samples from wild birds in Mongolia (where HPAIV has been circulating, n = 1,832) and Europe (where HPAIV has been rare or absent, n = 497) to a panel of reference viruses including classical Eurasian H5 (of low pathogenicity), and five HPAIV H5N1 antigens of the Asian lineage A/Goose/Guangdong/1/96. Antibody titres were detected against at least one of the test antigens for 182 Mongolian serum samples (total seroprevalence of 0.10, n = 1,832, 95% adjusted Wald confidence limits of 0.09–0.11) and 25 of the European sera tested (total seroprevalence of 0.05, n = 497, 95% adjusted Wald confidence limits of 0.03–0.07). A bias in antibody titres to HPAIV antigens was found in the Mongolian sample set (22/182) that was absent in the European sera (0/25). Although the interpretation of serological data from wild birds is complicated by the possibility of exposure to multiple strains, and variability in the timing of exposure, these findings suggest that a proportion of the Mongolian population had survived exposure to HPAIV, and that serological assays may enhance the targeting of traditional HPAIV surveillance toward populations where isolation of HPAIV is more likely. PMID:25502318

  4. Serological evidence for non-lethal exposures of Mongolian wild birds to highly pathogenic avian influenza H5N1 virus.

    PubMed

    Gilbert, Martin; Koel, Björn F; Bestebroer, Theo M; Lewis, Nicola S; Smith, Derek J; Fouchier, Ron A M

    2014-01-01

    Surveillance for highly pathogenic avian influenza viruses (HPAIV) in wild birds is logistically demanding due to the very low rates of virus detection. Serological approaches may be more cost effective as they require smaller sample sizes to identify exposed populations. We hypothesized that antigenic differences between classical Eurasian H5 subtype viruses (which have low pathogenicity in chickens) and H5N1 viruses of the Goose/Guangdong/96 H5 lineage (which are HPAIV) may be used to differentiate populations where HPAIVs have been circulating, from those where they have not. To test this we performed hemagglutination inhibition assays to compare the reactivity of serum samples from wild birds in Mongolia (where HPAIV has been circulating, n = 1,832) and Europe (where HPAIV has been rare or absent, n = 497) to a panel of reference viruses including classical Eurasian H5 (of low pathogenicity), and five HPAIV H5N1 antigens of the Asian lineage A/Goose/Guangdong/1/96. Antibody titres were detected against at least one of the test antigens for 182 Mongolian serum samples (total seroprevalence of 0.10, n = 1,832, 95% adjusted Wald confidence limits of 0.09-0.11) and 25 of the European sera tested (total seroprevalence of 0.05, n = 497, 95% adjusted Wald confidence limits of 0.03-0.07). A bias in antibody titres to HPAIV antigens was found in the Mongolian sample set (22/182) that was absent in the European sera (0/25). Although the interpretation of serological data from wild birds is complicated by the possibility of exposure to multiple strains, and variability in the timing of exposure, these findings suggest that a proportion of the Mongolian population had survived exposure to HPAIV, and that serological assays may enhance the targeting of traditional HPAIV surveillance toward populations where isolation of HPAIV is more likely.

  5. PA-X-associated early alleviation of the acute lung injury contributes to the attenuation of a highly pathogenic H5N1 avian influenza virus in mice.

    PubMed

    Hu, Jiao; Mo, Yiqun; Gao, Zhao; Wang, Xiaoquan; Gu, Min; Liang, Yanyan; Cheng, Xin; Hu, Shunlin; Liu, Wenbo; Liu, Huimou; Chen, Sujuan; Liu, Xiaowen; Peng, Daxing; Liu, Xiufan

    2016-08-01

    PA-X is a novel discovered accessory protein encoded by the PA mRNA. Our previous study demonstrated that PA-X decreases the virulence of a highly pathogenic H5N1 strain A/Chicken/Jiangsu/k0402/2010 in mice. However, the underlying mechanism of virulence attenuation associated with PA-X is still unknown. In this study, we compared two PA-X-deficient mutant viruses and the parental virus in terms of induction of pathology and manipulation of host response in the mouse lung, stimulation of cell death and PA nuclear accumulation. We first found that down-regulated PA-X expression markedly aggravated the acute lung injury of the infected mice early on day 1 post-infection (p.i.). We then determined that loss of PA-X expression induced higher levels of cytokines, chemokines and complement-derived peptides (C3a and C5a) in the lung, especially at early time point's p.i. In addition, in vitro assays showed that the PA-X-deficient viruses enhanced cell death and increased expression of reactive oxygen species (ROS) in mammalian cells. Moreover, we also found that PA nuclear accumulation of the PA-X-null viruses accelerated in MDCK cells. These results demonstrate that PA-X decreases the level of complement components, ROS, cell death and inflammatory response, which may together contribute to the alleviated lung injury and the attenuation of the virulence of H5N1 virus in mice.

  6. Immunogenetic factors associated with severe respiratory illness caused by zoonotic H1N1 and H5N1 influenza viruses.

    PubMed

    Juno, Jennifer; Fowke, Keith R; Keynan, Yoav

    2012-01-01

    Following the 2009 H1N1 pandemic and ongoing sporadic avian-to-human transmission of H5N1 viruses, an emphasis has been placed on better understanding the determinants and pathogenesis of severe influenza infections. Much of the current literature has focused on viral genetics and its impact on host immunity as well as novel risk factors for severe infection (particularly within the H1N1 pandemic). An understanding of the host genetic determinants of susceptibility and severe respiratory illness, however, is currently lacking. By better defining the role of genetic variability in influenza infection and identifying key polymorphisms that impair the host immune response or correlate with protection, we will be able to better identify at-risk populations and new targets for therapeutic interventions and vaccines. This paper will summarize known immunogenetic factors associated with susceptibility or severity of both pH1N1 and H5N1 infections and will also identify genetic pathways and polymorphisms of high relevance for future study.

  7. Differential immune response of mallard duck peripheral blood mononuclear cells to two highly pathogenic avian influenza H5N1 viruses with distinct pathogenicity in mallard ducks.

    PubMed

    Cui, Zhu; Hu, Jiao; He, Liang; Li, Qunhui; Gu, Min; Wang, Xiaoquan; Hu, Shunlin; Liu, Huimou; Liu, Wenbo; Liu, Xiaowen; Liu, Xiufan

    2014-02-01

    CK10 and GS10 are two H5N1 highly pathogenic influenza viruses of similar genetic background but differ in their pathogenicity in mallard ducks. CK10 is highly pathogenic whereas GS10 is low pathogenic. In this study, strong inflammatory response in terms of the expression level of several cytokines was observed in mallard duck peripheral blood mononuclear cells (PBMC) infected with CK10 while mild response was triggered in those by GS10 infection. Two remarkable and intense peaks of immune response were induced by CK10 infection within 24 hours (at 8 and 24 hours post infection, respectively) without reducing the virus replication. Our observations indicated that sustained and intense innate immune responses may be central to the high pathogenicity caused by CK10 in ducks.

  8. Production of H5N1 Influenza Virus Matrix Protein 2 Ectodomain Protein Bodies in Tobacco Plants and in Insect Cells as a Candidate Universal Influenza Vaccine.

    PubMed

    Mbewana, Sandiswa; Mortimer, Elizabeth; Pêra, Francisco F P G; Hitzeroth, Inga Isabel; Rybicki, Edward P

    2015-01-01

    The spread of influenza A viruses is partially controlled and prevented by vaccination. The matrix protein 2 ectodomain (M2e) is the most conserved sequence in influenza A viruses, and is therefore a good potential target for a vaccine to protect against multiple virus subtypes. We explored the feasibility of an M2e-based universal influenza A vaccine candidate based on the highly pathogenic avian influenza A virus, H5N1. A synthetic M2e gene was human- and plant-codon optimized and fused in-frame with a sequence encoding the N-terminal proline-rich domain (Zera(®)) of the γ-zein protein of maize. Zera(®)M2e was expressed transiently in Nicotiana benthamiana and Sf21 baculovirus/insect cell expression systems, and Zera(®)M2e protein bodies (PBs) were successfully produced in both expression systems. The plant-produced Zera(®)M2e PBs were purified and injected into Balb/c mice. Western blot analysis using insect cell-produced Zera(®)M2e PBs and multiple tandem M2e sequences (5xM2e) fused with the avian influenza H5N1 transmembrane and cytosolic tail (5xM2e_tHA) confirmed the presence of M2e-specific antibodies in immunized mice sera. The immunogenicity of the Zera(®)M2e indicates that our plant-produced protein has potential as an inexpensive universal influenza A vaccine. PMID:26697423

  9. Production of H5N1 Influenza Virus Matrix Protein 2 Ectodomain Protein Bodies in Tobacco Plants and in Insect Cells as a Candidate Universal Influenza Vaccine

    PubMed Central

    Mbewana, Sandiswa; Mortimer, Elizabeth; Pêra, Francisco F. P. G.; Hitzeroth, Inga Isabel; Rybicki, Edward P.

    2015-01-01

    The spread of influenza A viruses is partially controlled and prevented by vaccination. The matrix protein 2 ectodomain (M2e) is the most conserved sequence in influenza A viruses, and is therefore a good potential target for a vaccine to protect against multiple virus subtypes. We explored the feasibility of an M2e-based universal influenza A vaccine candidate based on the highly pathogenic avian influenza A virus, H5N1. A synthetic M2e gene was human- and plant-codon optimized and fused in-frame with a sequence encoding the N-terminal proline-rich domain (Zera®) of the γ-zein protein of maize. Zera®M2e was expressed transiently in Nicotiana benthamiana and Sf21 baculovirus/insect cell expression systems, and Zera®M2e protein bodies (PBs) were successfully produced in both expression systems. The plant-produced Zera®M2e PBs were purified and injected into Balb/c mice. Western blot analysis using insect cell-produced Zera®M2e PBs and multiple tandem M2e sequences (5xM2e) fused with the avian influenza H5N1 transmembrane and cytosolic tail (5xM2e_tHA) confirmed the presence of M2e-specific antibodies in immunized mice sera. The immunogenicity of the Zera®M2e indicates that our plant-produced protein has potential as an inexpensive universal influenza A vaccine. PMID:26697423

  10. Transmission rate and reproductive number of the H5N1 highly pathogenic avian influenza virus during the December 2005-July 2008 epidemic in Nigeria.

    PubMed

    Bett, B; Henning, J; Abdu, P; Okike, I; Poole, J; Young, J; Randolph, T F; Perry, B D

    2014-02-01

    We quantified the between-village transmission rate, β (the rate of transmission of H5N1 HPAI virus per effective contact), and the reproductive number, Re (the average number of outbreaks caused by one infectious village during its entire infectious period), of H5N1 highly pathogenic avian influenza (HPAI) virus in Nigeria using outbreak data collected between December 2005 and July 2008. We classified the outbreaks into two phases to assess the effectiveness of the control measures implemented. Phase 1 (December 2005-October 2006) represents the period when the Federal Government of Nigeria managed the HPAI surveillance and response measures, while Phase 2 (November 2006-July 2008) represents the time during which the Nigeria Avian Influenza Control and Human Pandemic Preparedness project (NAICP), funded by a World Bank credit of US$ 50 million, had taken over the management of most of the interventions. We used a total of 204 outbreaks from 176 villages that occurred in 78 local government areas of 25 states. The compartmental susceptible-infectious model was used as the analytical tool. Means and 95% percentile confidence intervals were obtained using bootstrapping techniques. The overall mean β (assuming a duration of infectiousness, T, of 12 days) was 0.07/day (95% percentile confidence interval: 0.06-0.09). The first and second phases of the epidemic had comparable β estimates of 0.06/day (0.04-0.09) and 0.08/day (0.06-0.10), respectively. The Re of the virus associated with these β and T estimates was 0.9 (0.7-1.1); the first and second phases of the epidemic had Re of 0.84 (0.5-1.2) and 0.9 (0.6-1.2), respectively. We conclude that the intervention measures implemented in the second phase of the epidemic had comparable effects to those implemented during the first phase and that the Re of the epidemic was low, indicating that the Nigeria H5N1 HPAI epidemic was unstable.

  11. Generation of an infectious clone of duck enteritis virus (DEV) and of a vectored DEV expressing hemagglutinin of H5N1 avian influenza virus.

    PubMed

    Wang, Jichun; Osterrieder, Nikolaus

    2011-07-01

    We report on the generation of an infectious bacterial artificial chromosome (BAC) clone of duck enteritis virus (DEV) and a vectored DEV vaccine expressing hemagglutinin (H5) of high pathogenicity H5N1 avian influenza virus (AIV). For generation of the DEV BAC, we inserted mini-F vector sequences by homologous recombination in lieu of the UL44 (gC) gene of DEV isolate 2085. DNA of the resulting in recombinant virus v2085-GFPΔgC was electroporated into Escherichia coli and a full-length DEV BAC clone (p2085) was recovered. Transfection of p2085 into chicken embryo cells resulted in DEV-specific plaques exhibiting green autofluorescence. A gC-negative mutant, v2085ΔgC, was generated by deleting mini-F vector sequences by using Cre-Lox recombination, and a revertant virus v2085ΔgC-R was constructed by co-transfection of p2085 with UL44 sequences. Finally, AIV H5 was inserted into p2085, and high-level H5 expression of the v2085_H5 virus was detected by indirect immunofluorescence and western blotting. Plaque area measurements showed that v2085ΔgC plaques were significantly increased (12%) over those of parental 2085 virus or the v2085ΔgC-R revertant virus (ANOVA, P<0.05), while plaque areas of the H5- or GFP-expressing DEV mutants were significantly smaller. There was no significant difference between DEV with respect to virus titers determined after trypsinization titration of infected cells, while virus titers of infected-cell supernatants revealed significant reductions in case of the gC-negative viruses of more than 700-fold when compared to parental 2085 or v2085ΔgC-R. Cell-associated virus titers of gC-negative DEV also showed significant reduction of 50-500-fold (ANOVA, P<0.05). We conclude that (i) absence of DEV gC results in increased plaque sizes in vitro, (ii) gC plays a role in DEV egress, and (iii) generation of an infectious DEV clone allows rapid generation of vectored vaccines.

  12. Comparison of 3 kinds of Toll-like receptor ligands for inactivated avian H5N1 influenza virus intranasal immunization in chicken.

    PubMed

    Liang, Jinfeng; Fu, Jia; Kang, Haihong; Lin, Jian; Yu, Qinghua; Yang, Qian

    2013-10-01

    To evaluate the effects of co-administration of inactivated avian influenza H5N1 virus (IAIV) and different Toll-like receptor (TLR) ligands in chickens, 10-d-old chickens were immunized intranasally with IAIV and TLR ligand [Bacillus subtilis spores, polyinosinic-polycytidylic acid, and CpG oligodeoxynucleotides (CpG-ODN), respectively]. The results showed that both anti-avian influenza virus (AIV) specific secretory IgA level in respiratory tract and anti-AIV specific IgG level in serum significantly increased, as well as the expressions of IL-12, interferon-γ, IL-6, and TLR in the nasal cavity and trachea after intranasal immunization with IAIV and TLR ligand. Among the used TLR ligands, B. subtilis spores as the adjuvant for nasal IAIV had the strongest effect on the expression of IL-6 and IL-12 (P < 0.01), whereas the CpG-ODN could present an advantageous effect on the induction of anti-AIV specific IgG and neutralization antibodies (P < 0.01). The chickens that were previously co-administrated with IAIV and B. subtilis spores could survive at an improved rate upon challenge by live AIV H5N1 virus. Our study suggested that B. subtilis spores, polyinosinic-polycytidylic acid, or CpG-ODN all could effectively enhance the local and systemic immune responses to IAIV in chickens. Considering of the effects and cost of these TLR ligands, we prospected that B. subtilis spores might serve as a more affordable and efficacious mucosal adjuvant for nasal IAIV in chickens. PMID:24046412

  13. Structural and functional changes in pulmonary macrophages and lungs of mice infected with influenza virus A/H5N1 A/goose/Krasnoozerskoye/627/05.

    PubMed

    Kovner, A V; Anikina, A G; Potapova, O V; Sharkova, T V; Cherdanceva, L A; Shkurupy, V A; Shestopalov, A M

    2012-06-01

    C57Bl/6 mice were intranasally infected with influenza virus A/H5N1 A/goose/Krasnoozerskoye/627/05. The mortality rate of animals reached 70% on day 14 of the disease. The lungs of animals were characterized by necroses, destruction of vessels, hemorrhagic and thrombotic complications, edematous syndrome, and early fibrosis of the interstitium. On days 6-10 after infection, fibrosis was found in the zones of postnecrotic inflammatory infiltration. The expression of lysozyme and myeloperoxidase by pulmonary macrophages was initially increased, but decreased on day 10 of the study. The number of cathepsin D-expressing macrophages was elevated up to the 10th day of examination.

  14. Genetic and biological characterization of avian influenza H5N1 viruses isolated from wild birds and poultry in Western Siberia.

    PubMed

    Sharshov, Kirill; Romanovskaya, Alesia; Uzhachenko, Roman; Durymanov, Alexander; Zaykovskaya, Anna; Kurskaya, Olga; Ilinykh, Philipp; Silko, Nikita; Kulak, Mikhail; Alekseev, Alexander; Zolotykh, Sergei; Shestopalov, Alexander; Drozdov, Iliya

    2010-07-01

    Three viruses included in the study were isolated from dead birds (A/duck/Omsk/1822/2006, A/chicken/Reshoty/02/2006, and A/duck/Tuva/01/2006), whereas the virus A/common gull/Chany/P/2006 was isolated from an apparently healthy gull during outbreaks of highly pathogenic avian influenza in Russia in 2006. The intravenous pathogenicity index (IVPI) of viruses A/duck/Omsk/1822/2006, A/chicken/Reshoty/02/2006, and A/duck/Tuva/01/2006 ranged from 2.7 to 3.0, while the virus A/common gull/Chany/P/2006 had a markedly lower IVPI of 1.7. The virus A/common gull/Chany/P/2006 had a unique pattern of six amino acid substitutions in the regions of viral proteins crucial for virulence of H5N1 viruses. We hypothesize that these substitutions may affect the pathogenicity of A/common gull/Chany/P/2006.

  15. A non-VH1-69 heterosubtypic neutralizing human monoclonal antibody protects mice against H1N1 and H5N1 viruses.

    PubMed

    De Marco, Donata; Clementi, Nicola; Mancini, Nicasio; Solforosi, Laura; Moreno, Guisella J; Sun, Xiangjie; Tumpey, Terrence M; Gubareva, Larisa V; Mishin, Vasiliy; Clementi, Massimo; Burioni, Roberto

    2012-01-01

    Influenza viruses are among the most important human pathogens and are responsible for annual epidemics and sporadic, potentially devastating pandemics. The humoral immune response plays an important role in the defense against these viruses, providing protection mainly by producing antibodies directed against the hemagglutinin (HA) glycoprotein. However, their high genetic variability allows the virus to evade the host immune response and the potential protection offered by seasonal vaccines. The emergence of resistance to antiviral drugs in recent years further limits the options available for the control of influenza. The development of alternative strategies for influenza prophylaxis and therapy is therefore urgently needed. In this study, we describe a human monoclonal antibody (PN-SIA49) that recognizes a highly conserved epitope located on the stem region of the HA and able to neutralize a broad spectrum of influenza viruses belonging to different subtypes (H1, H2 and H5). Furthermore, we describe its protective activity in mice after lethal challenge with H1N1 and H5N1 viruses suggesting a potential application in the treatment of influenza virus infections. PMID:22496802

  16. Pathogenicity of Genetically Similar, H5N1 Highly Pathogenic Avian Influenza Virus Strains in Chicken and the Differences in Sensitivity among Different Chicken Breeds

    PubMed Central

    Matsuu, Aya; Kobayashi, Tomoko; Patchimasiri, Tuangthong; Shiina, Takashi; Suzuki, Shingo; Chaichoune, Kridsada; Ratanakorn, Parntep; Hiromoto, Yasuaki; Abe, Haruka; Parchariyanon, Sujira; Saito, Takehiko

    2016-01-01

    Differences in the pathogenicity of genetically closely related H5N1 highly pathogenic avian influenza viruses (HPAIVs) were evaluated in White Leghorn chickens. These viruses varied in the clinical symptoms they induced, including lethality, virus shedding, and replication in host tissues. A comparison of the host responses in the lung, brain, and spleen suggested that the differences in viral replication efficiency were related to the host cytokine response at the early phase of infection, especially variations in the proinflammatory cytokine IL-6. Based on these findings, we inoculated the virus that showed the mildest pathogenicity among the five tested, A/pigeon/Thailand/VSMU-7-NPT/2004, into four breeds of Thai indigenous chicken, Phadu-Hung-Dang (PHD), Chee, Dang, and Luang-Hung-Khao (LHK), to explore effects of genetic background on host response. Among these breeds, Chee, Dang, and LHK showed significantly longer survival times than White Leghorns. Virus shedding from dead Thai indigenous chickens was significantly lower than that from White Leghorns. Although polymorphisms were observed in the Mx and MHC class I genes, there was no significant association between the polymorphisms in these loci and resistance to HPAIV. PMID:27078641

  17. Pathogenicity of Genetically Similar, H5N1 Highly Pathogenic Avian Influenza Virus Strains in Chicken and the Differences in Sensitivity among Different Chicken Breeds.

    PubMed

    Matsuu, Aya; Kobayashi, Tomoko; Patchimasiri, Tuangthong; Shiina, Takashi; Suzuki, Shingo; Chaichoune, Kridsada; Ratanakorn, Parntep; Hiromoto, Yasuaki; Abe, Haruka; Parchariyanon, Sujira; Saito, Takehiko

    2016-01-01

    Differences in the pathogenicity of genetically closely related H5N1 highly pathogenic avian influenza viruses (HPAIVs) were evaluated in White Leghorn chickens. These viruses varied in the clinical symptoms they induced, including lethality, virus shedding, and replication in host tissues. A comparison of the host responses in the lung, brain, and spleen suggested that the differences in viral replication efficiency were related to the host cytokine response at the early phase of infection, especially variations in the proinflammatory cytokine IL-6. Based on these findings, we inoculated the virus that showed the mildest pathogenicity among the five tested, A/pigeon/Thailand/VSMU-7-NPT/2004, into four breeds of Thai indigenous chicken, Phadu-Hung-Dang (PHD), Chee, Dang, and Luang-Hung-Khao (LHK), to explore effects of genetic background on host response. Among these breeds, Chee, Dang, and LHK showed significantly longer survival times than White Leghorns. Virus shedding from dead Thai indigenous chickens was significantly lower than that from White Leghorns. Although polymorphisms were observed in the Mx and MHC class I genes, there was no significant association between the polymorphisms in these loci and resistance to HPAIV.

  18. The multibasic cleavage site of the hemagglutinin of highly pathogenic A/Vietnam/1203/2004 (H5N1) avian influenza virus acts as a virulence factor in a host-specific manner in mammals.

    PubMed

    Suguitan, Amorsolo L; Matsuoka, Yumiko; Lau, Yuk-Fai; Santos, Celia P; Vogel, Leatrice; Cheng, Lily I; Orandle, Marlene; Subbarao, Kanta

    2012-03-01

    Highly pathogenic avian influenza (HPAI) viruses of the H5 and H7 subtypes typically possess multiple basic amino acids around the cleavage site (MBS) of their hemagglutinin (HA) protein, a recognized virulence motif in poultry. To determine the importance of the H5 HA MBS as a virulence factor in mammals, recombinant wild-type HPAI A/Vietnam/1203/2004 (H5N1) viruses that possessed (H5N1) or lacked (ΔH5N1) the H5 HA MBS were generated and evaluated for their virulence in BALB/c mice, ferrets, and African green monkeys (AGMs) (Chlorocebus aethiops). The presence of the H5 HA MBS was associated with lethality, significantly higher virus titers in the respiratory tract, virus dissemination to extrapulmonary organs, lymphopenia, significantly elevated levels of proinflammatory cytokines and chemokines, and inflammation in the lungs of mice and ferrets. In AGMs, neither H5N1 nor ΔH5N1 virus was lethal and neither caused clinical symptoms. The H5 HA MBS was associated with mild enhancement of replication and delayed virus clearance. Thus, the contribution of H5 HA MBS to the virulence of the HPAI H5N1 virus varies among mammalian hosts and is most significant in mice and ferrets and less remarkable in nonhuman primates. PMID:22205751

  19. Pre-exposing Canada Geese (Branta canadensis) to a low-pathogenic H1N1 avian influenza virus protects them against H5N1 HPAI virus challenge.

    PubMed

    Berhane, Yohannes; Embury-Hyatt, Carissa; Leith, Marsha; Kehler, Helen; Suderman, Matthew; Pasick, John

    2014-01-01

    In previous studies we examined the role of Canada Geese (Branta canadensis) in the epidemiology of Eurasian highly pathogenic avian influenza (HPAI) H5N1. To expand on this and better understand how pre-exposure to heterosubtypic low-pathogenic avian influenza (LPAI) viruses might influence the outcome of H5N1 HPAI infection, we pre-exposed naïve juvenile Canada Geese to different North American wild-bird-origin LPAI viruses. We selected H1, H2, and H6 hemagglutinin subtype viruses based on their higher-order evolutionary relatedness to the H5 hemagglutinin. Pre-exposing Canada Geese to either H2N3 or H6N5 viruses did not protect them against a lethal H5N1 HPAI virus challenge. In addition, H5N1 was transmitted to naïve control birds that were placed among both groups resulting in death by 5 days postcontact. In contrast, Canada Geese that were pre-exposed to H1N1 were protected against a lethal H5N1 challenge, shed minimal amounts of the virus into the environment, and did not transmit the infection to naïve contact birds. None of the H1N1, H2N3, or H6N5 pre-exposure sera neutralized H5N1 in vitro; however, sera from H1N1-infected birds reduced virus plaque size but not number when compared with H2N3, H6N5, or negative sera, suggesting that antibodies directed against the neuraminidase may have had a role in the protective effects observed.

  20. Human microRNAs profiling in response to influenza A viruses (subtypes pH1N1, H3N2, and H5N1)

    PubMed Central

    Makkoch, Jarika; Poomipak, Witthaya; Saengchoowong, Suthat; Khongnomnan, Kritsada; Praianantathavorn, Kesmanee; Jinato, Thananya; Poovorawan, Yong

    2016-01-01

    MicroRNAs (miRNAs) play an important role in regulation of gene silencing and are involved in many cellular processes including inhibition of infected viral replication. This study investigated cellular miRNA expression profiles operating in response to influenza virus in early stage of infection which might be useful for understanding and control of viral infection. A549 cells were infected with different subtypes of influenza virus (pH1N1, H3N2 and H5N1). After 24 h post-infection, miRNAs were extracted and then used for DNA library construction. All DNA libraries with different indexes were pooled together with equal concentration, followed by high-throughput sequencing based on MiSeq platform. The miRNAs were identified and counted from sequencing data by using MiSeq reporter software. The miRNAs expressions were classified into up and downregulated miRNAs compared to those found in non-infected cells. Mostly, each subtype of influenza A virus triggered the upregulated responses in miRNA expression profiles. Hsa-miR-101, hsa-miR-193b, hsa-miR-23b, and hsa-miR-30e* were upregulated when infected with all three subtypes of influenza A virus. Target prediction results showed that virus infection can trigger genes in cellular process, metabolic process, developmental process and biological regulation. This study provided some insights into the cellular miRNA profiling in response to various subtypes of influenza A viruses in circulation and which have caused outbreaks in human population. The regulated miRNAs might be involved in virus–host interaction or host defense mechanism, which should be investigated for effective antiviral therapeutic interventions. PMID:26518627

  1. [Study on the histopathology of cats inoculated with H5N1 subtype high pathogenic avian influenza virus originated from tigers].

    PubMed

    Chang, Shuang; Ding, Zhuang; Yang, Song-Tao; Gao, Yu-Wei; Zou, Xiao-Huan; Wang, Tie-Cheng; Xia, Xian-Zhu

    2007-11-01

    In this study, the HPAIV A/Tiger/Harbin/01/2002 (H5N1) used was originated from tigers and propagated in SPF embryonated hen eggs. TCID5, of the virus was 10(-7.36)/0. 05mL on MDCK cell. The cats were inoculated through bronchus route and then, the cats of dead and control were collected for histopathological and immunohistochemistry examination. Meanwhile, the emulsion supernatant fluid of organs and the pharyngeal swab samples of the dead cats were collected for RT-PCR, survived cats and the control cats were tested for the presence of HI antibody by standard method. The results indicated that the damage of lungs from the dead cats were most obvious, the wide range of red consolidation focus emerged on the lobus pulmonis, the fused focus of infection caused injury of lungs. Histology under the microscope revealed diffuse alveolar damage, confluence phlegmasia pathology, infiltration of lymphomonocytes, sackful of infiltration of macrophages and manipulus protein-like effusion in the alveolar. By immunohistochemistry, the positively stained virus particles were found on the epithelial cells of bronchus and alveolus, and also in the endochylema of lymphomonocytes. The specific electophoretic band of 464bp amplified by RT-PCR from samples of pharyngeal swabs, lungs, kidneys, hearts and brains was as same as the theory value. HI antibody titers of the survived cat were 1:32.

  2. A Single Mutation in the PB1-F2 of H5N1 (HK/97) and 1918 Influenza A Viruses Contributes to Increased Virulence

    PubMed Central

    Conenello, Gina M; Zamarin, Dmitriy; Perrone, Lucy A; Tumpey, Terrence; Palese, Peter

    2007-01-01

    The proapoptotic PB1-F2 protein of influenza A viruses has been shown to contribute to pathogenesis in the mouse model. Expression of full-length PB1-F2 increases the pathogenesis of the influenza A virus, causing weight loss, slower viral clearance, and increased viral titers in the lungs. After comparing viruses from the Hong Kong 1997 H5N1 outbreak, one amino acid change (N66S) was found in the PB1-F2 sequence at position 66 that correlated with pathogenicity. This same amino acid change (N66S) was also found in the PB1-F2 protein of the 1918 pandemic A/Brevig Mission/18 virus. Two isogenic recombinant chimeric viruses were created with an influenza A/WSN/33 virus background containing the PB1 segment from the HK/156/97: WH and WH N66S. In mice infected with WH N66S virus there was increased pathogenicity as measured by weight loss and decreased survival, and a 100-fold increase in virus replication when compared to mice infected with the WH virus. The 1918 pandemic strain A/Brevig Mission/18 was reconstructed with a pathogenicity-reducing mutation in PB1-F2 (S66N). The resultant 1918 S66N virus was attenuated in mice having a 3-log lower 50% lethal dose and caused less morbidity and mortality in mice than the wild-type virus. Viral lung titers were also decreased in 1918 S66N–infected mice compared with wild-type 1918 virus–infected mice. In addition, both viruses with an S at position 66 (WH N66S and wt 1918) induced elevated levels of cytokines in the lungs of infected mice. Together, these data show that a single amino acid substitution in PB1-F2 can result in increased viral pathogenicity and could be one of the factors contributing to the high lethality seen with the 1918 pandemic virus. PMID:17922571

  3. Investigating a crow die-off in January-February 2011 during the introduction of a new clade of highly pathogenic avian influenza virus H5N1 into Bangladesh.

    PubMed

    Khan, Salah Uddin; Berman, Lashondra; Haider, Najmul; Gerloff, Nancy; Rahman, Md Z; Shu, Bo; Rahman, Mustafizur; Dey, Tapan Kumar; Davis, Todd C; Das, Bidhan Chandra; Balish, Amanda; Islam, Ausraful; Teifke, Jens P; Zeidner, Nord; Lindstrom, Steven; Klimov, Alexander; Donis, Ruben O; Luby, Stephen P; Shivaprasad, H L; Mikolon, Andrea B

    2014-03-01

    We investigated unusual crow mortality in Bangladesh during January-February 2011 at two sites. Crows of two species, Corvus splendens and C. macrorhynchos, were found sick and dead during the outbreaks. In selected crow roosts, morbidity was ~1 % and mortality was ~4 % during the investigation. Highly pathogenic avian influenza virus H5N1 clade 2.3.2.1 was isolated from dead crows. All isolates were closely related to A/duck/India/02CA10/2011 (H5N1) with 99.8 % and A/crow/Bangladesh/11rs1984-15/2011 (H5N1) virus with 99 % nucleotide sequence identity in their HA genes. The phylogenetic cluster of Bangladesh viruses suggested a common ancestor with viruses found in poultry from India, Myanmar and Nepal. Histopathological changes and immunohistochemistry staining in brain, pancreas, liver, heart, kidney, bursa of Fabricius, rectum, and cloaca were consistent with influenza virus infection. Through our limited investigation in domesticated birds near the crow roosts, we did not identify any samples that tested positive for influenza virus A/H5N1. However, environmental samples collected from live-bird markets near an outbreak site during the month of the outbreaks tested very weakly positive for influenza virus A/H5N1 in clade 2.3.2.1-specific rRT-PCR. Continuation of surveillance in wild and domestic birds may identify evolution of new avian influenza virus and associated public-health risks.

  4. Evaluation of the antigenic relatedness and cross-protective immunity of the neuraminidase between human influenza A (H1N1) virus and highly pathogenic avian influenza A (H5N1) virus.

    PubMed

    Lu, Xiuhua; Liu, Feng; Zeng, Hui; Sheu, Tiffany; Achenbach, Jenna E; Veguilla, Vic; Gubareva, Larisa V; Garten, Rebecca; Smith, Catherine; Yang, Hua; Stevens, James; Xu, Xiyan; Katz, Jacqueline M; Tumpey, Terrence M

    2014-04-01

    To determine the genetic and antigenic relatedness as well as the cross-protective immunity of human H1N1 and avian H5N1 influenza virus neuraminidase (NA), we immunized rabbits with either a baculovirus-expressed recombinant NA from A/Beijing/262/95 (BJ/262) H1N1 or A/Hong Kong/483/97 (HK/483) H5N1 virus. Cross-reactive antibody responses were evaluated by multiple serological assays and cross-protection against H5N1 virus challenge was evaluated in mice. In a neuraminidase inhibition (NI) test, the antisera exhibited substantial inhibition of NA activity of the homologous virus, but failed to inhibit the NA activity of heterologous virus. However, these antisera exhibited low levels of cross-reactivity measured by plaque size reduction, replication inhibition, single radial hemolysis, and ELISA assays. Passive immunization with HK/483 NA-specific antisera significantly reduced virus replication and disease, and afforded almost complete protection against lethal homologous virus challenge in mice. However, passive immunization with BJ/262 (H1N1) NA-specific antisera was ineffective at providing cross-protection against lethal H5N1 virus challenge and only slightly reduced weight loss. Substantial amino acid variation among the NA antigenic sites was observed between BJ/262 and HK/483 virus, which was consistent with the lack of cross-reactive NI activity by the antibody and limited cross-protective immunity in mice. These results show a strong correlation between the lack of cross-protective immunity and low structural similarities of NA from a human seasonal H1N1 virus and an avian H5N1 influenza virus.

  5. Disease Severity Is Associated with Differential Gene Expression at the Early and Late Phases of Infection in Nonhuman Primates Infected with Different H5N1 Highly Pathogenic Avian Influenza Viruses

    PubMed Central

    Muramoto, Yukiko; Shoemaker, Jason E.; Le, Mai Quynh; Itoh, Yasushi; Tamura, Daisuke; Sakai-Tagawa, Yuko; Imai, Hirotaka; Uraki, Ryuta; Takano, Ryo; Kawakami, Eiryo; Ito, Mutsumi; Okamoto, Kiyoko; Ishigaki, Hirohito; Mimuro, Hitomi; Sasakawa, Chihiro; Matsuoka, Yukiko; Noda, Takeshi; Fukuyama, Satoshi; Ogasawara, Kazumasa; Kitano, Hiroaki

    2014-01-01

    ABSTRACT Occasional transmission of highly pathogenic avian H5N1 influenza viruses to humans causes severe pneumonia with high mortality. To better understand the mechanisms via which H5N1 viruses induce severe disease in humans, we infected cynomolgus macaques with six different H5N1 strains isolated from human patients and compared their pathogenicity and the global host responses to the virus infection. Although all H5N1 viruses replicated in the respiratory tract, there was substantial heterogeneity in their replicative ability and in the disease severity induced, which ranged from asymptomatic to fatal. A comparison of global gene expression between severe and mild disease cases indicated that interferon-induced upregulation of genes related to innate immunity, apoptosis, and antigen processing/presentation in the early phase of infection was limited in severe disease cases, although interferon expression was upregulated in both severe and mild cases. Furthermore, coexpression analysis of microarray data, which reveals the dynamics of host responses during the infection, demonstrated that the limited expression of these genes early in infection led to a failure to suppress virus replication and to the hyperinduction of genes related to immunity, inflammation, coagulation, and homeostasis in the late phase of infection, resulting in a more severe disease. Our data suggest that the attenuated interferon-induced activation of innate immunity, apoptosis, and antigen presentation in the early phase of H5N1 virus infection leads to subsequent severe disease outcome. IMPORTANCE Highly pathogenic avian H5N1 influenza viruses sometimes transmit to humans and cause severe pneumonia with ca. 60% lethality. The continued circulation of these viruses poses a pandemic threat; however, their pathogenesis in mammals is not fully understood. We, therefore, investigated the pathogenicity of six H5N1 viruses and compared the host responses of cynomolgus macaques to the virus

  6. A reinforced composite structure composed of polydiacetylene assemblies deposited on polystyrene microspheres and its application to H5N1 virus detection

    PubMed Central

    Dong, Wenjie; Luo, Jing; He, Hongxuan; Jiang, Long

    2013-01-01

    In this study, we immobilized polydiacetylene vesicles (PDAVs) onto the surface of polystyrene (PS) microspheres (1 μm in diameter) by using both electrical charge and conjugated forces to form a reinforced composite structure. These reinforced complexes could be easily washed, separated by centrifugation, and resuspended by gentle agitation. After passing through a narrow 200 μm-diameter channel, the composite structures maintained their original shape, demonstrating their resilience and potential for use in microfluidic technologies. The number of PDAVs in the composite structure could be mediated by changing the extent of layer deposition, which affected the sensitivity of detection. It showed that PDAVs did not change their blue color after addition of detecting probes such as anti-H5N1, which was of great importance in the fabrication and modification of stable color-changeable biosensors based on PDAVs. By conjugating anti-H5N1 antibodies to the PS@PDAV via N -hydroxysuccinimide and 1-ethyl3-(3-dimethylaminopropyl) carbodiimide chemistry, a stable blue complex, anti-H5N1 microsphere (PS@PDAV-anti-H5N1) was formed. A target antigen of H5N1 (HAQ [H5N1 strain A/ environment/Qinghai/1/2008{H5N1} in clade 0]) was detected by PS@PDAV-anti-H5N1. At an optimal PDAV deposition level of three layers, the limit of detection was determined to be approximately 3 0 ng/mL of HAQ by using optical spectrum measurement and visual inspection, meeting the needs of fast and simple color-changeable detection. However, a much lower limitation of detection (1 ng/mL) was able to be obtained using laser-scanning confocal microscopy, which could be compared with the results obtained with other sophisticated equipment. PMID:23403826

  7. Domestic ducks and H5N1 influenza epidemic, Thailand.

    PubMed

    Songserm, Thaweesak; Jam-on, Rungroj; Sae-Heng, Numdee; Meemak, Noppadol; Hulse-Post, Diane J; Sturm-Ramirez, Katharine M; Webster, Robert G

    2006-04-01

    In addition to causing 12 human deaths and 17 cases of human infection, the 2004 outbreak of H5N1 influenza virus in Thailand resulted in the death or slaughter of 60 million domestic fowl and the disruption of poultry production and trade. After domestic ducks were recognized as silent carriers of H5N1 influenza virus, government teams went into every village to cull flocks in which virus was detected; these team efforts markedly reduced H5N1 infection. Here we examine the pathobiology and epidemiology of H5N1 influenza virus in the 4 systems of duck raising used in Thailand in 2004. No influenza viruses were detected in ducks raised in "closed" houses with high biosecurity. However, H5N1 influenza virus was prevalent among ducks raised in "open" houses, free-ranging (grazing) ducks, and backyard ducks.

  8. DNA probe modified with 3-iron bis(dicarbollide) for electrochemical determination of DNA sequence of Avian Influenza Virus H5N1.

    PubMed

    Grabowska, Iwona; Stachyra, Anna; Góra-Sochacka, Anna; Sirko, Agnieszka; Olejniczak, Agnieszka B; Leśnikowski, Zbigniew J; Radecki, Jerzy; Radecka, Hanna

    2014-01-15

    In this work, we report on oligonucleotide probes bearing metallacarborane [3-iron bis(dicarbollide)] redox label, deposited on gold electrode for electrochemical determination of DNA sequence derived from Avian Influenza Virus (AIV), type H5N1. The oligonucleotide probes containing 5'-terminal NH2 group were covalently attached to the electrode, via NHS/EDC coupling to 3-mercaptopropionic acid SAM, previously deposited on the surface of gold. The changes in redox activity of Fe(III) centre of the metallacarborane complex before and after hybridization process was used as analytical signal. The signals generated upon hybridization with targets such as complementary or non-complementary 20-mer ssDNA or various PCR products consisting of 180-190 bp (dsDNA) were recorded by Osteryoung square-wave voltammetry (OSWV). The developed system was very sensitive towards targets containing sequence complementary to the probe with the detection limit estimated as 0.03 fM (S/N=3.0) and 0.08 fM (S/N=3.0) for 20-mer ssDNA and for dsDNA (PCR product), respectively. The non-complementary targets generated very weak responses. Furthermore, the proposed genosensor was suitable for discrimination of PCR products with different location of the complementarity region.

  9. Multiple introductions of a reassortant H5N1 avian influenza virus of clade 2.3.2.1c with PB2 gene of H9N2 subtype into Indian poultry.

    PubMed

    Tosh, Chakradhar; Nagarajan, Shanmugasundaram; Kumar, Manoj; Murugkar, Harshad V; Venkatesh, Govindarajulu; Shukla, Shweta; Mishra, Amit; Mishra, Pranav; Agarwal, Sonam; Singh, Bharati; Dubey, Prashant; Tripathi, Sushil; Kulkarni, Diwakar D

    2016-09-01

    Highly pathogenic avian influenza (HPAI) H5N1 viruses are a threat to poultry in Asia, Europe, Africa and North America. Here, we report isolation and characterization of H5N1 viruses isolated from ducks and turkeys in Kerala, Chandigarh and Uttar Pradesh, India between November 2014 and March 2015. Genetic and phylogenetic analyses of haemagglutinin gene identified that the virus belonged to a new clade 2.3.2.1c which has not been detected earlier in Indian poultry. The virus possessed molecular signature for high pathogenicity to chickens, which was corroborated by intravenous pathogenicity index of 2.96. The virus was a reassortant which derives its PB2 gene from H9N2 virus isolated in China during 2007-2013. However, the neuraminidase and internal genes are of H5N1 subtype. Phylogenetic and network analysis revealed that after detection in China in 2013/2014, the virus moved to Europe, West Africa and other Asian countries including India. The analyses further indicated multiple introductions of H5N1 virus in Indian poultry and internal spread in Kerala. One of the outbreaks in ducks in Kerala is linked to the H5N1 virus isolated from wild birds in Dubai suggesting movement of virus probably through migration of wild birds. However, the outbreaks in ducks in Chandigarh and Uttar Pradesh were from an unknown source in Asia which also contributed gene pools to the outbreaks in Europe and West Africa. The widespread incidence of the novel H5N1 HPAI is similar to the spread of clade 2.2 ("Qinghai-like") virus in 2005, and should be monitored to avoid threat to animal and public health. PMID:27174088

  10. High-Yield Expression of M2e Peptide of Avian Influenza Virus H5N1 in Transgenic Duckweed Plants.

    PubMed

    Firsov, Aleksey; Tarasenko, Irina; Mitiouchkina, Tatiana; Ismailova, Natalya; Shaloiko, Lyubov; Vainstein, Alexander; Dolgov, Sergey

    2015-07-01

    Avian influenza is a major viral disease in poultry. Antigenic variation of this virus hinders vaccine development. However, the extracellular domain of the virus-encoded M2 protein (peptide M2e) is nearly invariant in all influenza A strains, enabling the development of a broad-range vaccine against them. Antigen expression in transgenic plants is becoming a popular alternative to classical expression methods. Here we expressed M2e from avian influenza virus A/chicken/Kurgan/5/2005(H5N1) in nuclear-transformed duckweed plants for further development of avian influenza vaccine. The N-terminal fragment of M2, including M2e, was selected for expression. The M2e DNA sequence fused in-frame to the 5' end of β-glucuronidase was cloned into pBI121 under the control of CaMV 35S promoter. The resulting plasmid was successfully used for duckweed transformation, and western analysis with anti-β-glucuronidase and anti-M2e antibodies confirmed accumulation of the target protein (M130) in 17 independent transgenic lines. Quantitative ELISA of crude protein extracts from these lines showed M130-β-glucuronidase accumulation ranging from 0.09-0.97 mg/g FW (0.12-1.96 % of total soluble protein), equivalent to yields of up to 40 μg M2e/g plant FW. This relatively high yield holds promise for the development of a duckweed-based expression system to produce an edible vaccine against avian influenza.

  11. Experimental infection of bar-headed geese (Anser indicus) and ruddy shelducks (Tadorna ferruginea) with a clade 2.3.2 H5N1 highly pathogenic avian influenza virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Since 2005, clade 2.2 H5N1 highly pathogenic avian influenza (HPAI) viruses have caused infections and disease involving numerous species of wild waterfowl in Eurasia and Africa. However, outbreaks associated with clade 2.3.2 viruses have increased since 2009, and viruses within this clade have beco...

  12. Pathogenesis of the 1918 pandemic and H5N1 influenza virus infection in a guinea pig model: The antiviral potential of exogenous alpha-interferon to reduce virus shedding

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Although highly pathogenic avian influenza H5N1 viruses have yet to acquire the ability to transmit efficiently among humans, the geographic expansion, and continued outbreaks in humans and avian species underscore the need for more effective influenza vaccines and antivirals. Additional small anim...

  13. Varied pathogenicity of a Hong Kong-origin H5N1 avian influenza virus in four passerine species and budgerigars.

    PubMed

    Perkins, L E L; Swayne, D E

    2003-01-01

    This investigation assessed the ability of the zoonotic A/chicken/Hong Kong/220/97 (chicken/Hong Kong) (H5N1) highly pathogenic avian influenza virus to infect and cause disease in zebra finches (Taeniopygia guttata), house finches (Carpodacus mexicanus), house sparrows (Passer domesticus), European starlings (Sternus vulgaris), and budgerigars (Melopsittacus undulatus) after intranasal administration. Zebra finches were the most severely affected of the five species, demonstrating anorexia, depression, and 100% mortality within 5 days of inoculation. Gross lesions in this species were absent or only mild. But histologic lesions and the corresponding viral antigen were observed in multiple organs, especially in the nasal cavity, brain, pancreas, spleen, adrenal glands, and ovary. Significant morbidity and mortality also were observed in both house finches and budgerigars. Affected birds of these two species demonstrated anorexia, depression, and neurologic signs and typically were moribund or dead within 2 days of the onset of clinical signs. Gross lesions were mild or absent in house finches and budgerigars. Histologically, the brain and pancreas were the most consistently and severely affected organs in house finches. The brain was the most affected organ in budgerigars. Unlike these three species, house sparrows suffered only mild transient depression, had no mortality, and lacked gross lesions. Viral antigen and microscopic lesions were observed only in the heart and testicle of a minority of birds of this species. Starlings demonstrated neither clinical disease nor mortality and lacked gross and histologic lesions. Viral antigen was not observed in any of the collected tissues from starlings. These results indicate that there is significant variation in the pathogenicity of the chicken/Hong Kong virus for different species of birds, including species within the same order. In addition, neurotropism is a recurrent feature among birds that eventually succumb to

  14. Outbreaks of highly pathogenic avian influenza H5N1 clade 2.3.2.1c in hunting falcons and kept wild birds in Dubai implicate intercontinental virus spread.

    PubMed

    Naguib, Mahmoud M; Kinne, Jörg; Chen, Honglin; Chan, Kwok-Hung; Joseph, Sunitha; Wong, Po-Chun; Woo, Patrick C Y; Wernery, Renate; Beer, Martin; Wernery, Ulrich; Harder, Timm C

    2015-11-01

    Highly pathogenic avian influenza viruses (HPAIVs) of subtype H5N1 have continued to perpetuate with divergent genetic variants in poultry within Asia since 2003. Further dissemination of Asian-derived H5 HPAIVs to Europe, Africa and, most recently, to the North American continent has occurred. We report an outbreak of HPAIV H5N1 among falcons kept for hunting and other wild bird species bred as falcon prey in Dubai, United Arab Emirates, during the autumn of 2014. The causative agent was identified as avian influenza virus subtype H5N1, clade 2.3.2.1c, by genetic and phylogenetic analyses. High mortality in infected birds was in accordance with systemic pathomorphological and histological alterations in affected falcons. Genetic analysis showed the HPAIV H5N1 of clade 2.3.2.1c is a reassortant in which the PB2 segment was derived from an Asian-origin H9N2 virus lineage. The Dubai H5N1 viruses were closely related to contemporary H5N1 HPAIVs from Nigeria, Burkina-Faso, Romania and Bulgaria. Median-joining network analysis of 2.3.2.1c viruses revealed that the Dubai outbreak was an episode of a westward spread of these viruses on a larger scale from unidentified Asian sources. The incursion into Dubai, possibly via infected captive hunting falcons returning from hunting trips to central Asian countries, preceded outbreaks in Nigeria and other West African countries. The alarmingly enhanced geographical mobility of clade 2.3.2.1.c and clade 2.3.4.4 viruses may represent another wave of transcontinental dissemination of Asian-origin HPAIV H5 viruses, such as the outbreak at Qinghai Lake caused by clade 2.2 (‘Qinghai’ lineage) in 2005.

  15. Influenza virus subpopulations: exchange of lethal H5N1 virus NS for H1N1 virus NS triggers de novo generation of defective-interfering particles and enhances interferon-inducing particle efficiency.

    PubMed

    Ngunjiri, John M; Buchek, Gregory M; Mohni, Kareem N; Sekellick, Margaret J; Marcus, Philip I

    2013-03-01

    Reassortment of influenza A viruses is known to affect viability, replication efficiency, antigenicity, host range, and virulence, and can generate pandemic strains. In this study, we demonstrated that the specific exchange of the NS gene segment from highly pathogenic A/HK/156/97 (H5N1) [E92 or E92D NS1] virus for the cognate NS gene segment of A/PR/834(H1N1) [D92 NS1] virus did not cause a significant change in the sizes of infectious particle subpopulations. However, it resulted in 2 new phenotypic changes: (1) de novo generation of large subpopulations of defective-interfering particles (DIPs); and (2) enhancement of interferon (IFN)-inducing particle efficiency leading to an order of magnitude or higher quantum (peak) yield of IFN in both avian and mammalian cells. These changes were attributed to loss of function of the H5N1-NS gene products. Most notably, the NS exchange obliterated the usual IFN-induction-suppressing capacity associated with expression of full-size NS1 proteins, and hence functionally mimicked deletions in the NS1 gene. The loss of NS1-mediated suppression of IFN induction, de novo generation of DIPs, and the concomitant enhancement of IFN-inducing particle efficiency suggest that in an attenuated background, the H5N1-NS could be used to formulate a self-adjuvanting live attenuated influenza vaccine similar to viruses with deletions in the NS1 gene.

  16. Independent-Trajectories Thermodynamic-Integration Free-Energy Changes for Biomolecular Systems: Determinants of H5N1 Avian Influenza Virus Neuraminidase Inhibition by Peramivir

    PubMed Central

    2009-01-01

    Free-energy changes are essential physicochemical quantities for understanding most biochemical processes. Yet, the application of accurate thermodynamic-integration (TI) computation to biological and macromolecular systems is limited by finite-sampling artifacts. In this paper, we employ independent-trajectories thermodynamic-integration (IT-TI) computation to estimate improved free-energy changes and their uncertainties for (bio)molecular systems. IT-TI aids sampling statistics of the thermodynamic macrostates for flexible associating partners by ensemble averaging of multiple, independent simulation trajectories. We study peramivir (PVR) inhibition of the H5N1 avian influenza virus neuraminidase flexible receptor (N1). Binding site loops 150 and 119 are highly mobile, as revealed by N1-PVR 20-ns molecular dynamics. Due to such heterogeneous sampling, standard TI binding free-energy estimates span a rather large free-energy range, from a 19% underestimation to a 29% overestimation of the experimental reference value (−62.2 ± 1.8 kJ mol−1). Remarkably, our IT-TI binding free-energy estimate (−61.1 ± 5.4 kJ mol−1) agrees with a 2% relative difference. In addition, IT-TI runs provide a statistics-based free-energy uncertainty for the process of interest. Using ∼800 ns of overall sampling, we investigate N1-PVR binding determinants by IT-TI alchemical modifications of PVR moieties. These results emphasize the dominant electrostatic contribution, particularly through the N1 E277−PVR guanidinium interaction. Future drug development may be also guided by properly tuning ligand flexibility and hydrophobicity. IT-TI will allow estimation of relative free energies for systems of increasing size, with improved reliability by employing large-scale distributed computing. PMID:19461872

  17. 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)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Goose/Guangdong (Gs/GD)-lineage H5N1 highly pathogenic avian influenza (HPAI) viruses continue to circulate and cause great economic losses in poultry in Asia, the Middle East, and Africa. Recently, the Gs/GD-lineage H5N8 HPAI virus belonging to clade 2.3.4.4 and its reassortants have caused out...

  18. 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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 ...

  19. Differences in pathogenicity, response to vaccination, and innate immune responses in different types of ducks infected with a virulent H5N1 highly pathogenic avian influenza virus from Vietnam

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Wild ducks are reservoirs of avian influenza viruses in nature, and usually don’t show signs of disease. However, some Asian lineage H5N1 highly pathogenic avian influenza (HPAI) viruses can cause disease and death in both wild and domestic ducks. The objective of this study was to compare the cli...