Genetically Diverse Low Pathogenicity Avian Influenza A Virus Subtypes Co-Circulate among Poultry in Bangladesh

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Gerloff, Nancy A.; Khan, Salah Uddin; Zanders, Natosha; Balish, Amanda; Haider, Najmul; Islam, Ausraful; Chowdhury, Sukanta; Rahman, Mahmudur Ziaur; Haque, Ainul; Hosseini, Parviez; Gurley, Emily S.; Luby, Stephen P.; Wentworth, David E.; Donis, Ruben O.; Sturm-Ramirez, Katharine; Davis, C. Todd

2016-01-01

Influenza virus surveillance, poultry outbreak investigations and genomic sequencing were assessed to understand the ecology and evolution of low pathogenicity avian influenza (LPAI) A viruses in Bangladesh from 2007 to 2013. We analyzed 506 avian specimens collected from poultry in live bird markets and backyard flocks to identify influenza A viruses. Virus isolation-positive specimens (n = 50) were subtyped and their coding-complete genomes were sequenced. The most frequently identified subtypes among LPAI isolates were H9N2, H11N3, H4N6, and H1N1. Less frequently detected subtypes included H1N3, H2N4, H3N2, H3N6, H3N8, H4N2, H5N2, H6N1, H6N7, and H7N9. Gene sequences were compared to publicly available sequences using phylogenetic inference approaches. Among the 14 subtypes identified, the majority of viral gene segments were most closely related to poultry or wild bird viruses commonly found in Southeast Asia, Europe, and/or northern Africa. LPAI subtypes were distributed over several geographic locations in Bangladesh, and surface and internal protein gene segments clustered phylogenetically with a diverse number of viral subtypes suggesting extensive reassortment among these LPAI viruses. H9N2 subtype viruses differed from other LPAI subtypes because genes from these viruses consistently clustered together, indicating this subtype is enzootic in Bangladesh. The H9N2 strains identified in Bangladesh were phylogenetically and antigenically related to previous human-derived H9N2 viruses detected in Bangladesh representing a potential source for human infection. In contrast, the circulating LPAI H5N2 and H7N9 viruses were both phylogenetically and antigenically unrelated to H5 viruses identified previously in humans in Bangladesh and H7N9 strains isolated from humans in China. In Bangladesh, domestic poultry sold in live bird markets carried a wide range of LPAI virus subtypes and a high diversity of genotypes. These findings, combined with the seven year

Low-pathogenic influenza A viruses in North American diving ducks contribute to the emergence of a novel highly pathogenic influenza A(H7N8) virus

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Xu, Yifei; Ramey, Andrew M.; Bowman, Andrew S; DeLiberto, Thomas J.; Killian, Mary Lea; Krauss, Scott; Nolting, Jacqueline M.; Torchetti, Mia Kim; Reeves, Andrew B.; Webby, Richard J.; Stallknecht, David E.; Wan, Xiu-Feng

2017-01-01

Introductions of low-pathogenic avian influenza (LPAI) viruses of subtypes H5 and H7 into poultry from wild birds have the potential to mutate to highly pathogenic avian influenza (HPAI) viruses, but such viruses' origins are often unclear. In January 2016, a novel H7N8 HPAI virus caused an outbreak in turkeys in Indiana, USA. To determine the virus's origin, we sequenced the genomes of 441 wild-bird origin influenza A viruses (IAVs) from North America and subjected them to evolutionary analyses. The results showed that the H7N8 LPAI virus most likely circulated among diving ducks in the Mississippi flyway during autumn 2015 and was subsequently introduced to Indiana turkeys, in which it evolved high pathogenicity. Preceding the outbreak, an isolate with six gene segments (PB2, PB1, PA, HA, NA, and NS) sharing >99% sequence identity with those of H7N8 turkey isolates was recovered from a diving duck sampled in Kentucky, USA. H4N8 IAVs from other diving ducks possessed five H7N8-like gene segments (PB2, PB1, NA, MP, and NS; >98% sequence identity). Our findings suggest that viral gene constellations circulating among diving ducks can contribute to the emergence of IAVs that affect poultry. Therefore, diving ducks may serve an important and understudied role in the maintenance, diversification, and transmission of IAVs in the wild-bird reservoir.

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

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Teng Zhao

2016-11-01

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

Preliminary Epidemiology of Human Infections with Highly Pathogenic Avian Influenza A(H7N9) Virus, China, 2017.

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Zhou, Lei; Tan, Yi; Kang, Min; Liu, Fuqiang; Ren, Ruiqi; Wang, Yali; Chen, Tao; Yang, Yiping; Li, Chao; Wu, Jie; Zhang, Hengjiao; Li, Dan; Greene, Carolyn M; Zhou, Suizan; Iuliano, A Danielle; Havers, Fiona; Ni, Daxin; Wang, Dayan; Feng, Zijian; Uyeki, Timothy M; Li, Qun

2017-08-01

We compared the characteristics of cases of highly pathogenic avian influenza (HPAI) and low pathogenic avian influenza (LPAI) A(H7N9) virus infections in China. HPAI A(H7N9) case-patients were more likely to have had exposure to sick and dead poultry in rural areas and were hospitalized earlier than were LPAI A(H7N9) case-patients.

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

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

Prevalence of avian influenza virus in wild birds before and after the HPAI H5N8 outbreak in 2014 in South Korea.

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Shin, Jeong-Hwa; Woo, Chanjin; Wang, Seung-Jun; Jeong, Jipseol; An, In-Jung; Hwang, Jong-Kyung; Jo, Seong-Deok; Yu, Seung Do; Choi, Kyunghee; Chung, Hyen-Mi; Suh, Jae-Hwa; Kim, Seol-Hee

2015-07-01

Since 2003, highly pathogenic avian influenza (HPAI) virus outbreaks have occurred five times in Korea, with four HPAI H5N1 outbreaks and one HPAI H5N8 outbreak. Migratory birds have been suggested to be the first source of HPAI in Korea. Here, we surveyed migratory wild birds for the presence of AI and compared regional AI prevalence in wild birds from September 2012 to April 2014 for birds having migratory pathways in South Korea. Finally, we investigated the prevalence of AI in migratory birds before and after HPAI H5N8 outbreaks. Overall, we captured 1617 migratory wild birds, while 18,817 feces samples and 74 dead birds were collected from major wild bird habitats. A total of 21 HPAI viruses were isolated from dead birds, and 86 low pathogenic AI (LPAI) viruses were isolated from captured birds and from feces samples. Spatiotemporal distribution analysis revealed that AI viruses were spread southward until December, but tended to shift north after January, consistent with the movement of migratory birds in South Korea. Furthermore, we found that LPAI virus prevalences within wild birds were notably higher in 2013-2014 than the previous prevalence during the northward migration season. The data from our study demonstrate the importance of the surveillance of AI in wild birds. Future studies including in-depth genetic analysis in combination with evaluation of the movement and ecology of migratory birds might help us to bridge the gaps in our knowledge and better explain, predict, and ultimately prevent future HPAI outbreaks.

Insights into genetic diversity and biological propensities of potentially zoonotic avian influenza H9N2 viruses circulating in Egypt.

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Naguib, Mahmoud M; Arafa, Abdel-Satar; Parvin, Rokshana; Beer, Martin; Vahlenkamp, Thomas; Harder, Timm C

2017-11-01

Low pathogenic avian influenza (LPAI) H9N2 viruses have established endemic status in Egyptian poultry populations since 2012. Recently, four cases of human H9N2 virus infections in Egypt demonstrated the zoonotic potential of these viruses. Egyptian H9N2 viruses obtained from 2011 to 2014 phylogenetically grouped into three clusters (1-3) within subclade B of the G1 lineage. Antigenically, a close clustering of the Egyptian H9N2 viruses with other recent G1-B like H9N2 strains and a significant antigenic distance from viruses outside the G1-B lineage was evident. Recent Egyptian LPAIV H9N2 showed a tendency to increased binding with erythrocytes expressing α 2,6-linked sialic acid which correlated with the Q226L amino acid substitution at the receptor binding unit of the hemagglutinin (Q234L, H9 numbering). Sequence analyses of the N2 neuraminidase (NA) revealed substitutions in the NA hemadsorption site similar to the N2 of prepandemic H3N2/1968, but no distinct antigenic or functional characteristics of the H9N2 NA associated with increased zoonotic potential could be identified. Copyright © 2017 Elsevier Inc. All rights reserved.

Molecular characteristic and pathogenicity of Indonesian H5N1 clade 2.3.2 viruses

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Dharmayanti NLPI

2013-06-01

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

Signal Immune Reactions of Macrophages Differentiated from THP-1 Monocytes to Infection with Pandemic H1N1PDM09 Virus and H5N2 and H9N2 Avian Influenza A Virus.

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Sokolova, T M; Poloskov, V V; Shuvalov, A N; Rudneva, I A; Timofeeva, T A

2018-03-01

In culture of THP-1 cells differentiated into macrophages with PMA (THP-PMA macrophages) infected with influenza viruses of subtypes H1, H5 and H9, we measured the expression of TLR7 and RIG1 receptor genes, sensors of viral RNA and ribonucleoprotein, and the levels of production of inflammatory cytokines IL-1β, TNFα, IL-10, and IFNα. The sensitivity and inflammatory response of THP-PMA macrophages to pandemic influenza A virus H1N1pdm09 and avian influenza H5N2 and H9N2 viruses correlate with the intracellular level of their viral RNA and activation of the RIG1 gene. Abortive infection is accompanied by intensive macrophage secretion of TNFα, IL-1β, and toxic factors inducing cell death. Activity of endosomal TLR7 receptor gene changed insignificantly in 24 h after infection and significantly decreased in 48 and 72 h under the action of H5N2 and H9N2, which correlated with manifestation of the cytopathogenic effect of these viruses. H5N2 and H9N2 avian viruses in THP-PMA macrophages are strong activators of the expression of the gene of the cytoplasmic RIG1 receptor 24 and 48 h after infection, and the pandemic virus H1N1pdm09 is a weak stimulator of RIG1 gene. Avian influenza H5N2 and H9N2 viruses are released by rapid induction of the inflammatory response in macrophages. At the late stages of infection, we observed a minor increase in IL-10 secretion in macrophages and, probably, the polarization of a part of the population in type M2. The studied influenza A viruses are weak inductors of IFN in THP-PMA macrophages. In the culture medium of THP-PMA macrophages infected with H9N2 and H5N2 viruses, MTT test revealed high levels of toxic factors causing the death of Caco-2 cells. In contrast to avian viruses, pandemic virus H1N1pdm09 did not induce production of toxic factors.

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

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

2016-01-01

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

Infection of mice with a human influenza A/H3N2 virus induces protective immunity against lethal infection with influenza A/H5N1 virus.

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Kreijtz, J H C M; Bodewes, R; van den Brand, J M A; de Mutsert, G; Baas, C; van Amerongen, G; Fouchier, R A M; Osterhaus, A D M E; Rimmelzwaan, G F

2009-08-06

The transmission of highly pathogenic avian influenza (HPAI) A viruses of the H5N1 subtype from poultry to man and the high case fatality rate fuels the fear for a pandemic outbreak caused by these viruses. However, prior infections with seasonal influenza A/H1N1 and A/H3N2 viruses induce heterosubtypic immunity that could afford a certain degree of protection against infection with the HPAI A/H5N1 viruses, which are distantly related to the human influenza A viruses. To assess the protective efficacy of such heterosubtypic immunity mice were infected with human influenza virus A/Hong Kong/2/68 (H3N2) 4 weeks prior to a lethal infection with HPAI virus A/Indonesia/5/05 (H5N1). Prior infection with influenza virus A/Hong Kong/2/68 reduced clinical signs, body weight loss, mortality and virus replication in the lungs as compared to naive mice infected with HPAI virus A/Indonesia/5/05. Priming by infection with respiratory syncytial virus, a non-related virus did not have a beneficial effect on the outcome of A/H5N1 infections, indicating that adaptive immune responses were responsible for the protective effect. In mice primed by infection with influenza A/H3N2 virus cytotoxic T lymphocytes (CTL) specific for NP(366-374) epitope ASNENMDAM and PA(224-232) SCLENFRAYV were observed. A small proportion of these CTL was cross-reactive with the peptide variant derived from the influenza A/H5N1 virus (ASNENMEVM and SSLENFRAYV respectively) and upon challenge infection with the influenza A/H5N1 virus cross-reactive CTL were selectively expanded. These CTL, in addition to those directed to conserved epitopes, shared by the influenza A/H3N2 and A/H5N1 viruses, most likely contributed to accelerated clearance of the influenza A/H5N1 virus infection. Although also other arms of the adaptive immune response may contribute to heterosubtypic immunity, the induction of virus-specific CTL may be an attractive target for development of broad protective vaccines. Furthermore the

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

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

2016-11-28

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

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

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Hana Apsari Pawestri

2016-07-01

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

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

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Hana Apsari Pawestri

2016-07-01

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

Evaluation of twenty rapid antigen tests for the detection of human influenza A H5N1, H3N2, H1N1, and B viruses.

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Taylor, Janette; McPhie, Kenneth; Druce, Julian; Birch, Chris; Dwyer, Dominic E

2009-11-01

Twenty rapid antigen assays were compared for their ability to detect influenza using dilutions of virus culture supernatants from human isolates of influenza A H5N1 (clade 1 and 2 strains), H3N2 and H1N1 viruses, and influenza B. There was variation amongst the rapid antigen assays in their ability to detect different influenza viruses. Six of the 12 assays labeled as distinguishing between influenza A and B had comparable analytical sensitivities for detecting both influenza A H5N1 strains, although their ability to detect influenza A H3N2 and H1N1 strains varied. The two assays claiming H5 specificity did not detect either influenza A H5N1 strains, and the two avian influenza-specific assays detected influenza A H5N1, but missed some influenza A H3N2 virus supernatants. Clinical trials of rapid antigen tests for influenza A H5N1 are limited. For use in a pandemic where novel influenza strains are circulating (such as the current novel influenza A H1N1 09 virus), rapid antigen tests should ideally have comparable sensitivity and specificity for the new strains as for co-circulating seasonal influenza strains.

Avian influenza viruses in humans.

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Malik Peiris, J S

2009-04-01

Past pandemics arose from low pathogenic avian influenza (LPAI) viruses. In more recent times, highly pathogenic avian influenza (HPAI) H5N1, LPAI H9N2 and both HPAI and LPAI H7 viruses have repeatedly caused zoonotic disease in humans. Such infections did not lead to sustained human-to-human transmission. Experimental infection of human volunteers and seroepidemiological studies suggest that avian influenza viruses of other subtypes may also infect humans. Viruses of the H7 subtype appear to have a predilection to cause conjunctivitis and influenza-like illness (ILI), although HPAI H7N7 virus has also caused fatal respiratory disease. Low pathogenic H9N2 viruses have caused mild ILI and its occurrence may be under-recognised for this reason. In contrast, contemporary HPAI H5N1 viruses are exceptional in their virulence for humans and differ from human seasonal influenza viruses in their pathogenesis. Patients have a primary viral pneumonia progressing to acute respiratory distress syndrome (ARDS) and multiple organ dysfunction syndrome. Over 380 human cases have been confirmed to date, with an overall case fatality of 63%. The zoonotic transmission of avian influenza is a rare occurrence, butthe greater public health concern is the adaptation of such viruses to efficient human transmission, which could lead to a pandemic. A better understanding of the ecology of avian influenza viruses and the biological determinants of transmissibility and pathogenicity in humans is important for pandemic preparedness.

Virus pathotype and deep sequencing of the HA gene of a low pathogenicity H7N1 avian influenza virus causing mortality in Turkeys.

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Munir Iqbal

Full Text Available Low pathogenicity avian influenza (LPAI viruses of the H7 subtype generally cause mild disease in poultry. However the evolution of a LPAI virus into highly pathogenic avian influenza (HPAI virus results in the generation of a virus that can cause severe disease and death. The classification of these two pathotypes is based, in part, on disease signs and death in chickens, as assessed in an intravenous pathogenicity test, but the effect of LPAI viruses in turkeys is less well understood. During an investigation of LPAI virus infection of turkeys, groups of three-week-old birds inoculated with A/chicken/Italy/1279/99 (H7N1 showed severe disease signs and died or were euthanised within seven days of infection. Virus was detected in many internal tissues and organs from culled birds. To examine the possible evolution of the infecting virus to a highly pathogenic form in these turkeys, sequence analysis of the haemagglutinin (HA gene cleavage site was carried out by analysing multiple cDNA amplicons made from swabs and tissue sample extracts employing Sanger and Next Generation Sequencing. In addition, a RT-PCR assay to detect HPAI virus was developed. There was no evidence of the presence of HPAI virus in either the virus used as inoculum or from swabs taken from infected birds. However, a small proportion (<0.5% of virus carried in individual tracheal or liver samples did contain a molecular signature typical of a HPAI virus at the HA cleavage site. All the signature sequences were identical and were similar to HPAI viruses collected during the Italian epizootic in 1999/2000. We assume that the detection of HPAI virus in tissue samples following infection with A/chicken/Italy/1279/99 reflected amplification of a virus present at very low levels within the mixed inoculum but, strikingly, we observed no new HPAI virus signatures in the amplified DNA analysed by deep-sequencing.

Structural and antigenic variation among diverse clade 2 H5N1 viruses.

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David A Shore

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

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.

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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.

Internal Gene Cassette from a Genotype S H9N2 Avian Influenza Virus Attenuates the Pathogenicity of H5 Viruses in Chickens and Mice

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Xiaoli Hao

2017-10-01

Full Text Available H9N2 avian influenza virus (AIV of genotype S frequently donate internal genes to facilitate the generation of novel reassortants such as H7N9, H10N8, H5N2 and H5N6 AIVs, posing an enormous threat to both human health and poultry industry. However, the pathogenicity and transmission of reassortant H5 viruses with internal gene cassette of genotype S H9N2-origin in chickens and mice remain unknown. In this study, four H5 reassortants carrying the HA and NA genes from different clades of H5 viruses and the remaining internal genes from an H9N2 virus of the predominant genotype S were generated by reverse genetics. We found that all four H5 reassortant viruses showed attenuated virulence in both chickens and mice, thus leading to increased the mean death times compared to the corresponding parental viruses. Consistently, the polymerase activity and replication ability in mammalian and avian cells, and the cytokine responses in the lungs of chickens and mice were also decreased when compared to their respective parental viruses. Moreover, these reassortants transmitted from birds to birds by direct contact but not by an airborne route. Our data indicate that the internal genes as a whole cassette from genotype S H9N2 viruses play important roles in reducing the pathogenicity of the H5 recombinants in chickens and mice, and might contribute to the circulation in avian or mammalian hosts.

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

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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 ...

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

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Bertran, Kateri; Balzli, Charles; Lee, Dong-Hun; Suarez, David L; Kapczynski, Darrell R; Swayne, David E

2017-11-01

During December 2014-June 2015, the U.S. experienced a high pathogenicity avian influenza (HPAI) outbreak caused by clade 2.3.4.4 H5Nx Goose/Guangdong lineage viruses with devastating consequences for the poultry industry. Three vaccines, developed based on updating existing registered vaccines or currently licensed technologies, were evaluated for possible use: an inactivated reverse genetics H5N1 vaccine (rgH5N1) and an RNA particle vaccine (RP-H5), both containing the hemagglutinin gene of clade 2.3.4.4 strain, and a recombinant herpesvirus turkey vectored vaccine (rHVT-H5) containing the hemagglutinin gene of clade 2.2 strain. The efficacy of the three vaccines, alone or in combination, was assessed in White Leghorn chickens against clade 2.3.4.4 H5N2 HPAI virus challenge. In Study 1, single (rHVT-H5) and prime-boost (rHVT-H5+rgH5N1 or rHVT-H5+RP-H5) vaccination strategies protected chickens with high levels of protective immunity and significantly reduced virus shedding. In Study 2, single vaccination with either rgH5N1 or RP-H5 vaccines provided clinical protection in adult chickens and significantly reduced virus shedding. In Study 3, double rgH5N1 vaccination protected adult chickens from clinical signs and mortality when challenged 20weeks post-boost, with high levels of long-lasting protective immunity and significantly reduced virus shedding. These studies support the use of genetically related vaccines, possibly in combination with a broad protective priming vaccine, for emergency vaccination programs against clade 2.3.4.4 H5Nx HPAI virus in young and adult layer chickens. Published by Elsevier Ltd.

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

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

2015-04-01

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

Novel genotypes of H9N2 influenza A viruses isolated from poultry in Pakistan containing NS genes similar to highly pathogenic H7N3 and H5N1 viruses.

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Munir Iqbal

2009-06-01

Full Text Available The impact of avian influenza caused by H9N2 viruses in Pakistan is now significantly more severe than in previous years. Since all gene segments contribute towards the virulence of avian influenza virus, it was imperative to investigate the molecular features and genetic relationships of H9N2 viruses prevalent in this region. Analysis of the gene sequences of all eight RNA segments from 12 viruses isolated between 2005 and 2008 was undertaken. The hemagglutinin (HA sequences of all isolates were closely related to H9N2 viruses isolated from Iran between 2004 and 2007 and contained leucine instead of glutamine at position 226 in the receptor binding pocket, a recognised marker for the recognition of sialic acids linked alpha2-6 to galactose. The neuraminidase (NA of two isolates contained a unique five residue deletion in the stalk (from residues 80 to 84, a possible indication of greater adaptation of these viruses to the chicken host. The HA, NA, nucleoprotein (NP, and matrix (M genes showed close identity with H9N2 viruses isolated during 1999 in Pakistan and clustered in the A/Quail/Hong Kong/G1/97 virus lineage. In contrast, the polymerase genes clustered with H9N2 viruses from India, Iran and Dubai. The NS gene segment showed greater genetic diversity and shared a high level of similarity with NS genes from either H5 or H7 subtypes rather than with established H9N2 Eurasian lineages. These results indicate that during recent years the H9N2 viruses have undergone extensive genetic reassortment which has led to the generation of H9N2 viruses of novel genotypes in the Indian sub-continent. The novel genotypes of H9N2 viruses may play a role in the increased problems observed by H9N2 to poultry and reinforce the continued need to monitor H9N2 infections for their zoonotic potential.

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

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Jhung, Michael A; Nelson, Deborah I

2015-02-06

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

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

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

2015-07-01

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

Reassortant H9N2 influenza viruses containing H5N1-like PB1 genes isolated from black-billed magpies in Southern China.

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Guoying Dong

Full Text Available H9N2 influenza A viruses have become endemic in different types of terrestrial poultry and wild birds in Asia, and are occasionally transmitted to humans and pigs. To evaluate the role of black-billed magpies (Pica pica in the evolution of influenza A virus, we conducted two epidemic surveys on avian influenza viruses in wild black-billed magpies in Guangxi, China in 2005 and characterized three isolated black-billed magpie H9N2 viruses (BbM viruses. Phylogenetic analysis indicated that three BbM viruses were almost identical with 99.7 to 100% nucleotide homology in their whole genomes, and were reassortants containing BJ94-like (Ck/BJ/1/94 HA, NA, M, and NS genes, SH/F/98-like (Ck/SH/F/98 PB2, PA, and NP genes, and H5N1-like (Ck/YN/1252/03, clade 1 PB1 genes. Genetic analysis showed that BbM viruses were most likely the result of multiple reassortments between co-circulating H9N2-like and H5N1-like viruses, and were genetically different from other H9N2 viruses because of the existence of H5N1-like PB1 genes. Genotypical analysis revealed that BbM viruses evolved from diverse sources and belonged to a novel genotype (B46 discovered in our recent study. Molecular analysis suggested that BbM viruses were likely low pathogenic reassortants. However, results of our pathogenicity study demonstrated that BbM viruses replicated efficiently in chickens and a mammalian mouse model but were not lethal for infected chickens and mice. Antigenic analysis showed that BbM viruses were antigenic heterologous with the H9N2 vaccine strain. Our study is probably the first report to document and characterize H9N2 influenza viruses isolated from black-billed magpies in southern China. Our results suggest that black-billed magpies were susceptible to H9N2 influenza viruses, which raise concerns over possible transmissions of reassortant H9N2 viruses among poultry and wild birds.

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

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

2013-01-01

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

Influenza A(H9N2) Virus, Myanmar, 2014-2015.

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Lin, Thant Nyi; Nonthabenjawan, Nutthawan; Chaiyawong, Supassama; Bunpapong, Napawan; Boonyapisitsopa, Supanat; Janetanakit, Taveesak; Mon, Pont Pont; Mon, Hla Hla; Oo, Kyaw Naing; Oo, Sandi Myint; Mar Win, Mar; Amonsin, Alongkorn

2017-06-01

Routine surveillance of influenza A virus was conducted in Myanmar during 2014-2015. Influenza A(H9N2) virus was isolated in Shan State, upper Myanmar. Whole-genome sequencing showed that H9N2 virus from Myanmar was closely related to H9N2 virus of clade 4.2.5 from China.

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

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Indriani R

2015-03-01

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

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

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Vivi Setiawaty

2013-01-01

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

Risk of Human Infections With Highly Pathogenic H5N2 and Low Pathogenic H7N1 Avian Influenza Strains During Outbreaks in Ostriches in South Africa.

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Venter, Marietjie; Treurnicht, Florette K; Buys, Amelia; Tempia, Stefano; Samudzi, Rudo; McAnerney, Johanna; Jacobs, Charlene A; Thomas, Juno; Blumberg, Lucille

2017-09-15

Risk factors for human infection with highly pathogenic (HP) and low-pathogenic (LP) avian influenza (AI) H5N2 and H7N1 were investigated during outbreaks in ostriches in the Western Cape province, South Africa. Serum surveys were conducted for veterinarians, farmworkers, and laboratory and abattoir workers involved in 2 AI outbreaks in the Western Cape province: (1) controlling and culling of 42000 ostriches during (HPAI)H5N2 outbreaks in ostriches (2011) (n = 207); (2) movement control during (LPAI)H7N1 outbreaks in 2012 (n = 66). A third serosurvey was conducted on state veterinarians from across the country in 2012 tasked with disease control in general (n = 37). Antibodies to H5 and H7 were measured by means of hemagglutination inhibition and microneutralization assays, with microneutralization assay titers >40 considered positive. Two of 207 (1%) participants were seropositive for H5 and 4 of 207 (2%) for H7 in 2011, compared with 1 of 66 (1.5%) and 8 of 66 (13%) in 2012. Although individuals in all professions tested seropositive, abattoir workers (10 of 97; 10.3%) were significantly more at risk of influenza A(H7N1) infection (P = .001) than those in other professions (2 of 171;1.2%). Among state veterinarians, 4 of 37(11%) were seropositive for H7 and 1 of 37 (2.7%) for H5. Investigations of (LP)H7N1-associated fatalities in wild birds and quarantined exotic birds in Gauteng, AI outbreaks in poultry in KwaZulu-Natal, and ostriches in Western Cape province provide possible exposure events. (LPAI)H7N1 strains pose a greater infection-risk than (HPAI)H5N2 strains to persons involved in control of outbreaks in infected birds, with ostrich abattoir workers at highest risk. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

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

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Ahn, Insung; Son, Hyeon S

2007-07-01

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

Genetic and biological characterization of three poultry-origin H5N6 avian influenza viruses with all internal genes from genotype S H9N2 viruses.

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Liu, Kaituo; Gu, Min; Hu, Shunlin; Gao, Ruyi; Li, Juan; Shi, Liwei; Sun, Wenqi; Liu, Dong; Gao, Zhao; Xu, Xiulong; Hu, Jiao; Wang, Xiaoquan; Liu, Xiaowen; Chen, Sujuan; Peng, Daxin; Jiao, Xinan; Liu, Xiufan

2018-04-01

During surveillance for avian influenza viruses, three H5N6 viruses were isolated in chickens obtained from live bird markets in eastern China, between January 2015 and April 2016. Sequence analysis revealed a high genomic homology between these poultry isolates and recent human H5N6 variants whose internal genes were derived from genotype S H9N2 avian influenza viruses. Glycan binding assays revealed that all avian H5N6 viruses were capable of binding to both human-type SAα-2,6Gal receptors and avian-type SAα-2,3Gal receptors. Their biological characteristics were further studied in BALB/c mice, specific-pathogen-free chickens, and mallard ducks. All three isolates had low pathogenicity in mice but were highly pathogenic to chickens, as evidenced by 100% mortality 36-120 hours post infection at a low dose of 10 3.0 EID 50 and through effective contact transmission. Moreover, all three poultry H5N6 isolates caused asymptomatic infections in ducks, which may serve as a reservoir host for their maintenance and dissemination; these migrating waterfowl could cause a potential global pandemic. Our study suggests that continuous epidemiological surveillance in poultry should be implemented for the early prevention of future influenza outbreaks.

Natural Reassortants of Potentially Zoonotic Avian Influenza Viruses H5N1 and H9N2 from Egypt Display Distinct Pathogenic Phenotypes in Experimentally Infected Chickens and Ferrets.

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Naguib, Mahmoud M; Ulrich, Reiner; Kasbohm, Elisa; Eng, Christine L P; Hoffmann, Donata; Grund, Christian; Beer, Martin; Harder, Timm C

2017-12-01

The cocirculation of zoonotic highly pathogenic avian influenza virus (HPAIV) of subtype H5N1 and avian influenza virus (AIV) of subtype H9N2 among poultry in Egypt for at least 6 years should render that country a hypothetical hot spot for the emergence of reassortant, phenotypically altered viruses, yet no reassortants have been detected in Egypt. The present investigations proved that reassortants of the Egyptian H5N1 clade 2.2.1.2 virus and H9N2 virus of the G1-B lineage can be generated by coamplification in embryonated chicken eggs. Reassortants were restricted to the H5N1 subtype and acquired between two and all six of the internal segments of the H9N2 virus. Five selected plaque-purified reassortant clones expressed a broad phenotypic spectrum both in vitro and in vivo Two groups of reassortants were characterized to have retarded growth characteristics in vitro compared to the H5N1 parent virus. One clone provoked reduced mortality in inoculated chickens, although the characteristics of a highly pathogenic phenotype were retained. Enhanced zoonotic properties were not predicted for any of these clones, and this prediction was confirmed by ferret inoculation experiments: neither the H5N1 parent virus nor two selected clones induced severe clinical symptoms or were transmitted to sentinel ferrets by contact. While the emergence of reassortants of Egyptian HPAIV of subtype H5N1 with internal gene segments of cocirculating H9N2 viruses is possible in principle, the spread of such viruses is expected to be governed by their fitness to outcompete the parental viruses in the field. The eventual spread of attenuated phenotypes, however, would negatively impact syndrome surveillance on poultry farms and might foster enzootic virus circulation. IMPORTANCE Despite almost 6 years of the continuous cocirculation of highly pathogenic avian influenza virus H5N1 and avian influenza virus H9N2 in poultry in Egypt, no reassortants of the two subtypes have been reported

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

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Hawkins, Michelle G; Crossley, Beate M; Osofsky, Anna; Webby, Richard J; Lee, Chang-Won; Suarez, David L; Hietala, Sharon K

2006-01-15

A 3-month-old red-lored Amazon parrot (Amazona autumnalis autumnalis) was evaluated for severe lethargy. Avian influenza virus hemagglutinin subtype H5N2 with low pathogenicity was characterized by virus isolation, real-time reverse transcriptase PCR assay, chicken intravenous pathogenicity index, and reference sera. The virus was also determined to be closely related to a virus lineage that had been reported only in Mexico and Central America. The chick was admitted to the hospital and placed in quarantine. Supportive care treatment was administered. Although detection of H5 avian influenza virus in birds in the United States typically results in euthanasia of infected birds, an alternative strategy with strict quarantine measures and repeated diagnostic testing was used. The chick recovered from the initial clinical signs after 4 days and was released from quarantine 9 weeks after initial evaluation after 2 consecutive negative virus isolation and real-time reverse transcriptase PCR assay results. To the authors' knowledge, this is the first report of H5N2 avian influenza A virus isolated from a psittacine bird and represents the first introduction of this virus into the United States, most likely by illegal importation of psittacine birds. Avian influenza A virus should be considered as a differential diagnosis for clinical signs of gastrointestinal tract disease in psittacine birds, especially in birds with an unknown history of origin. Although infection with avian influenza virus subtype H5 is reportable, destruction of birds is not always required.

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

International Nuclear Information System (INIS)

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

2009-01-01

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

Live bird markets of Bangladesh: H9N2 viruses and the near absence of highly pathogenic H5N1 influenza.

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Nicholas J Negovetich

2011-04-01

Full Text Available 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.

Activation of type I and III interferon signalling pathways occurs in lung epithelial cells infected with low pathogenic avian influenza viruses.

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Richard Sutejo

Full Text Available The host response to the low pathogenic avian influenza (LPAI H5N2, H5N3 and H9N2 viruses were examined in A549, MDCK, and CEF cells using a systems-based approach. The H5N2 and H5N3 viruses replicated efficiently in A549 and MDCK cells, while the H9N2 virus replicated least efficiently in these cell types. However, all LPAI viruses exhibited similar and higher replication efficiencies in CEF cells. A comparison of the host responses of these viruses and the H1N1/WSN virus and low passage pH1N1 clinical isolates was performed in A549 cells. The H9N2 and H5N2 virus subtypes exhibited a robust induction of Type I and Type III interferon (IFN expression, sustained STAT1 activation from between 3 and 6 hpi, which correlated with large increases in IFN-stimulated gene (ISG expression by 10 hpi. In contrast, cells infected with the pH1N1 or H1N1/WSN virus showed only small increases in Type III IFN signalling, low levels of ISG expression, and down-regulated expression of the IFN type I receptor. JNK activation and increased expression of the pro-apoptotic XAF1 protein was observed in A549 cells infected with all viruses except the H1N1/WSN virus, while MAPK p38 activation was only observed in cells infected with the pH1N1 and the H5 virus subtypes. No IFN expression and low ISG expression levels were generally observed in CEF cells infected with either AIV, while increased IFN and ISG expression was observed in response to the H1N1/WSN infection. These data suggest differences in the replication characteristics and antivirus signalling responses both among the different LPAI viruses, and between these viruses and the H1N1 viruses examined. These virus-specific differences in host cell signalling highlight the importance of examining the host response to avian influenza viruses that have not been extensively adapted to mammalian tissue culture.

Transmission of Avian Influenza Virus (H3N2) to Dogs

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Song, Daesub; Kang, Bokyu; Lee, Chulseung; Jung, Kwonil; Ha, Gunwoo; Kang, Dongseok; Park, Seongjun; Park, Bongkyun; Oh, Jinsik

2008-01-01

In South Korea, where avian influenza virus subtypes H3N2, H5N1, H6N1, and H9N2 circulate or have been detected, 3 genetically similar canine influenza virus (H3N2) strains of avian origin (A/canine/Korea/01/2007, A/canine/Korea/02/2007, and A/canine/Korea/03/2007) were isolated from dogs exhibiting severe respiratory disease. To determine whether the novel canine influenza virus of avian origin was transmitted among dogs, we experimentally infected beagles with this influenza virus (H3N2) is...

The onset of virus shedding and clinical signs in chickens infected with high-pathogenicity and low-pathogenicity avian influenza viruses.

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Spickler, Anna R; Trampel, Darrell W; Roth, James A

2008-12-01

Some avian influenza viruses may be transmissible to mammals by ingestion. Cats and dogs have been infected by H5N1 avian influenza viruses when they ate raw poultry, and two human H5N1 infections were linked to the ingestion of uncooked duck blood. The possibility of zoonotic influenza from exposure to raw poultry products raises concerns about flocks with unrecognized infections. The present review examines the onset of virus shedding and the development of clinical signs for a variety of avian influenza viruses in chickens. In experimentally infected birds, some high-pathogenicity avian influenza (HPAI) and low-pathogenicity avian influenza (LPAI) viruses can occur in faeces and respiratory secretions as early as 1 to 2 days after inoculation. Some HPAI viruses have also been found in meat 1 day after inoculation and in eggs after 3 days. There is no evidence that LPAI viruses can be found in meat, and the risk of their occurrence in eggs is poorly understood. Studies in experimentally infected birds suggest that clinical signs usually develop within a few days of virus shedding; however, some models and outbreak descriptions suggest that clinical signs may not become evident for a week or more in some H5 or H7 HPAI-infected flocks. During this time, avian influenza viruses might be found in poultry products. LPAI viruses can be shed in asymptomatically infected or minimally affected flocks, but these viruses are unlikely to cause significant human disease.

Passive immunotherapy for influenza A H5N1 virus infection with equine hyperimmune globulin F(ab'2 in mice

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Li Yanbin

2006-03-01

Full Text Available Abstract Background Avian influenza virus H5N1 has demonstrated considerable pandemic potential. Currently, no effective vaccines for H5N1 infection are available, so passive immunotherapy may be an alternative strategy. To investigate the possible therapeutic effect of antibody against highly pathogenic H5N1 virus on a mammal host, we prepared specific equine anti-H5N1 IgGs from horses vaccinated with inactivated H5N1 virus, and then obtained the F(ab'2 fragments by pepsin digestion of IgGs. Methods The horses were vaccinated with inactivated H5N1 vaccine to prepare anti-H5N1 IgGs. The F(ab'2 fragments were purified from anti-H5N1 hyperimmune sera by a protocol for 'enhanced pepsin digestion'. The protective effect of the F(ab'2 fragments against H5N1 virus infection was determined in cultured MDCK cells by cytopathic effect (CPE assay and in a BALB/c mouse model by survival rate assay. Results By the protocol for 'enhanced pepsin digestion', total 16 g F(ab'2 fragments were finally obtained from one liter equine antisera with the purity of over 90%. The H5N1-specific F(ab'2 fragments had a HI titer of 1:1024, and the neutralization titre of F(ab'2 reached 1: 2048. The in vivo assay showed that 100 μg of the F(ab'2 fragments could protect BALB/c mice infected with a lethal dose of influenza H5N1 virus. Conclusion The availability of highly purified H5N1-specific F(ab'2 fragments may be promising for treatment of influenza H5N1 infection. Our work has provided experimental support for the application of the therapeutic equine immunoglobulin in future large primate or human trials.

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).

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

2016-03-01

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

Comparison of temporal and spatial dynamics of seasonal H3N2, pandemic H1N1 and highly pathogenic avian influenza H5N1 virus infections in ferrets.

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Judith M A van den Brand

Full Text Available Humans may be infected by different influenza A viruses--seasonal, pandemic, and zoonotic--which differ in presentation from mild upper respiratory tract disease to severe and sometimes fatal pneumonia with extra-respiratory spread. Differences in spatial and temporal dynamics of these infections are poorly understood. Therefore, we inoculated ferrets with seasonal H3N2, pandemic H1N1 (pH1N1, and highly pathogenic avian H5N1 influenza virus and performed detailed virological and pathological analyses at time points from 0.5 to 14 days post inoculation (dpi, as well as describing clinical signs and hematological parameters. H3N2 infection was restricted to the nose and peaked at 1 dpi. pH1N1 infection also peaked at 1 dpi, but occurred at similar levels throughout the respiratory tract. H5N1 infection occurred predominantly in the alveoli, where it peaked for a longer period, from 1 to 3 dpi. The associated lesions followed the same spatial distribution as virus infection, but their severity peaked between 1 and 6 days later. Neutrophil and monocyte counts in peripheral blood correlated with inflammatory cell influx in the alveoli. Of the different parameters used to measure lower respiratory tract disease, relative lung weight and affected lung tissue allowed the best quantitative distinction between the virus groups. There was extra-respiratory spread to more tissues--including the central nervous system--for H5N1 infection than for pH1N1 infection, and to none for H3N2 infection. This study shows that seasonal, pandemic, and zoonotic influenza viruses differ strongly in the spatial and temporal dynamics of infection in the respiratory tract and extra-respiratory tissues of ferrets.

Isolation and genetic characterization of a novel 2.2.1.2a H5N1 virus from a vaccinated meat-turkeys flock in Egypt.

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Salaheldin, Ahmed H; Veits, Jutta; Abd El-Hamid, Hatem S; Harder, Timm C; Devrishov, Davud; Mettenleiter, Thomas C; Hafez, Hafez M; Abdelwhab, Elsayed M

2017-03-09

Vaccination of poultry to control highly pathogenic avian influenza virus (HPAIV) H5N1 is used in several countries. HPAIV H5N1 of clade 2.2.1 which is endemic in Egypt has diversified into two genetic clades. Clade 2.2.1.1 represents antigenic drift variants in vaccinated commercial poultry while clade 2.2.1.2 variants are detected in humans and backyard poultry. Little is known about H5N1 infection in vaccinated turkeys under field conditions. Here, we describe an HPAI H5N1 outbreak in a vaccinated meat-turkey flock in Egypt. Birds were vaccinated with inactivated H5N2 and H5N1 vaccines at 8 and 34 days of age, respectively. At 72 nd day of age (38 days post last vaccination), turkeys exhibited mild respiratory signs, cyanosis of snood and severe congestion of the internal organs. Survivors had a reduction in feed consumption and body gain. A mortality of ~29% cumulated within 10 days after the onset of clinical signs. Laboratory diagnosis using RT-qPCRs revealed presence of H5N1 but was negative for H7 and H9 subtypes. A substantial antigenic drift against different serum samples from clade 2.2.1.1 and clade 2.3.4.4 was observed. Based on full genome sequence analysis the virus belonged to clade 2.2.1.2 but clustered with recent H5N1 viruses from 2015 in poultry in Israel, Gaza and Egypt in a novel subclade designated here 2.2.1.2a which is distinct from 2014/2015 2.2.1.2 viruses. These viruses possess 2.2.1.2 clade-specific genetic signatures and also mutations in the HA similar to those in clade 2.2.1.1 that enabled evasion from humoral immune response. Taken together, this manuscript describes a recent HPAI H5N1 outbreak in vaccinated meat-turkeys in Egypt after infection with a virus representing novel distinct 2.2.1.2a subclade. Infection with HPAIV H5N1 in commercial turkeys resulted in significant morbidity and mortality despite of vaccination using H5 vaccines. The isolated virus showed antigenic drift and clustered in a novel cluster designated here

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

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

2017-06-15

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

Effect of neuraminidase inhibitor-resistant mutations on pathogenicity of clade 2.2 A/Turkey/15/06 (H5N1) influenza virus in ferrets.

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Ilyushina, Natalia A; Seiler, Jon P; Rehg, Jerold E; Webster, Robert G; Govorkova, Elena A

2010-05-27

The acquisition of neuraminidase (NA) inhibitor resistance by H5N1 influenza viruses has serious clinical implications, as this class of drugs can be an essential component of pandemic control measures. The continuous evolution of the highly pathogenic H5N1 influenza viruses results in the emergence of natural NA gene variations whose impact on viral fitness and NA inhibitor susceptibility are poorly defined. We generated seven genetically stable recombinant clade 2.2 A/Turkey/15/06-like (H5N1) influenza viruses carrying NA mutations located either in the framework residues (E119A, H274Y, N294S) or in close proximity to the NA enzyme active site (V116A, I117V, K150N, Y252H). NA enzyme inhibition assays showed that NA mutations at positions 116, 117, 274, and 294 reduced susceptibility to oseltamivir carboxylate (IC(50)s increased 5- to 940-fold). Importantly, the E119A NA mutation (previously reported to confer resistance in the N2 NA subtype) was stable in the clade 2.2 H5N1 virus background and induced cross-resistance to oseltamivir carboxylate and zanamivir. We demonstrated that Y252H NA mutation contributed for decreased susceptibility of clade 2.2 H5N1 viruses to oseltamivir carboxylate as compared to clade 1 viruses. The enzyme kinetic parameters (V(max), K(m) and K(i)) of the avian-like N1 NA glycoproteins were highly consistent with their IC(50) values. None of the recombinant H5N1 viruses had attenuated virulence in ferrets inoculated with 10(6) EID(50) dose. Most infected ferrets showed mild clinical disease signs that differed in duration. However, H5N1 viruses carrying the E119A or the N294S NA mutation were lethal to 1 of 3 inoculated animals and were associated with significantly higher virus titers (Pinfluenza drugs that target different virus/host factors and can limit the emergence of resistance.

Effect of neuraminidase inhibitor-resistant mutations on pathogenicity of clade 2.2 A/Turkey/15/06 (H5N1 influenza virus in ferrets.

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Natalia A Ilyushina

2010-05-01

Full Text Available The acquisition of neuraminidase (NA inhibitor resistance by H5N1 influenza viruses has serious clinical implications, as this class of drugs can be an essential component of pandemic control measures. The continuous evolution of the highly pathogenic H5N1 influenza viruses results in the emergence of natural NA gene variations whose impact on viral fitness and NA inhibitor susceptibility are poorly defined. We generated seven genetically stable recombinant clade 2.2 A/Turkey/15/06-like (H5N1 influenza viruses carrying NA mutations located either in the framework residues (E119A, H274Y, N294S or in close proximity to the NA enzyme active site (V116A, I117V, K150N, Y252H. NA enzyme inhibition assays showed that NA mutations at positions 116, 117, 274, and 294 reduced susceptibility to oseltamivir carboxylate (IC(50s increased 5- to 940-fold. Importantly, the E119A NA mutation (previously reported to confer resistance in the N2 NA subtype was stable in the clade 2.2 H5N1 virus background and induced cross-resistance to oseltamivir carboxylate and zanamivir. We demonstrated that Y252H NA mutation contributed for decreased susceptibility of clade 2.2 H5N1 viruses to oseltamivir carboxylate as compared to clade 1 viruses. The enzyme kinetic parameters (V(max, K(m and K(i of the avian-like N1 NA glycoproteins were highly consistent with their IC(50 values. None of the recombinant H5N1 viruses had attenuated virulence in ferrets inoculated with 10(6 EID(50 dose. Most infected ferrets showed mild clinical disease signs that differed in duration. However, H5N1 viruses carrying the E119A or the N294S NA mutation were lethal to 1 of 3 inoculated animals and were associated with significantly higher virus titers (P<0.01 and inflammation in the lungs compared to the wild-type virus. Our results suggest that highly pathogenic H5N1 variants carrying mutations within the NA active site that decrease susceptibility to NA inhibitors may possess increased

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

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Chan Chris CS

2010-01-01

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

Influenza A H5N1 clade 2.3.4 virus with a different antiviral susceptibility profile replaced clade 1 virus in humans in northern Vietnam.

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Mai T Q Le

2008-10-01

Full Text Available Prior to 2007, highly pathogenic avian influenza (HPAI H5N1 viruses isolated from poultry and humans in Vietnam were consistently reported to be clade 1 viruses, susceptible to oseltamivir but resistant to amantadine. Here we describe the re-emergence of human HPAI H5N1 virus infections in Vietnam in 2007 and the characteristics of the isolated viruses.Respiratory specimens from patients suspected to be infected with avian influenza in 2007 were screened by influenza and H5 subtype specific polymerase chain reaction. Isolated H5N1 strains were further characterized by genome sequencing and drug susceptibility testing. Eleven poultry outbreak isolates from 2007 were included in the sequence analysis. Eight patients, all of them from northern Vietnam, were diagnosed with H5N1 in 2007 and five of them died. Phylogenetic analysis of H5N1 viruses isolated from humans and poultry in 2007 showed that clade 2.3.4 H5N1 viruses replaced clade 1 viruses in northern Vietnam. Four human H5N1 strains had eight-fold reduced in-vitro susceptibility to oseltamivir as compared to clade 1 viruses. In two poultry isolates the I117V mutation was found in the neuraminidase gene, which is associated with reduced susceptibility to oseltamivir. No mutations in the M2 gene conferring amantadine resistance were found.In 2007, H5N1 clade 2.3.4 viruses replaced clade 1 viruses in northern Vietnam and were susceptible to amantadine but showed reduced susceptibility to oseltamivir. Combination antiviral therapy with oseltamivir and amantadine for human cases in Vietnam is recommended.

Exposure to a low pathogenic A/H7N2 virus in chickens protects against highly pathogenic A/H7N1 virus but not against subsequent infection with A/H5N1.

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Júlia Vergara-Alert

Full Text Available Recent evidences have demonstrated that the presence of low pathogenic avian influenza viruses (LPAIV may play an important role in host ecology and transmission of avian influenza viruses (AIV. While some authors have clearly demonstrated that LPAIV can mutate to render highly pathogenic avian influenza viruses (HPAIV, others have shown that their presence could provide the host with enough immunological memory to resist re-infections with HPAIV. In order to experimentally study the role of pre-existing host immunity, chickens previously infected with H7N2 LPAIV were subsequently challenged with H7N1 HPAIV. Pre-infection of chickens with H7N2 LAPIV conferred protection against the lethal challenge with H7N1 HPAIV, dramatically reducing the viral shedding, the clinical signs and the pathological outcome. Correlating with the protection afforded, sera from chickens primed with H7N2 LPAIV reacted with the H7-AIV subtype in hemagglutination inhibition assay and specifically with the N2-neuraminidase antigen. Conversely, subsequent exposure to H5N1 HPAIV resulted in a two days-delay on the onset of disease but all chickens died by 7 days post-challenge. Lack of protection correlated with the absence of H5-hemagglutining inhibitory antibodies prior to H5N1 HPAIV challenge. Our data suggest that in naturally occurring outbreaks of HPAIV, birds with pre-existing immunity to LPAIV could survive lethal infections with HA-homologous HPAIV but not subsequent re-infections with HA-heterologous HPAIV. These results could be useful to better understand the dynamics of AIV in chickens and might help in future vaccine formulations.

Genetic characterization of avian influenza subtype H4N6 and H4N9 from live bird market, Thailand

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Kitikoon Pravina

2011-03-01

Full Text Available Abstract A one year active surveillance program for influenza A viruses among avian species in a live-bird market (LBM in Bangkok, Thailand was conducted in 2009. Out of 970 samples collected, influenza A virus subtypes H4N6 (n = 2 and H4N9 (n = 1 were isolated from healthy Muscovy ducks. All three viruses were characterized by whole genome sequencing with subsequent phylogenetic analysis and genetic comparison. Phylogenetic analysis of all eight viral genes showed that the viruses clustered in the Eurasian lineage of influenza A viruses. Genetic analysis showed that H4N6 and H4N9 viruses display low pathogenic avian influenza characteristics. The HA cleavage site and receptor binding sites were conserved and resembled to LPAI viruses. This study is the first to report isolation of H4N6 and H4N9 viruses from birds in LBM in Thailand and shows the genetic diversity of the viruses circulating in the LBM. In addition, co-infection of H4N6 and H4N9 in the same Muscovy duck was observed.

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

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Annemarie Bouma

2009-01-01

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

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

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

2013-01-01

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

A duplex real-time RT-PCR assay for detecting H5N1 avian influenza virus and pandemic H1N1 influenza virus

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Qin E-de

2010-06-01

Full Text Available Abstract A duplex real-time reverse transcriptase polymerase chain reaction (RT-PCR assay was improved for simultaneous detection of highly pathogenic H5N1 avian influenza virus and pandemic H1N1 (2009 influenza virus, which is suitable for early diagnosis of influenza-like patients and for epidemiological surveillance. The sensitivity of this duplex real-time RT-PCR assay was 0.02 TCID50 (50% tissue culture infective dose for H5N1 and 0.2 TCID50 for the pandemic H1N1, which was the same as that of each single-target RT-PCR for pandemic H1N1 and even more sensitive for H5N1 with the same primers and probes. No cross reactivity of detecting other subtype influenza viruses or respiratory tract viruses was observed. Two hundred and thirty-six clinical specimens were tested by comparing with single real-time RT-PCR and result from the duplex assay was 100% consistent with the results of single real-time RT-PCR and sequence analysis.

A duplex real-time RT-PCR assay for detecting H5N1 avian influenza virus and pandemic H1N1 influenza virus

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Kang, Xiao-ping; Jiang, Tao; Li, Yong-qiang; Lin, Fang; Liu, Hong; Chang, Guo-hui; Zhu, Qing-yu; Qin, E-de; Qin, Cheng-feng; Yang, Yin-hui

2010-01-01

Abstract A duplex real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay was improved for simultaneous detection of highly pathogenic H5N1 avian influenza virus and pandemic H1N1 (2009) influenza virus, which is suitable for early diagnosis of influenza-like patients and for epidemiological surveillance. The sensitivity of this duplex real-time RT-PCR assay was 0.02 TCID50 (50% tissue culture infective dose) for H5N1 and 0.2 TCID50 for the pandemic H1N1, which was the same a...

H5N1 Influenza A Virus PB1-F2 Relieves HAX-1-Mediated Restriction of Avian Virus Polymerase PA in Human Lung Cells.

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Mazel-Sanchez, B; Boal-Carvalho, I; Silva, F; Dijkman, R; Schmolke, M

2018-06-01

Highly pathogenic influenza A viruses (IAV) from avian hosts were first reported to directly infect humans 20 years ago. However, such infections are rare events, and our understanding of factors promoting or restricting zoonotic transmission is still limited. One accessory protein of IAV, PB1-F2, was associated with pathogenicity of pandemic and zoonotic IAV. This short (90-amino-acid) peptide does not harbor an enzymatic function. We thus identified host factors interacting with H5N1 PB1-F2, which could explain its importance for virulence. PB1-F2 binds to HCLS1-associated protein X1 (HAX-1), a recently identified host restriction factor of the PA subunit of IAV polymerase complexes. We demonstrate that the PA of a mammal-adapted H1N1 IAV is resistant to HAX-1 imposed restriction, while the PA of an avian-origin H5N1 IAV remains sensitive. We also showed HAX-1 sensitivity for PAs of A/Brevig Mission/1/1918 (H1N1) and A/Shanghai/1/2013 (H7N9), two avian-origin zoonotic IAV. Inhibition of H5N1 polymerase by HAX-1 can be alleviated by its PB1-F2 through direct competition. Accordingly, replication of PB1-F2-deficient H5N1 IAV is attenuated in the presence of large amounts of HAX-1. Mammal-adapted H1N1 and H3N2 viruses do not display this dependence on PB1-F2 for efficient replication in the presence of HAX-1. We propose that PB1-F2 plays a key role in zoonotic transmission of avian H5N1 IAV into humans. IMPORTANCE Aquatic and shore birds are the natural reservoir of influenza A viruses from which the virus can jump into a variety of bird and mammal host species, including humans. H5N1 influenza viruses are a good model for this process. They pose an ongoing threat to human and animal health due to their high mortality rates. However, it is currently unclear what restricts these interspecies jumps on the host side or what promotes them on the virus side. Here we show that a short viral peptide, PB1-F2, helps H5N1 bird influenza viruses to overcome a human restriction

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

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

2013-01-01

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

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

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Jörg Rohde

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

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

DEFF Research Database (Denmark)

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

2012-01-01

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

Comparative pathology of pigs infected with Korean H1N1, H1N2, or H3N2 swine influenza A viruses

OpenAIRE

Lyoo, Kwang-Soo; Kim, Jeong-Ki; Jung, Kwonil; Kang, Bo-Kyu; Song, Daesub

2014-01-01

Background The predominant subtypes of swine influenza A virus (SIV) in Korea swine population are H1N1, H1N2, and H3N2. The viruses are genetically close to the classical U.S. H1N1 and triple-reassortant H1N2 and H3N2 viruses, respectively. Comparative pathogenesis caused by Korean H1N1, H1N2, and H3N2 SIV was evaluated in this study. Findings The H3N2 infected pigs had severe scores of gross and histopathological lesions at post-inoculation days (PID) 2, and this then progressively decrease...

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

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

2018-04-17

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

Comparative pathogenesis of an avian H5N2 and a swine H1N1 influenza virus in pigs.

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Annebel De Vleeschauwer

2009-08-01

Full Text Available Pigs are considered intermediate hosts for the transmission of avian influenza viruses (AIVs to humans but the basic organ pathogenesis of AIVs in pigs has been barely studied. We have used 42 four-week-old influenza naive pigs and two different inoculation routes (intranasal and intratracheal to compare the pathogenesis of a low pathogenic (LP H5N2 AIV with that of an H1N1 swine influenza virus. The respiratory tract and selected extra-respiratory tissues were examined for virus replication by titration, immunofluorescence and RT-PCR throughout the course of infection. Both viruses caused a productive infection of the entire respiratory tract and epithelial cells in the lungs were the major target. Compared to the swine virus, the AIV produced lower virus titers and fewer antigen positive cells at all levels of the respiratory tract. The respiratory part of the nasal mucosa in particular showed only rare AIV positive cells and this was associated with reduced nasal shedding of the avian compared to the swine virus. The titers and distribution of the AIV varied extremely between individual pigs and were strongly affected by the route of inoculation. Gross lung lesions and clinical signs were milder with the avian than with the swine virus, corresponding with lower viral loads in the lungs. The brainstem was the single extra-respiratory tissue found positive for virus and viral RNA with both viruses. Our data do not reject the theory of the pig as an intermediate host for AIVs, but they suggest that AIVs need to undergo genetic changes to establish full replication potential in pigs. From a biomedical perspective, experimental LP H5 AIV infection of pigs may be useful to examine heterologous protection provided by H5 vaccines or other immunization strategies, as well as for further studies on the molecular pathogenesis and neurotropism of AIVs in mammals.

Isolation of H5N6, H7N9 and H9N2 avian influenza A viruses from air sampled at live poultry markets in China, 2014 and 2015.

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Zhou, Jie; Wu, Jie; Zeng, Xianqiao; Huang, Guofeng; Zou, Lirong; Song, Yingchao; Gopinath, Divya; Zhang, Xin; Kang, Min; Lin, Jinyan; Cowling, Benjamin J; Lindsley, William G; Ke, Changwen; Peiris, Joseph Sriyal Malik; Yen, Hui-Ling

2016-09-01

Zoonotic infections by avian influenza viruses occur at the human-poultry interface, but the modes of transmission have not been fully investigated. We assessed the potential for airborne and fomite transmission at live poultry markets in Guangzhou city and in Hong Kong Special Administrative Region (SAR), China, during 2014 and 2015. Viral genome and infectious avian influenza A viruses of H5N6, H7N9, and H9N2 subtypes were detected predominantly from particles larger or equal to 1 μm in diameter in the air sampled with cyclone-based bioaerosol samplers at the live poultry markets in Guangzhou. Influenza A(H9N2) viruses were ubiquitously isolated every month during the study period from air and environmental swabs, and different lineages of H9N2 virus were isolated from markets where chickens and minor land-based poultry were sold. The use of de-feathering devices increased the quantity of virus-laden airborne particles while market closure reduced the amount of such particles. The results highlight the possibility of airborne transmission of avian influenza viruses among poultry or from poultry to humans within such settings. This may explain epidemiological observations in which some patients with H7N9 infection reported being in markets but no direct contact with live poultry or poultry stalls. This article is copyright of The Authors, 2016.

Victims and vectors: highly pathogenic avian influenza H5N1 and the ecology of wild birds

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Takekawa, John Y.; Prosser, Diann J.; Newman, Scott H.; Muzaffar, Sabir Bin; Hill, Nichola J.; Yan, Baoping; Xiao, Xiangming; Lei, Fumin; Li, Tianxian; Schwarzbach, Steven E.; Howell, Judd A.

2010-01-01

The emergence of highly pathogenic avian influenza (HPAI) viruses has raised concerns about the role of wild birds in the spread and persistence of the disease. In 2005, an outbreak of the highly pathogenic subtype H5N1 killed more than 6,000 wild waterbirds at Qinghai Lake, China. Outbreaks have continued to periodically occur in wild birds at Qinghai Lake and elsewhere in Central China and Mongolia. This region has few poultry but is a major migration and breeding area for waterbirds in the Central Asian Flyway, although relatively little is known about migratory movements of different species and connectivity of their wetland habitats. The scientific debate has focused on the role of waterbirds in the epidemiology, maintenance and spread of HPAI H5N1: to what extent are they victims affected by the disease, or vectors that have a role in disease transmission? In this review, we summarise the current knowledge of wild bird involvement in the ecology of HPAI H5N1. Specifically, we present details on: (1) origin of HPAI H5N1; (2) waterbirds as LPAI reservoirs and evolution into HPAI; (3) the role of waterbirds in virus spread and persistence; (4) key biogeographic regions of outbreak; and (5) applying an ecological research perspective to studying AIVs in wild waterbirds and their ecosystems.

The replication of Bangladeshi H9N2 avian influenza viruses carrying genes from H7N3 in mammals.

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Shanmuganatham, Karthik K; Jones, Jeremy C; Marathe, Bindumadhav M; Feeroz, Mohammed M; Jones-Engel, Lisa; Walker, David; Turner, Jasmine; Rabiul Alam, S M; Kamrul Hasan, M; Akhtar, Sharmin; Seiler, Patrick; McKenzie, Pamela; Krauss, Scott; Webby, Richard J; Webster, Robert G

2016-04-20

H9N2 avian influenza viruses are continuously monitored by the World Health Organization because they are endemic; they continually reassort with H5N1, H7N9 and H10N8 viruses; and they periodically cause human infections. We characterized H9N2 influenza viruses carrying internal genes from highly pathogenic H7N3 viruses, which were isolated from chickens or quail from live-bird markets in Bangladesh between 2010 and 2013. All of the H9N2 viruses used in this study carried mammalian host-specific mutations. We studied their replication kinetics in normal human bronchoepithelial cells and swine tracheal and lung explants, which exhibit many features of the mammalian airway epithelium and serve as a mammalian host model. All H9N2 viruses replicated to moderate-to-high titers in the normal human bronchoepithelial cells and swine lung explants, but replication was limited in the swine tracheal explants. In Balb/c mice, the H9N2 viruses were nonlethal, replicated to moderately high titers and the infection was confined to the lungs. In the ferret model of human influenza infection and transmission, H9N2 viruses possessing the Q226L substitution in hemagglutinin replicated well without clinical signs and spread via direct contact but not by aerosol. None of the H9N2 viruses tested were resistant to the neuraminidase inhibitors. Our study shows that the Bangladeshi H9N2 viruses have the potential to infect humans and highlights the importance of monitoring and characterizing this influenza subtype to better understand the potential risk these viruses pose to humans.

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

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Huaiying Xu

2015-02-01

Full Text Available Infection of poultry with diverse lineages of H5N2 avian influenza viruses has been documented for over three decades in different parts of the world, with limited outbreaks caused by this highly pathogenic avian influenza virus. In the present study, three avian H5N2 influenza viruses, A/chicken/Shijiazhuang/1209/2013, A/chicken/Chiping/0321/2014, and A/chicken/Laiwu/0313/2014, were isolated from chickens with clinical symptoms of avian influenza. Complete genomic and phylogenetic analyses demonstrated that all three isolates are novel recombinant viruses with hemagglutinin (HA and matrix (M genes derived from H5N1, and remaining genes derived from H9N2-like viruses. The HA cleavage motif in all three strains (PQIEGRRRKR/GL is characteristic of a highly pathogenic avian influenza virus strain. These results indicate the occurrence of H5N2 recombination and highlight the importance of continued surveillance of the H5N2 subtype virus and reformulation of vaccine strains.

Natural co-infection of influenza A/H3N2 and A/H1N1pdm09 viruses resulting in a reassortant A/H3N2 virus.

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Rith, Sareth; Chin, Savuth; Sar, Borann; Y, Phalla; Horm, Srey Viseth; Ly, Sovann; Buchy, Philippe; Dussart, Philippe; Horwood, Paul F

2015-12-01

Despite annual co-circulation of different subtypes of seasonal influenza, co-infections between different viruses are rarely detected. These co-infections can result in the emergence of reassortant progeny. We document the detection of an influenza co-infection, between influenza A/H3N2 with A/H1N1pdm09 viruses, which occurred in a 3 year old male in Cambodia during April 2014. Both viruses were detected in the patient at relatively high viral loads (as determined by real-time RT-PCR CT values), which is unusual for influenza co-infections. As reassortment can occur between co-infected influenza A strains we isolated plaque purified clonal viral populations from the clinical material of the patient infected with A/H3N2 and A/H1N1pdm09. Complete genome sequences were completed for 7 clonal viruses to determine if any reassorted viruses were generated during the influenza virus co-infection. Although most of the viral sequences were consistent with wild-type A/H3N2 or A/H1N1pdm09, one reassortant A/H3N2 virus was isolated which contained an A/H1N1pdm09 NS1 gene fragment. The reassortant virus was viable and able to infect cells, as judged by successful passage in MDCK cells, achieving a TCID50 of 10(4)/ml at passage number two. There is no evidence that the reassortant virus was transmitted further. The co-infection occurred during a period when co-circulation of A/H3N2 and A/H1N1pdm09 was detected in Cambodia. It is unclear how often influenza co-infections occur, but laboratories should consider influenza co-infections during routine surveillance activities. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

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

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

2013-01-01

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

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

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Misako Nakayama

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

Prophylactic and therapeutic efficacy of avian antibodies against influenza virus H5N1 and H1N1 in mice.

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Huan H Nguyen

Full Text Available 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

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

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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

2015-08-11

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.

Antigenic variation of H1N1, H1N2 and H3N2 swine influenza viruses in Japan and Vietnam.

Science.gov (United States)

Takemae, Nobuhiro; Nguyen, Tung; Ngo, Long Thanh; Hiromoto, Yasuaki; Uchida, Yuko; Pham, Vu Phong; Kageyama, Tsutomu; Kasuo, Shizuko; Shimada, Shinichi; Yamashita, Yasutaka; Goto, Kaoru; Kubo, Hideyuki; Le, Vu Tri; Van Vo, Hung; Do, Hoa Thi; Nguyen, Dang Hoang; Hayashi, Tsuyoshi; Matsuu, Aya; Saito, Takehiko

2013-04-01

The antigenicity of the influenza A virus hemagglutinin is responsible for vaccine efficacy in protecting pigs against swine influenza virus (SIV) infection. However, the antigenicity of SIV strains currently circulating in Japan and Vietnam has not been well characterized. We examined the antigenicity of classical H1 SIVs, pandemic A(H1N1)2009 (A(H1N1)pdm09) viruses, and seasonal human-lineage SIVs isolated in Japan and Vietnam. A hemagglutination inhibition (HI) assay was used to determine antigenic differences that differentiate the recent Japanese H1N2 and H3N2 SIVs from the H1N1 and H3N2 domestic vaccine strains. Minor antigenic variation between pig A(H1N1)pdm09 viruses was evident by HI assay using 13 mAbs raised against homologous virus. A Vietnamese H1N2 SIV, whose H1 gene originated from a human strain in the mid-2000s, reacted poorly with post-infection ferret serum against human vaccine strains from 2000-2010. These results provide useful information for selection of optimal strains for SIV vaccine production.

In vitro reassortment between endemic H1N2 and 2009 H1N1 pandemic swine influenza viruses generates attenuated viruses.

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Ben M Hause

Full Text Available The pandemic H1N1 (pH1N1 influenza virus was first reported in humans in the spring of 2009 and soon thereafter was identified in numerous species, including swine. Reassortant viruses, presumably arising from the co-infection of pH1N1 and endemic swine influenza virus (SIV, were subsequently identified from diagnostic samples collected from swine. In this study, co-infection of swine testicle (ST cells with swine-derived endemic H1N2 (MN745 and pH1N1 (MN432 yielded two reassortant H1N2 viruses (R1 and R2, both possessing a matrix gene derived from pH1N1. In ST cells, the reassortant viruses had growth kinetics similar to the parental H1N2 virus and reached titers approximately 2 log(10 TCID(50/mL higher than the pH1N1 virus, while in A549 cells these viruses had similar growth kinetics. Intranasal challenge of pigs with H1N2, pH1N1, R1 or R2 found that all viruses were capable of infecting and transmitting between direct contact pigs as measured by real time reverse transcription PCR of nasal swabs. Lung samples were also PCR-positive for all challenge groups and influenza-associated microscopic lesions were detected by histology. Interestingly, infectious virus was detected in lung samples for pigs challenged with the parental H1N2 and pH1N1 at levels significantly higher than either reassortant virus despite similar levels of viral RNA. Results of our experiment suggested that the reassortant viruses generated through in vitro cell culture system were attenuated without gaining any selective growth advantage in pigs over the parental lineages. Thus, reassortant influenza viruses described in this study may provide a good system to study genetic basis of the attenuation and its mechanism.

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

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

2016-01-01

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

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

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

2003-01-01

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

Evolution of highly pathogenic avian H5N1 influenza viruses

Energy Technology Data Exchange (ETDEWEB)

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

2009-01-01

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

Evaluation of the zoonotic potential of multiple subgroups of clade 2.3.4.4 influenza A (H5N8) virus.

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Lee, Yu-Na; Lee, Eun-Kyoung; Song, Byung-Min; Heo, Gyeong-Beom; Woo, Sang-Hee; Cheon, Sun-Ha; Lee, Youn-Jeong

2018-03-01

Clade 2.3.4.4 H5N8 highly pathogenic avian influenza viruses (HPAIVs) have spread worldwide. Phylogenetic analysis identified two genetic groups of the H5N8 HPAIVs in South Korea; group A evolved further into four subgroups. Here, we examined the zoonotic potential, both in vivo and in vitro, of genetically distinct subgroups of H5N8 HPAIVs isolated in South Korea. When compared with other subgroups, A/mallard/Korea/H2102/2015 (H2102) virus caused relatively severe disease in mice at high doses. In ferrets, all H5N8 viruses replicated restrictively in the respiratory tract and did not induce significant clinical signs of influenza infection. In vitro studies, all viruses displayed a hemagglutinin phenotype that was poorly adapted for infection of mammals, although the H2102 virus exhibited higher replication kinetics at 33°C than the others. Although H5N8 HPAIVs have not yet acquired all the characteristics required for adaptation to mammals, their ability to evolve continuously underscores the need for timely risk assessment. Copyright © 2018 Elsevier Inc. All rights reserved.

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

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Hameed Sajid

2009-03-01

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

Effect of Neuraminidase Inhibitor–Resistant Mutations on Pathogenicity of Clade 2.2 A/Turkey/15/06 (H5N1) Influenza Virus in Ferrets

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Ilyushina, Natalia A.; Seiler, Jon P.; Rehg, Jerold E.; Webster, Robert G.; Govorkova, Elena A.

2010-01-01

The acquisition of neuraminidase (NA) inhibitor resistance by H5N1 influenza viruses has serious clinical implications, as this class of drugs can be an essential component of pandemic control measures. The continuous evolution of the highly pathogenic H5N1 influenza viruses results in the emergence of natural NA gene variations whose impact on viral fitness and NA inhibitor susceptibility are poorly defined. We generated seven genetically stable recombinant clade 2.2 A/Turkey/15/06-like (H5N...

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

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

2017-01-01

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

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

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

2009-01-29

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

Novel reassortant of swine influenza H1N2 virus in Germany.

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Zell, Roland; Motzke, Susann; Krumbholz, Andi; Wutzler, Peter; Herwig, Volker; Dürrwald, Ralf

2008-01-01

European porcine H1N2 influenza viruses arose after multiple reassortment steps involving a porcine influenza virus with avian-influenza-like internal segments and human H1N1 and H3N2 viruses in 1994. In Germany, H1N2 swine influenza viruses first appeared in 2000. Two German H1N2 swine influenza virus strains isolated from pigs with clinical symptoms of influenza are described. They were characterized by the neutralization test, haemagglutination inhibition (HI) test and complete sequencing of the viral genomes. The data demonstrate that these viruses represent a novel H1N2 reassortant. The viruses showed limited neutralization by sera raised against heterologous A/sw/Bakum/1,832/00-like H1N2 viruses. Sera pools from recovered pigs showed a considerably lower HI reaction, indicative of diagnostic difficulties in using the HI test to detect these viruses with A/sw/Bakum/1,832/00-like H1N2 antigens. Genome sequencing revealed the novel combination of the human-like HAH1 gene of European porcine H1N2 influenza viruses and the NAN2 gene of European porcine H3N2 viruses.

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

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

2018-01-01

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

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

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El-Zoghby Elham F

2012-11-01

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

A historical perspective of influenza A(H1N2) virus.

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Komadina, Naomi; McVernon, Jodie; Hall, Robert; Leder, Karin

2014-01-01

The emergence and transition to pandemic status of the influenza A(H1N1)A(H1N1)pdm09) virus in 2009 illustrated the potential for previously circulating human viruses to re-emerge in humans and cause a pandemic after decades of circulating among animals. Within a short time of the initial emergence of A(H1N1)pdm09 virus, novel reassortants were isolated from swine. In late 2011, a variant (v) H3N2 subtype was isolated from humans, and by 2012, the number of persons infected began to increase with limited person-to-person transmission. During 2012 in the United States, an A(H1N2)v virus was transmitted to humans from swine. During the same year, Australia recorded its first H1N2 subtype infection among swine. The A(H3N2)v and A(H1N2)v viruses contained the matrix protein from the A(H1N1)pdm09 virus, raising the possibility of increased transmissibility among humans and underscoring the potential for influenza pandemics of novel swine-origin viruses. We report on the differing histories of A(H1N2) viruses among humans and animals.

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

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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.

Novel H5N8 clade 2.3.4.4 highly pathogenic avian influenza virus in wild awuatic birds, Russia, 2016

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H5N1 high pathogenicity avian influenza virus (HPAIV) emerged in 1996 in Guangdong China (Gs/GD) and has evolved into multiple genetic clades. Since 2008, HPAIV H5 clade 2.3.4 with N2, N5 and N8 neuraminidase subtypes have been identified in mainland China and outbreak of HPAIV H5N8 clade 2.3.4.4 ou...

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

OpenAIRE

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

2007-01-01

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

Direct isolation of H1N2 recombinant virus from a throat swab of a patient simultaneously infected with H1N1 and H3N2 influenza A viruses.

OpenAIRE

Nishikawa, F; Sugiyama, T

1983-01-01

Two H1N2 recombinant viruses were isolated by a plaquing method from a throat swab of a patient who was simultaneously infected with H1N1 and H3N2 influenza viruses during the Tokyo epidemic of 1981. This is the first direct evidence that recombination of influenza viruses occurred in the human body.

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

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

2017-07-01

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

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

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

2000-01-01

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

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

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Peirong eJiao

2014-11-01

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

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

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

2011-09-28

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

Acid Stability of the Hemagglutinin Protein Regulates H5N1 Influenza Virus Pathogenicity

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

2012-12-10

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

Single-dose mucosal immunization with a candidate universal influenza vaccine provides rapid protection from virulent H5N1, H3N2 and H1N1 viruses.

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Graeme E Price

2010-10-01

Full Text Available The sudden emergence of novel influenza viruses is a global public health concern. Conventional influenza vaccines targeting the highly variable surface glycoproteins hemagglutinin and neuraminidase must antigenically match the emerging strain to be effective. In contrast, "universal" vaccines targeting conserved viral components could be used regardless of viral strain or subtype. Previous approaches to universal vaccination have required protracted multi-dose immunizations. Here we evaluate a single dose universal vaccine strategy using recombinant adenoviruses (rAd expressing the conserved influenza virus antigens matrix 2 and nucleoprotein.In BALB/c mice, administration of rAd via the intranasal route was superior to intramuscular immunization for induction of mucosal responses and for protection against highly virulent H1N1, H3N2, or H5N1 influenza virus challenge. Mucosally vaccinated mice not only survived, but had little morbidity and reduced lung virus titers. Protection was observed as early as 2 weeks post-immunization, and lasted at least 10 months, as did antibodies and lung T cells with activated phenotypes. Virus-specific IgA correlated with but was not essential for protection, as demonstrated in studies with IgA-deficient animals.Mucosal administration of NP and M2-expressing rAd vectors provided rapid and lasting protection from influenza viruses in a subtype-independent manner. Such vaccines could be used in the interval between emergence of a new virus strain and availability of strain-matched vaccines against it. This strikingly effective single-dose vaccination thus represents a candidate off-the-shelf vaccine for emergency use during an influenza pandemic.

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

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

2017-01-01

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

Comparative pathology of pigs infected with Korean H1N1, H1N2, or H3N2 swine influenza A viruses.

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Lyoo, Kwang-Soo; Kim, Jeong-Ki; Jung, Kwonil; Kang, Bo-Kyu; Song, Daesub

2014-09-24

The predominant subtypes of swine influenza A virus (SIV) in Korea swine population are H1N1, H1N2, and H3N2. The viruses are genetically close to the classical U.S. H1N1 and triple-reassortant H1N2 and H3N2 viruses, respectively. Comparative pathogenesis caused by Korean H1N1, H1N2, and H3N2 SIV was evaluated in this study. The H3N2 infected pigs had severe scores of gross and histopathological lesions at post-inoculation days (PID) 2, and this then progressively decreased. Both the H1N1 and H1N2 infected pigs lacked gross lesions at PID 2, but they showed moderate to severe pneumonia on PID 4, 7 and 14. The pigs infected with H1N1 had significant scores of gross and histopathological lesions when compared with the other pigs infected with H1N2, H3N2, and mock at PID 14. Mean SIV antigen-positive scores were rarely detected for pigs infected with H1N2 and H3N2 from PID 7, whereas a significantly increased amount of viral antigens were found in the bronchioles and alveolar epithelium of the H1N1infected pigs at PID 14. We demonstrated that Korean SIV subtypes had different pulmonary pathologic patterns. The Korean H3N2 rapidly induced acute lung lesions such as broncho-interstitial pneumonia, while the Korean H1N1 showed longer course of infection as compared to other strains.

Efficacy of Live-Attenuated H9N2 Influenza Vaccine Candidates Containing NS1 Truncations against H9N2 Avian Influenza Viruses

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Sujuan Chen

2017-06-01

Full Text Available H9N2 avian influenza virus is a zoonotic agent with a broad host range that can contribute genetic information to H5 or H7N9 subtype viruses, which are significant threats to both humans and birds. Thus, there is a great need for a vaccine to control H9N2 avian influenza. Three mutant viruses of an H9N2 virus A/chicken/Taixing/10/2010 (rTX-NS1-73, rTX-NS1-100, and rTX-NS1-128 were constructed with different NS1 gene truncations and confirmed by western blot analysis. The genetic stability, pathogenicity, transmissibility, and host immune responses toward these mutants were evaluated. The mutant virus rTX-NS1-128 exhibited the most attenuated phenotype and lost transmissibility. The expression levels of interleukin 12 in the nasal and tracheal tissues from chickens immunized with rTX-NS1-128 were significantly upregulated on day 3 post-immunization and the IgA and IgG antibody levels were significantly increased on days 7, 14, and 21 post-immunization when compared to chickens that received an inactivated vaccine. rTX-NS1-128 also protected chickens from challenge by homologous and heterologous H9N2 avian influenza viruses. The results indicate that rTX-NS1-128 can be used as a potential live-attenuated vaccine against H9N2 avian influenza.

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

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

2012-01-01

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

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

Science.gov (United States)

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

2013-07-01

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

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

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Chunmao Zhang

2017-09-01

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

Influenza H5N1 and H1N1 virus replication and innate immune responses in bronchial epithelial cells are influenced by the state of differentiation.

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Renee W Y Chan

Full Text Available Influenza H5N1 virus continues to be enzootic in poultry and transmits zoonotically to humans. Although a swine-origin H1N1 virus has emerged to become pandemic, its virulence for humans remains modest in comparison to that seen in zoonotic H5N1 disease. As human respiratory epithelium is the primary target cells for influenza viruses, elucidating the viral tropism and host innate immune responses of influenza H5N1 virus in human bronchial epithelium may help to understand the pathogenesis. Here we established primary culture of undifferentiated and well differentiated normal human bronchial epithelial (NHBE cells and infected with highly pathogenic influenza H5N1 virus (A/Vietnam/3046/2004 and a seasonal influenza H1N1 virus (A/Hong Kong/54/1998, the viral replication kinetics and cytokine and chemokine responses were compared by qPCR and ELISA. We found that the in vitro culture of the well differentiated NHBE cells acquired the physiological properties of normal human bronchi tissue which express high level of alpha2-6-linked sialic acid receptors and human airway trypsin-like (HAT protease, in contrast to the low expression in the non-differentiated NHBE cells. When compared to H1N1 virus, the H5N1 virus replicated more efficiently and induced a stronger type I interferon response in the undifferentiated NHBE cells. In contrast, in well differentiated cultures, H5N1 virus replication was less efficient and elicited a lower interferon-beta response in comparison with H1N1 virus. Our data suggest that the differentiation of bronchial epithelial cells has a major influence in cells' permissiveness to human H1N1 and avian H5N1 viruses and the host innate immune responses. The reduced virus replication efficiency partially accounts for the lower interferon-beta responses in influenza H5N1 virus infected well differentiated NHBE cells. Since influenza infection in the bronchial epithelium will lead to tissue damage and associate with the

Evolution of highly pathogenic H5N1 avian influenza viruses in Vietnam between 2001 and 2007.

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Xiu-Feng Wan

Full Text Available Highly pathogenic avian influenza (HPAI H5N1 viruses have caused dramatic economic losses to the poultry industry of Vietnam and continue to pose a serious threat to public health. As of June 2008, Vietnam had reported nearly one third of worldwide laboratory confirmed human H5N1 infections. To better understand the emergence, spread and evolution of H5N1 in Vietnam we studied over 300 H5N1 avian influenza viruses isolated from Vietnam since their first detection in 2001. Our phylogenetic analyses indicated that six genetically distinct H5N1 viruses were introduced into Vietnam during the past seven years. The H5N1 lineage that evolved following the introduction in 2003 of the A/duck/Hong Kong/821/2002-like viruses, with clade 1 hemagglutinin (HA, continued to predominate in southern Vietnam as of May 2007. A virus with a clade 2.3.4 HA newly introduced into northern Vietnam in 2007, reassorted with pre-existing clade 1 viruses, resulting in the emergence of novel genotypes with neuraminidase (NA and/or internal gene segments from clade 1 viruses. A total of nine distinct genotypes have been present in Vietnam since 2001, including five that were circulating in 2007. At least four of these genotypes appear to have originated in Vietnam and represent novel H5N1 viruses not reported elsewhere. Geographic and temporal analyses of H5N1 infection dynamics in poultry suggest that the majority of viruses containing new genes were first detected in northern Vietnam and subsequently spread to southern Vietnam after reassorting with pre-existing local viruses in northern Vietnam. Although the routes of entry and spread of H5N1 in Vietnam remain speculative, enhanced poultry import controls and virologic surveillance efforts may help curb the entry and spread of new HPAI viral genes.

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

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

2009-01-01

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

PB2 mutations D701N and S714R promote adaptation of an influenza H5N1 virus to a mammalian host.

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Czudai-Matwich, Volker; Otte, Anna; Matrosovich, Mikhail; Gabriel, Gülsah; Klenk, Hans-Dieter

2014-08-01

Mutation D701N in the PB2 protein is known to play a prominent role in the adaptation of avian influenza A viruses to mammalian hosts. In contrast, little is known about the nearby mutations S714I and S714R, which have been observed in some avian influenza viruses highly pathogenic for mammals. We have generated recombinant H5N1 viruses with PB2 displaying the avian signature 701D or the mammalian signature 701N and serine, isoleucine, and arginine at position 714 and compared them for polymerase activity and virus growth in avian and mammalian cells, as well as for pathogenicity in mice. Mutation D701N led to an increase in polymerase activity and replication efficiency in mammalian cells and in mouse pathogenicity, and this increase was significantly enhanced when mutation D701N was combined with mutation S714R. Stimulation by mutation S714I was less distinct. These observations indicate that PB2 mutation S714R, in combination with the mammalian signature at position 701, has the potential to promote the adaptation of an H5N1 virus to a mammalian host. Influenza A/H5N1 viruses are avian pathogens that have pandemic potential, since they are spread over large parts of Asia, Africa, and Europe and are occasionally transmitted to humans. It is therefore of high scientific interest to understand the mechanisms that determine the host specificity and pathogenicity of these viruses. It is well known that the PB2 subunit of the viral polymerase is an important host range determinant and that PB2 mutation D701N plays an important role in virus adaptation to mammalian cells. In the present study, we show that mutation S714R is also involved in adaptation and that it cooperates with D701N in exposing a nuclear localization signal that mediates importin-α binding and entry of PB2 into the nucleus, where virus replication and transcription take place. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Rapid detection of the avian influenza virus H5N1 subtype in Egypt

African Journals Online (AJOL)

Dr

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

Novel genetic reassortants in H9N2 influenza A viruses and their diverse pathogenicity to mice

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Bi Yuhai

2011-11-01

Full Text Available Abstract Background H9N2 influenza A viruses have undergone extensive reassortments in different host species, and could lead to the epidemics or pandemics with the potential emergence of novel viruses. Methods To understand the genetic and pathogenic features of early and current circulating H9N2 viruses, 15 representative H9N2 viruses isolated from diseased chickens in northern China between 1998 and 2010 were characterized and compared with all Chinese H9N2 viruses available in the NCBI database. Then, the representative viruses of different genotypes were selected to study the pathogenicity in mice with the aim to investigate the adaptation and the potential pathogenicity of the novel H9N2 reassortants to mammals. Results Our results demonstrated that most of the 15 isolates were reassortants and generated four novel genotypes (B62-B65, which incorporated the gene segments from Eurasian H9N2 lineage, North American H9N2 branch, and H5N1 viruses. It was noteworthy that the newly identified genotype B65 has been prevalent in China since 2007, and more importantly, different H9N2 influenza viruses displayed a diverse pathogenicity to mice. The isolates of the 2008-2010 epidemic (genotypes B55 and B65 were lowly infectious, while two representative viruses of genotypes B0 and G2 isolated from the late 1990s were highly pathogenic to mice. In addition, Ck/SD/LY-1/08 (genotype 63, containing H5N1-like NP and PA genes was able to replicate well in mouse lungs with high virus titers but caused mild clinical signs. Conclusion Several lines of evidence indicated that the H9N2 influenza viruses constantly change their genetics and pathogenicity. Thus, the genetic evolution of H9N2 viruses and their pathogenicity to mammals should be closely monitored to prevent the emergence of novel pandemic viruses.

MVA-based H5N1 vaccine affords cross-clade protection in mice against influenza A/H5N1 viruses at low doses and after single immunization.

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Joost H C M Kreijtz

Full Text Available Human infections with highly pathogenic avian influenza viruses of the H5N1 subtype, frequently reported since 2003, result in high morbidity and mortality. It is feared that these viruses become pandemic, therefore the development of safe and effective vaccines is desirable. MVA-based H5N1 vaccines already proved to be effective when two immunizations with high doses were used. Dose-sparing strategies would increase the number of people that can be vaccinated when the amount of vaccine preparations that can be produced is limited. Furthermore, protective immunity is induced ideally after a single immunization. Therefore the minimal requirements for induction of protective immunity with a MVA-based H5N1 vaccine were assessed in mice. To this end, mice were vaccinated once or twice with descending doses of a recombinant MVA expressing the HA gene of influenza virus A/Vietnam/1194/04. The protective efficacy was determined after challenge infection with the homologous clade 1 virus and a heterologous virus derived from clade 2.1, A/Indonesia/5/05 by assessing weight loss, virus replication and histopathological changes. It was concluded that MVA-based vaccines allowed significant dose-sparing and afford cross-clade protection, also after a single immunization, which are favorable properties for an H5N1 vaccine candidate.

Generation, characterization and epitope mapping of two neutralizing and protective human recombinant antibodies against influenza A H5N1 viruses.

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Lina Sun

Full Text Available BACKGROUND: The development of new therapeutic targets and strategies to control highly pathogenic avian influenza (HPAI H5N1 virus infection in humans is urgently needed. Broadly cross-neutralizing recombinant human antibodies obtained from the survivors of H5N1 avian influenza provide an important role in immunotherapy for human H5N1 virus infection and definition of the critical epitopes for vaccine development. METHODOLOGY/PRINCIPAL FINDINGS: We have characterized two recombinant baculovirus-expressed human antibodies (rhAbs, AVFluIgG01 and AVFluIgG03, generated by screening a Fab antibody phage library derived from a patient recovered from infection with a highly pathogenic avian influenza A H5N1 clade 2.3 virus. AVFluIgG01 cross-neutralized the most of clade 0, clade 1, and clade 2 viruses tested, in contrast, AVFluIgG03 only neutralized clade 2 viruses. Passive immunization of mice with either AVFluIgG01 or AVFluIgG03 antibody resulted in protection from a lethal H5N1 clade 2.3 virus infection. Furthermore, through epitope mapping, we identify two distinct epitopes on H5 HA molecule recognized by these rhAbs and demonstrate their potential to protect against a lethal H5N1 virus infection in a mouse model. CONCLUSIONS/SIGNIFICANCE: Importantly, localization of the epitopes recognized by these two neutralizing and protective antibodies has provided, for the first time, insight into the human antibody responses to H5N1 viruses which contribute to the H5 immunity in the recovered patient. These results highlight the potential of a rhAbs treatment strategy for human H5N1 virus infection and provide new insight for the development of effective H5N1 pandemic vaccines.

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

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

2007-07-01

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

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

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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

2014-09-01

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. 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. 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. 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. 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. © 2014 The Authors. Influenza and Other Respiratory Viruses Published by John Wiley & Sons Ltd.

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

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Stefan J Halbherr

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

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

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Xue Wang

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

A reverse genetic analysis of human Influenza A virus H1N2

OpenAIRE

Anton, Aline

2010-01-01

Reassortment between influenza A viruses of different subtypes rarely appears. Even in a community where H1N1 and H3N2 viruses co-circulate, reassortment to produce persistent viruses of mixed gene segments does not readily occur. H1N2 viruses, that circulated between 2001-2003 were considered to have arisen through the reassortment of the two human influenza subtypes H1N1 and H3N2. Due to the fact they make such a rare appearance, H1N2 viruses used to have new characteristics compared to the...

Low pathogenicity avian influenza viruses infect chicken layers by different routes of inoculation.

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Pantin-Jackwood, Mary J; Smith, Diane M; Wasilenko, Jamie L; Spackman, Erica

2012-06-01

In order to develop better control measures against avian influenza, it is necessary to understand how the virus transmits in poultry. In a previous study in which the infectivity and transmissibility of the pandemic H1N1 influenza virus was examined in different poultry species, we found that no or minimal infection occurred in chicken and turkeys intranasally (IN) inoculated with the virus. However, we demonstrated that the virus can infect laying turkey hens by the intracloacal (IC) and intraoviduct (IO) routes, possibly explaining the drops in egg production observed in turkey breeder farms affected by the virus. Such novel routes of exposure have not been previously examined in chickens and could also explain outbreaks of low pathogenicity avian influenza (LPAI) that cause a decrease in egg production in chicken layers and breeders. In the present study, 46-wk-old specific-pathogen-free chicken layers were infected by the IN, IC, or IO routes with one of two LPAI viruses: a poultry origin virus, A/chicken/CA/1255/02 (H6N2), and a live bird market isolate, A/chicken/NJ/12220/97 (H9N2). Only hens IN inoculated with the H6N2 virus presented mild clinical signs consisting of depression and anorexia. However, a decrease in number of eggs laid was observed in all virus-inoculated groups when compared to control hens. Evidence of infection was found in all chickens inoculated with the H6N2 virus by any of the three routes and the virus transmitted to contact hens. On the other hand, only one or two hens from each of the groups inoculated with the H9N2 virus shed detectable levels of virus, or seroconverted and did not transmit the virus to contacts, regardless of the route of inoculation. In conclusion, LPAI viruses can also infect chickens through other routes besides the IN route, which is considered the natural route of exposure. However, as seen with the H9N2 virus, the infectivity of the virus did not increase when given by these alternate routes.

The pathogenecity of H5N1 highly pathogenic Avian Influenza (HPAI virus clade 2.3.2. in Indonesian indigenous chicken by contact tranmission with infected duck

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R. Damayanti

2017-05-01

Full Text Available An experimental transmission study was conducted using nine healthy Indonesian indigenous chickens placed together with two 30 days old ducks which were experimentally infected with H5N1 HPAI clade 2.3.2 virus in the Biosafety Laboratory Level 3 (BSL-3 facilities. The aim of the study was to find out the pathogenicity of H5N1 HPAI virus clade 2.3.2 in Indonesian indigenous chickens. The study showed that within twenty four hours rearing, the chickens were exhibited mild clinical signs and by 48 hours, all of the chickens died, whereas the ducks survived but with severe clinical signs. The H5N1 HPAI virus has been successfully isolated from chickens and ducks swabs, confirming that those animals were infected by the virus. Histologically, the infected chicken encountered with severe inflammation reaction namely non suppuratives encephalitis, tracheitis, myocarditis, interstitial pneumonia, hepatitis, proventriculitis, enteritis, pancreatitis, nephritis and bursitis. Necrotizing spleen and pancreas were also prominent. Viral antigen was detected by immunohistochemistry staining in various affected visceral organs. This suggests that Indonesian indigenous chickens were susceptible to H5N1 HPAI virus clade 2.3.2 and it can be transmitted easily to Indonesian indigenous chickens by contact transmission with infected ducks.

Influenza A (H3N2) Variant Virus

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... Swine Variant Pandemic Other Influenza A (H3N2) Variant Virus Language: English (US) Español Recommend on Facebook Tweet Share Compartir Influenza viruses that normally circulate in pigs are called “variant” ...

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

International Nuclear Information System (INIS)

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

2008-01-01

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

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

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

2017-03-21

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

Genetic and biological characterisation of an avian-like H1N2 swine influenza virus generated by reassortment of circulating avian-like H1N1 and H3N2 subtypes in Denmark

DEFF Research Database (Denmark)

Trebbien, Ramona; Bragstad, Karoline; Larsen, Lars Erik

2013-01-01

BACKGROUND: The influenza A virus subtypes H1N1, H1N2 and H3N2 are the most prevalent subtypes in swine. In 2003, a reassorted H1N2 swine influenza virus (SIV) subtype appeared and became prevalent in Denmark. In the present study, the reassortant H1N2 subtype was characterised genetically...... and the infection dynamics compared to an “avian-like” H1N1 virus by an experimental infection study. METHODS: Sequence analyses were performed of the H1N2 virus. Two groups of pigs were inoculated with the reassortant H1N2 virus and an “avian-like” H1N1 virus, respectively, followed by inoculation...... with the opposite subtype four weeks later. Measurements of HI antibodies and acute phase proteins were performed. Nasal virus excretion and virus load in lungs were determined by real-time RT-PCR. RESULTS: The phylogenetic analysis revealed that the reassorted H1N2 virus contained a European “avian-like” H1-gene...

Differential replication of avian influenza H9N2 viruses in human alveolar epithelial A549 cells

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Peiris Malik

2010-03-01

Full Text Available Abstract Avian influenza virus H9N2 isolates cause a mild influenza-like illness in humans. However, the pathogenesis of the H9N2 subtypes in human remains to be investigated. Using a human alveolar epithelial cell line A549 as host, we found that A/Quail/Hong Kong/G1/97 (H9N2/G1, which shares 6 viral "internal genes" with the lethal A/Hong Kong/156/97 (H5N1/97 virus, replicates efficiently whereas other H9N2 viruses, A/Duck/Hong Kong/Y280/97 (H9N2/Y280 and A/Chicken/Hong Kong/G9/97 (H9N2/G9, replicate poorly. Interestingly, we found that there is a difference in the translation of viral protein but not in the infectivity or transcription of viral genes of these H9N2 viruses in the infected cells. This difference may possibly be explained by H9N2/G1 being more efficient on viral protein production in specific cell types. These findings suggest that the H9N2/G1 virus like its counterpart H5N1/97 may be better adapted to the human host and replicates efficiently in human alveolar epithelial cells.

Genetic Characterization of H1N1 and H1N2 Influenza A Viruses Circulating in Ontario Pigs in 2012.

Science.gov (United States)

Grgić, Helena; Costa, Marcio; Friendship, Robert M; Carman, Susy; Nagy, Éva; Poljak, Zvonimir

2015-01-01

The objective of this study was to characterize H1N1 and H1N2 influenza A virus isolates detected during outbreaks of respiratory disease in pig herds in Ontario (Canada) in 2012. Six influenza viruses were included in analysis using full genome sequencing based on the 454 platform. In five H1N1 isolates, all eight segments were genetically related to 2009 pandemic virus (A(H1N1)pdm09). One H1N2 isolate had hemagglutinin (HA), polymerase A (PA) and non-structural (NS) genes closely related to A(H1N1)pdm09, and neuraminidase (NA), matrix (M), polymerase B1 (PB1), polymerase B2 (PB2), and nucleoprotein (NP) genes originating from a triple-reassortant H3N2 virus (tr H3N2). The HA gene of five Ontario H1 isolates exhibited high identity of 99% with the human A(H1N1)pdm09 [A/Mexico/InDRE4487/09] from Mexico, while one Ontario H1N1 isolate had only 96.9% identity with this Mexican virus. Each of the five Ontario H1N1 viruses had between one and four amino acid (aa) changes within five antigenic sites, while one Ontario H1N2 virus had two aa changes within two antigenic sites. Such aa changes in antigenic sites could have an effect on antibody recognition and ultimately have implications for immunization practices. According to aa sequence analysis of the M2 protein, Ontario H1N1 and H1N2 viruses can be expected to offer resistance to adamantane derivatives, but not to neuraminidase inhibitors.

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

International Nuclear Information System (INIS)

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

2012-01-01

Highlights: ► Vero cell-based HPAI H5N1 vaccine with stable high yield. ► Stable high yield derived from the YNVa H3N2 backbone. ► 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.

Novel reassortant influenza A(H1N2) virus derived from A(H1N1)pdm09 virus isolated from swine, Japan, 2012.

Science.gov (United States)

Kobayashi, Miho; Takayama, Ikuyo; Kageyama, Tsutomu; Tsukagoshi, Hiroyuki; Saitoh, Mika; Ishioka, Taisei; Yokota, Yoko; Kimura, Hirokazu; Tashiro, Masato; Kozawa, Kunihisa

2013-12-01

We isolated a novel influenza virus A(H1N2) strain from a pig on January 13, 2012, in Gunma Prefecture, Japan. Phylogenetic analysis showed that the strain was a novel type of double-reassortant virus derived from the swine influenza virus strains H1N1pdm09 and H1N2, which were prevalent in Gunma at that time.

Phylogenetic diversity and genotypical complexity of H9N2 influenza A viruses revealed by genomic sequence analysis.

Directory of Open Access Journals (Sweden)

Guoying Dong

Full Text Available H9N2 influenza A viruses have become established worldwide in terrestrial poultry and wild birds, and are occasionally transmitted to mammals including humans and pigs. To comprehensively elucidate the genetic and evolutionary characteristics of H9N2 influenza viruses, we performed a large-scale sequence analysis of 571 viral genomes from the NCBI Influenza Virus Resource Database, representing the spectrum of H9N2 influenza viruses isolated from 1966 to 2009. Our study provides a panoramic framework for better understanding the genesis and evolution of H9N2 influenza viruses, and for describing the history of H9N2 viruses circulating in diverse hosts. Panorama phylogenetic analysis of the eight viral gene segments revealed the complexity and diversity of H9N2 influenza viruses. The 571 H9N2 viral genomes were classified into 74 separate lineages, which had marked host and geographical differences in phylogeny. Panorama genotypical analysis also revealed that H9N2 viruses include at least 98 genotypes, which were further divided according to their HA lineages into seven series (A-G. Phylogenetic analysis of the internal genes showed that H9N2 viruses are closely related to H3, H4, H5, H7, H10, and H14 subtype influenza viruses. Our results indicate that H9N2 viruses have undergone extensive reassortments to generate multiple reassortants and genotypes, suggesting that the continued circulation of multiple genotypical H9N2 viruses throughout the world in diverse hosts has the potential to cause future influenza outbreaks in poultry and epidemics in humans. We propose a nomenclature system for identifying and unifying all lineages and genotypes of H9N2 influenza viruses in order to facilitate international communication on the evolution, ecology and epidemiology of H9N2 influenza viruses.

Different neuraminidase inhibitor susceptibilities of human H1N1, H1N2, and H3N2 influenza A viruses isolated in Germany from 2001 to 2005/2006.

Science.gov (United States)

Bauer, Katja; Richter, Martina; Wutzler, Peter; Schmidtke, Michaela

2009-04-01

In the flu season 2005/2006 amantadine-resistant human influenza A viruses (FLUAV) of subtype H3N2 circulated in Germany. This raises questions on the neuraminidase inhibitor (NAI) susceptibility of FLUAV. To get an answer, chemiluminescence-based neuraminidase inhibition assays were performed with 51 H1N1, H1N2, and H3N2 FLUAV isolated in Germany from 2001 to 2005/2006. According to the mean IC(50) values (0.38-0.91 nM for oseltamivir and 0.76-1.13 nM for zanamivir) most H1N1 and H3N2 FLUAV were NAI-susceptible. But, about four times higher zanamivir concentrations were necessary to inhibit neuraminidase activity of H1N2 viruses. Two H1N1 isolates were less susceptible to both drugs in NA inhibition as well as virus yield reduction assays. Results from sequence analysis of viral hemagglutinin and neuraminidase genes and evolutionary analysis of N2 gene revealed (i) different subclades for N2 in H1N2 and H3N2 FLUAV that could explain the differences in zanamivir susceptibility among these viruses and (ii) specific amino acid substitutions in the neuraminidase segment of the two less NAI-susceptible H1N1 isolates. One H3N2 was isolate proved to be a mixture of a NA deletion mutant and full-length NA viruses.

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

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Jacob Lum

2015-07-01

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

Influenza A H5N1 clade 2.3.4 virus with a different antiviral susceptibility profile replaced clade 1 virus in humans in northern Vietnam

NARCIS (Netherlands)

Le, Mai T. Q.; Wertheim, Heiman F. L.; Nguyen, Hien D.; Taylor, Walter; Hoang, Phuong V. M.; Vuong, Cuong D.; Nguyen, Hang L. K.; Nguyen, Ha H.; Nguyen, Thai Q.; Nguyen, Trung V.; van, Trang D.; Ngoc, Bich T.; Bui, Thinh N.; Nguyen, Binh G.; Nguyen, Liem T.; Luong, San T.; Phan, Phuc H.; Pham, Hung V.; Nguyen, Tung; Fox, Annette; Nguyen, Cam V.; Do, Ha Q.; Crusat, Martin; Farrar, Jeremy; Nguyen, Hien T.; de Jong, Menno D.; Horby, Peter

2008-01-01

BACKGROUND: Prior to 2007, highly pathogenic avian influenza (HPAI) H5N1 viruses isolated from poultry and humans in Vietnam were consistently reported to be clade 1 viruses, susceptible to oseltamivir but resistant to amantadine. Here we describe the re-emergence of human HPAI H5N1 virus infections

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

Science.gov (United States)

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

2014-03-01

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

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

Directory of Open Access Journals (Sweden)

Yuen Kit M

2009-10-01

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

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

Science.gov (United States)

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

2015-01-01

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

Genetic Characterization of H1N1 and H1N2 Influenza A Viruses Circulating in Ontario Pigs in 2012.

Directory of Open Access Journals (Sweden)

Helena Grgić

Full Text Available The objective of this study was to characterize H1N1 and H1N2 influenza A virus isolates detected during outbreaks of respiratory disease in pig herds in Ontario (Canada in 2012. Six influenza viruses were included in analysis using full genome sequencing based on the 454 platform. In five H1N1 isolates, all eight segments were genetically related to 2009 pandemic virus (A(H1N1pdm09. One H1N2 isolate had hemagglutinin (HA, polymerase A (PA and non-structural (NS genes closely related to A(H1N1pdm09, and neuraminidase (NA, matrix (M, polymerase B1 (PB1, polymerase B2 (PB2, and nucleoprotein (NP genes originating from a triple-reassortant H3N2 virus (tr H3N2. The HA gene of five Ontario H1 isolates exhibited high identity of 99% with the human A(H1N1pdm09 [A/Mexico/InDRE4487/09] from Mexico, while one Ontario H1N1 isolate had only 96.9% identity with this Mexican virus. Each of the five Ontario H1N1 viruses had between one and four amino acid (aa changes within five antigenic sites, while one Ontario H1N2 virus had two aa changes within two antigenic sites. Such aa changes in antigenic sites could have an effect on antibody recognition and ultimately have implications for immunization practices. According to aa sequence analysis of the M2 protein, Ontario H1N1 and H1N2 viruses can be expected to offer resistance to adamantane derivatives, but not to neuraminidase inhibitors.

Avian Influenza A (H5N1)

Centers for Disease Control (CDC) Podcasts

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

Genetic and biological characterisation of an avian-like H1N2 swine influenza virus generated by reassortment of circulating avian-like H1N1 and H3N2 subtypes in Denmark.

Science.gov (United States)

Trebbien, Ramona; Bragstad, Karoline; Larsen, Lars Erik; Nielsen, Jens; Bøtner, Anette; Heegaard, Peter M H; Fomsgaard, Anders; Viuff, Birgitte; Hjulsager, Charlotte Kristiane

2013-09-18

The influenza A virus subtypes H1N1, H1N2 and H3N2 are the most prevalent subtypes in swine. In 2003, a reassorted H1N2 swine influenza virus (SIV) subtype appeared and became prevalent in Denmark. In the present study, the reassortant H1N2 subtype was characterised genetically and the infection dynamics compared to an "avian-like" H1N1 virus by an experimental infection study. Sequence analyses were performed of the H1N2 virus. Two groups of pigs were inoculated with the reassortant H1N2 virus and an "avian-like" H1N1 virus, respectively, followed by inoculation with the opposite subtype four weeks later. Measurements of HI antibodies and acute phase proteins were performed. Nasal virus excretion and virus load in lungs were determined by real-time RT-PCR. The phylogenetic analysis revealed that the reassorted H1N2 virus contained a European "avian-like" H1-gene and a European "swine-like" N2-gene, thus being genetically distinct from most H1N2 viruses circulating in Europe, but similar to viruses reported in 2009/2010 in Sweden and Italy. Sequence analyses of the internal genes revealed that the reassortment probably arose between circulating Danish "avian-like" H1N1 and H3N2 SIVs. Infected pigs developed cross-reactive antibodies, and increased levels of acute phase proteins after inoculations. Pigs inoculated with H1N2 exhibited nasal virus excretion for seven days, peaking day 1 after inoculation two days earlier than H1N1 infected pigs and at a six times higher level. The difference, however, was not statistically significant. Pigs euthanized on day 4 after inoculation, had a high virus load in all lung lobes. After the second inoculation, the nasal virus excretion was minimal. There were no clinical sign except elevated body temperature under the experimental conditions. The "avian-like" H1N2 subtype, which has been established in the Danish pig population at least since 2003, is a reassortant between circulating swine "avian-like" H1N1 and H3N2. The Danish