Serveur d'exploration H2N2

Attention, ce site est en cours de développement !
Attention, site généré par des moyens informatiques à partir de corpus bruts.
Les informations ne sont donc pas validées.

Characterization of a Novel Reassortant Influenza A Virus (H2N2) from a Domestic Duck in Eastern China

Identifieur interne : 000008 ( Pmc/Corpus ); précédent : 000007; suivant : 000009

Characterization of a Novel Reassortant Influenza A Virus (H2N2) from a Domestic Duck in Eastern China

Auteurs : Mai-Juan Ma ; Xiao-Xian Yang ; Yan-Hua Qian ; Si-Yan Zhao ; Sha Hua ; Tie-Cheng Wang ; Shan-Hui Chen ; Guang-Yuan Ma ; Xiao-Yu Sang ; Lin-Na Liu ; Ai-Ping Wu ; Tai-Jiao Jiang ; Yu-Wei Gao ; Gregory C. Gray ; Teng Zhao ; Xia Ling ; Jing-Lin Wang ; Bing Lu ; Jun Qian ; Wu-Chun Cao

Source :

RBID : PMC:4274511

Abstract

While H2N2 viruses have been sporadically isolated from wild and domestic birds, H2N2 viruses have not been detected among human populations since 1968. Should H2N2 viruses adapt to domestic poultry they may pose a risk of infection to people, as most anyone born after 1968 would likely be susceptible to their infection. We report the isolation of a novel influenza A virus (H2N2) cultured in 2013 from a healthy domestic duck at a live poultry market in Wuxi City, China. Sequence data revealed that the novel H2N2 virus was similar to Eurasian avian lineage avian influenza viruses, the virus had been circulating for ≥ two years among poultry, had an increase in α2,6 binding affinity, and was not highly pathogenic. Approximately 9% of 100 healthy chickens sampled from the same area had elevated antibodies against the H2 antigen. Fortunately, there was sparse serological evidence that the virus was infecting poultry workers or had adapted to infect other mammals. These findings suggest that a novel H2N2 virus has been circulating among domestic poultry in Wuxi City, China and has some has increased human receptor affinity. It seems wise to conduct better surveillance for novel influenza viruses at Chinese live bird markets.


Url:
DOI: 10.1038/srep07588
PubMed: 25533850
PubMed Central: 4274511

Links to Exploration step

PMC:4274511

Le document en format XML

<record>
<TEI>
<teiHeader>
<fileDesc>
<titleStmt>
<title xml:lang="en">Characterization of a Novel Reassortant Influenza A Virus (H2N2) from a Domestic Duck in Eastern China</title>
<author>
<name sortKey="Ma, Mai Juan" sort="Ma, Mai Juan" uniqKey="Ma M" first="Mai-Juan" last="Ma">Mai-Juan Ma</name>
<affiliation>
<nlm:aff id="a1">
<institution>State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology</institution>
, Beijing, 100071, P. R. China</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Yang, Xiao Xian" sort="Yang, Xiao Xian" uniqKey="Yang X" first="Xiao-Xian" last="Yang">Xiao-Xian Yang</name>
<affiliation>
<nlm:aff id="a1">
<institution>State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology</institution>
, Beijing, 100071, P. R. China</nlm:aff>
</affiliation>
<affiliation>
<nlm:aff id="a2">
<institution>School of Public Health, Central South University</institution>
, Changsha, Hunan, 410000 P. R. China</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Qian, Yan Hua" sort="Qian, Yan Hua" uniqKey="Qian Y" first="Yan-Hua" last="Qian">Yan-Hua Qian</name>
<affiliation>
<nlm:aff id="a3">
<institution>Wuxi Center for Disease Control and Prevention</institution>
, Wuxi, 214023, P. R. China</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Zhao, Si Yan" sort="Zhao, Si Yan" uniqKey="Zhao S" first="Si-Yan" last="Zhao">Si-Yan Zhao</name>
<affiliation>
<nlm:aff id="a4">
<institution>Military Veterinary Research Institute, Academy of Military Medical Sciences</institution>
, Changchun, 130122, P. R. China</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Hua, Sha" sort="Hua, Sha" uniqKey="Hua S" first="Sha" last="Hua">Sha Hua</name>
<affiliation>
<nlm:aff id="a5">
<institution>Key Laboratory of Protein and Peptide Pharmaceutical, National Laboratory of Biomacromolecules, Institute of Biophysics</institution>
, Chinese Academy of Sciences, Beijing, 100101,
<country>P. R. China</country>
</nlm:aff>
</affiliation>
<affiliation>
<nlm:aff id="a6">
<institution>University of Chinese Academy of Sciences</institution>
, Beijing, 100049,
<country>China</country>
</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Wang, Tie Cheng" sort="Wang, Tie Cheng" uniqKey="Wang T" first="Tie-Cheng" last="Wang">Tie-Cheng Wang</name>
<affiliation>
<nlm:aff id="a4">
<institution>Military Veterinary Research Institute, Academy of Military Medical Sciences</institution>
, Changchun, 130122, P. R. China</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Chen, Shan Hui" sort="Chen, Shan Hui" uniqKey="Chen S" first="Shan-Hui" last="Chen">Shan-Hui Chen</name>
<affiliation>
<nlm:aff id="a3">
<institution>Wuxi Center for Disease Control and Prevention</institution>
, Wuxi, 214023, P. R. China</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Ma, Guang Yuan" sort="Ma, Guang Yuan" uniqKey="Ma G" first="Guang-Yuan" last="Ma">Guang-Yuan Ma</name>
<affiliation>
<nlm:aff id="a3">
<institution>Wuxi Center for Disease Control and Prevention</institution>
, Wuxi, 214023, P. R. China</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Sang, Xiao Yu" sort="Sang, Xiao Yu" uniqKey="Sang X" first="Xiao-Yu" last="Sang">Xiao-Yu Sang</name>
<affiliation>
<nlm:aff id="a7">
<institution>Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences</institution>
, Harbin, 150001,
<country>China</country>
</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Liu, Lin Na" sort="Liu, Lin Na" uniqKey="Liu L" first="Lin-Na" last="Liu">Lin-Na Liu</name>
<affiliation>
<nlm:aff id="a4">
<institution>Military Veterinary Research Institute, Academy of Military Medical Sciences</institution>
, Changchun, 130122, P. R. China</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Wu, Ai Ping" sort="Wu, Ai Ping" uniqKey="Wu A" first="Ai-Ping" last="Wu">Ai-Ping Wu</name>
<affiliation>
<nlm:aff id="a5">
<institution>Key Laboratory of Protein and Peptide Pharmaceutical, National Laboratory of Biomacromolecules, Institute of Biophysics</institution>
, Chinese Academy of Sciences, Beijing, 100101,
<country>P. R. China</country>
</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Jiang, Tai Jiao" sort="Jiang, Tai Jiao" uniqKey="Jiang T" first="Tai-Jiao" last="Jiang">Tai-Jiao Jiang</name>
<affiliation>
<nlm:aff id="a5">
<institution>Key Laboratory of Protein and Peptide Pharmaceutical, National Laboratory of Biomacromolecules, Institute of Biophysics</institution>
, Chinese Academy of Sciences, Beijing, 100101,
<country>P. R. China</country>
</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Gao, Yu Wei" sort="Gao, Yu Wei" uniqKey="Gao Y" first="Yu-Wei" last="Gao">Yu-Wei Gao</name>
<affiliation>
<nlm:aff id="a4">
<institution>Military Veterinary Research Institute, Academy of Military Medical Sciences</institution>
, Changchun, 130122, P. R. China</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Gray, Gregory C" sort="Gray, Gregory C" uniqKey="Gray G" first="Gregory C." last="Gray">Gregory C. Gray</name>
<affiliation>
<nlm:aff id="a8">
<institution>Duke Infectious Disease & Duke Health Institute, Duke University Medical Center</institution>
, Durham, NC, 27710,
<country>USA</country>
</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Zhao, Teng" sort="Zhao, Teng" uniqKey="Zhao T" first="Teng" last="Zhao">Teng Zhao</name>
<affiliation>
<nlm:aff id="a1">
<institution>State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology</institution>
, Beijing, 100071, P. R. China</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Ling, Xia" sort="Ling, Xia" uniqKey="Ling X" first="Xia" last="Ling">Xia Ling</name>
<affiliation>
<nlm:aff id="a3">
<institution>Wuxi Center for Disease Control and Prevention</institution>
, Wuxi, 214023, P. R. China</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Wang, Jing Lin" sort="Wang, Jing Lin" uniqKey="Wang J" first="Jing-Lin" last="Wang">Jing-Lin Wang</name>
<affiliation>
<nlm:aff id="a1">
<institution>State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology</institution>
, Beijing, 100071, P. R. China</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Lu, Bing" sort="Lu, Bing" uniqKey="Lu B" first="Bing" last="Lu">Bing Lu</name>
<affiliation>
<nlm:aff id="a3">
<institution>Wuxi Center for Disease Control and Prevention</institution>
, Wuxi, 214023, P. R. China</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Qian, Jun" sort="Qian, Jun" uniqKey="Qian J" first="Jun" last="Qian">Jun Qian</name>
<affiliation>
<nlm:aff id="a4">
<institution>Military Veterinary Research Institute, Academy of Military Medical Sciences</institution>
, Changchun, 130122, P. R. China</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Cao, Wu Chun" sort="Cao, Wu Chun" uniqKey="Cao W" first="Wu-Chun" last="Cao">Wu-Chun Cao</name>
<affiliation>
<nlm:aff id="a1">
<institution>State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology</institution>
, Beijing, 100071, P. R. China</nlm:aff>
</affiliation>
<affiliation>
<nlm:aff id="a2">
<institution>School of Public Health, Central South University</institution>
, Changsha, Hunan, 410000 P. R. China</nlm:aff>
</affiliation>
</author>
</titleStmt>
<publicationStmt>
<idno type="wicri:source">PMC</idno>
<idno type="pmid">25533850</idno>
<idno type="pmc">4274511</idno>
<idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4274511</idno>
<idno type="RBID">PMC:4274511</idno>
<idno type="doi">10.1038/srep07588</idno>
<date when="2014">2014</date>
<idno type="wicri:Area/Pmc/Corpus">000008</idno>
<idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000008</idno>
</publicationStmt>
<sourceDesc>
<biblStruct>
<analytic>
<title xml:lang="en" level="a" type="main">Characterization of a Novel Reassortant Influenza A Virus (H2N2) from a Domestic Duck in Eastern China</title>
<author>
<name sortKey="Ma, Mai Juan" sort="Ma, Mai Juan" uniqKey="Ma M" first="Mai-Juan" last="Ma">Mai-Juan Ma</name>
<affiliation>
<nlm:aff id="a1">
<institution>State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology</institution>
, Beijing, 100071, P. R. China</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Yang, Xiao Xian" sort="Yang, Xiao Xian" uniqKey="Yang X" first="Xiao-Xian" last="Yang">Xiao-Xian Yang</name>
<affiliation>
<nlm:aff id="a1">
<institution>State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology</institution>
, Beijing, 100071, P. R. China</nlm:aff>
</affiliation>
<affiliation>
<nlm:aff id="a2">
<institution>School of Public Health, Central South University</institution>
, Changsha, Hunan, 410000 P. R. China</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Qian, Yan Hua" sort="Qian, Yan Hua" uniqKey="Qian Y" first="Yan-Hua" last="Qian">Yan-Hua Qian</name>
<affiliation>
<nlm:aff id="a3">
<institution>Wuxi Center for Disease Control and Prevention</institution>
, Wuxi, 214023, P. R. China</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Zhao, Si Yan" sort="Zhao, Si Yan" uniqKey="Zhao S" first="Si-Yan" last="Zhao">Si-Yan Zhao</name>
<affiliation>
<nlm:aff id="a4">
<institution>Military Veterinary Research Institute, Academy of Military Medical Sciences</institution>
, Changchun, 130122, P. R. China</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Hua, Sha" sort="Hua, Sha" uniqKey="Hua S" first="Sha" last="Hua">Sha Hua</name>
<affiliation>
<nlm:aff id="a5">
<institution>Key Laboratory of Protein and Peptide Pharmaceutical, National Laboratory of Biomacromolecules, Institute of Biophysics</institution>
, Chinese Academy of Sciences, Beijing, 100101,
<country>P. R. China</country>
</nlm:aff>
</affiliation>
<affiliation>
<nlm:aff id="a6">
<institution>University of Chinese Academy of Sciences</institution>
, Beijing, 100049,
<country>China</country>
</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Wang, Tie Cheng" sort="Wang, Tie Cheng" uniqKey="Wang T" first="Tie-Cheng" last="Wang">Tie-Cheng Wang</name>
<affiliation>
<nlm:aff id="a4">
<institution>Military Veterinary Research Institute, Academy of Military Medical Sciences</institution>
, Changchun, 130122, P. R. China</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Chen, Shan Hui" sort="Chen, Shan Hui" uniqKey="Chen S" first="Shan-Hui" last="Chen">Shan-Hui Chen</name>
<affiliation>
<nlm:aff id="a3">
<institution>Wuxi Center for Disease Control and Prevention</institution>
, Wuxi, 214023, P. R. China</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Ma, Guang Yuan" sort="Ma, Guang Yuan" uniqKey="Ma G" first="Guang-Yuan" last="Ma">Guang-Yuan Ma</name>
<affiliation>
<nlm:aff id="a3">
<institution>Wuxi Center for Disease Control and Prevention</institution>
, Wuxi, 214023, P. R. China</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Sang, Xiao Yu" sort="Sang, Xiao Yu" uniqKey="Sang X" first="Xiao-Yu" last="Sang">Xiao-Yu Sang</name>
<affiliation>
<nlm:aff id="a7">
<institution>Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences</institution>
, Harbin, 150001,
<country>China</country>
</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Liu, Lin Na" sort="Liu, Lin Na" uniqKey="Liu L" first="Lin-Na" last="Liu">Lin-Na Liu</name>
<affiliation>
<nlm:aff id="a4">
<institution>Military Veterinary Research Institute, Academy of Military Medical Sciences</institution>
, Changchun, 130122, P. R. China</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Wu, Ai Ping" sort="Wu, Ai Ping" uniqKey="Wu A" first="Ai-Ping" last="Wu">Ai-Ping Wu</name>
<affiliation>
<nlm:aff id="a5">
<institution>Key Laboratory of Protein and Peptide Pharmaceutical, National Laboratory of Biomacromolecules, Institute of Biophysics</institution>
, Chinese Academy of Sciences, Beijing, 100101,
<country>P. R. China</country>
</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Jiang, Tai Jiao" sort="Jiang, Tai Jiao" uniqKey="Jiang T" first="Tai-Jiao" last="Jiang">Tai-Jiao Jiang</name>
<affiliation>
<nlm:aff id="a5">
<institution>Key Laboratory of Protein and Peptide Pharmaceutical, National Laboratory of Biomacromolecules, Institute of Biophysics</institution>
, Chinese Academy of Sciences, Beijing, 100101,
<country>P. R. China</country>
</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Gao, Yu Wei" sort="Gao, Yu Wei" uniqKey="Gao Y" first="Yu-Wei" last="Gao">Yu-Wei Gao</name>
<affiliation>
<nlm:aff id="a4">
<institution>Military Veterinary Research Institute, Academy of Military Medical Sciences</institution>
, Changchun, 130122, P. R. China</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Gray, Gregory C" sort="Gray, Gregory C" uniqKey="Gray G" first="Gregory C." last="Gray">Gregory C. Gray</name>
<affiliation>
<nlm:aff id="a8">
<institution>Duke Infectious Disease & Duke Health Institute, Duke University Medical Center</institution>
, Durham, NC, 27710,
<country>USA</country>
</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Zhao, Teng" sort="Zhao, Teng" uniqKey="Zhao T" first="Teng" last="Zhao">Teng Zhao</name>
<affiliation>
<nlm:aff id="a1">
<institution>State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology</institution>
, Beijing, 100071, P. R. China</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Ling, Xia" sort="Ling, Xia" uniqKey="Ling X" first="Xia" last="Ling">Xia Ling</name>
<affiliation>
<nlm:aff id="a3">
<institution>Wuxi Center for Disease Control and Prevention</institution>
, Wuxi, 214023, P. R. China</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Wang, Jing Lin" sort="Wang, Jing Lin" uniqKey="Wang J" first="Jing-Lin" last="Wang">Jing-Lin Wang</name>
<affiliation>
<nlm:aff id="a1">
<institution>State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology</institution>
, Beijing, 100071, P. R. China</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Lu, Bing" sort="Lu, Bing" uniqKey="Lu B" first="Bing" last="Lu">Bing Lu</name>
<affiliation>
<nlm:aff id="a3">
<institution>Wuxi Center for Disease Control and Prevention</institution>
, Wuxi, 214023, P. R. China</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Qian, Jun" sort="Qian, Jun" uniqKey="Qian J" first="Jun" last="Qian">Jun Qian</name>
<affiliation>
<nlm:aff id="a4">
<institution>Military Veterinary Research Institute, Academy of Military Medical Sciences</institution>
, Changchun, 130122, P. R. China</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Cao, Wu Chun" sort="Cao, Wu Chun" uniqKey="Cao W" first="Wu-Chun" last="Cao">Wu-Chun Cao</name>
<affiliation>
<nlm:aff id="a1">
<institution>State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology</institution>
, Beijing, 100071, P. R. China</nlm:aff>
</affiliation>
<affiliation>
<nlm:aff id="a2">
<institution>School of Public Health, Central South University</institution>
, Changsha, Hunan, 410000 P. R. China</nlm:aff>
</affiliation>
</author>
</analytic>
<series>
<title level="j">Scientific Reports</title>
<idno type="eISSN">2045-2322</idno>
<imprint>
<date when="2014">2014</date>
</imprint>
</series>
</biblStruct>
</sourceDesc>
</fileDesc>
<profileDesc>
<textClass></textClass>
</profileDesc>
</teiHeader>
<front>
<div type="abstract" xml:lang="en">
<p>While H2N2 viruses have been sporadically isolated from wild and domestic birds, H2N2 viruses have not been detected among human populations since 1968. Should H2N2 viruses adapt to domestic poultry they may pose a risk of infection to people, as most anyone born after 1968 would likely be susceptible to their infection. We report the isolation of a novel influenza A virus (H2N2) cultured in 2013 from a healthy domestic duck at a live poultry market in Wuxi City, China. Sequence data revealed that the novel H2N2 virus was similar to Eurasian avian lineage avian influenza viruses, the virus had been circulating for ≥ two years among poultry, had an increase in α2,6 binding affinity, and was not highly pathogenic. Approximately 9% of 100 healthy chickens sampled from the same area had elevated antibodies against the H2 antigen. Fortunately, there was sparse serological evidence that the virus was infecting poultry workers or had adapted to infect other mammals. These findings suggest that a novel H2N2 virus has been circulating among domestic poultry in Wuxi City, China and has some has increased human receptor affinity. It seems wise to conduct better surveillance for novel influenza viruses at Chinese live bird markets.</p>
</div>
</front>
<back>
<div1 type="bibliography">
<listBibl>
<biblStruct>
<analytic>
<author>
<name sortKey="Medina, R A" uniqKey="Medina R">R. A. Medina</name>
</author>
<author>
<name sortKey="Garcia Sastre, A" uniqKey="Garcia Sastre A">A. Garcia-Sastre</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Webster, R G" uniqKey="Webster R">R. G. Webster</name>
</author>
<author>
<name sortKey="Bean, W J" uniqKey="Bean W">W. J. Bean</name>
</author>
<author>
<name sortKey="Gorman, O T" uniqKey="Gorman O">O. T. Gorman</name>
</author>
<author>
<name sortKey="Chambers, T M" uniqKey="Chambers T">T. M. Chambers</name>
</author>
<author>
<name sortKey="Kawaoka, Y" uniqKey="Kawaoka Y">Y. Kawaoka</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Lipatov, A S" uniqKey="Lipatov A">A. S. Lipatov</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Neumann, G" uniqKey="Neumann G">G. Neumann</name>
</author>
<author>
<name sortKey="Noda, T" uniqKey="Noda T">T. Noda</name>
</author>
<author>
<name sortKey="Kawaoka, Y" uniqKey="Kawaoka Y">Y. Kawaoka</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Gao, R" uniqKey="Gao R">R. Gao</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="To, K K" uniqKey="To K">K. K. To</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Claas, E C" uniqKey="Claas E">E. C. Claas</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Subbarao, K" uniqKey="Subbarao K">K. Subbarao</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Ma, W" uniqKey="Ma W">W. Ma</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Tong, S" uniqKey="Tong S">S. Tong</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Tong, S" uniqKey="Tong S">S. Tong</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Nabel, G J" uniqKey="Nabel G">G. J. Nabel</name>
</author>
<author>
<name sortKey="Wei, C J" uniqKey="Wei C">C. J. Wei</name>
</author>
<author>
<name sortKey="Ledgerwood, J E" uniqKey="Ledgerwood J">J. E. Ledgerwood</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Chen, G L" uniqKey="Chen G">G. L. Chen</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Hilleman, M R" uniqKey="Hilleman M">M. R. Hilleman</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Webster, R G" uniqKey="Webster R">R. G. Webster</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Steel, J" uniqKey="Steel J">J. Steel</name>
</author>
<author>
<name sortKey="Lowen, A C" uniqKey="Lowen A">A. C. Lowen</name>
</author>
<author>
<name sortKey="Mubareka, S" uniqKey="Mubareka S">S. Mubareka</name>
</author>
<author>
<name sortKey="Palese, P" uniqKey="Palese P">P. Palese</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Matrosovich, M N" uniqKey="Matrosovich M">M. N. Matrosovich</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Zhou, N" uniqKey="Zhou N">N. Zhou</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Shortridge, K F" uniqKey="Shortridge K">K. F. Shortridge</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Shu, L L" uniqKey="Shu L">L. L. Shu</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Bi, Y" uniqKey="Bi Y">Y. Bi</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Yu, H" uniqKey="Yu H">H. Yu</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Zhang, G" uniqKey="Zhang G">G. Zhang</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Bao, Y" uniqKey="Bao Y">Y. Bao</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Vines, A" uniqKey="Vines A">A. Vines</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Connor, R J" uniqKey="Connor R">R. J. Connor</name>
</author>
<author>
<name sortKey="Kawaoka, Y" uniqKey="Kawaoka Y">Y. Kawaoka</name>
</author>
<author>
<name sortKey="Webster, R G" uniqKey="Webster R">R. G. Webster</name>
</author>
<author>
<name sortKey="Paulson, J C" uniqKey="Paulson J">J. C. Paulson</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Jones, J C" uniqKey="Jones J">J. C. Jones</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Ito, T" uniqKey="Ito T">T. Ito</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Liu, M" uniqKey="Liu M">M. Liu</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Peng, X" uniqKey="Peng X">X. Peng</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Hilleman, M R" uniqKey="Hilleman M">M. R. Hilleman</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Liu, D" uniqKey="Liu D">D. Liu</name>
</author>
<author>
<name sortKey="Shi, W" uniqKey="Shi W">W. Shi</name>
</author>
<author>
<name sortKey="Gao, G F" uniqKey="Gao G">G. F. Gao</name>
</author>
</analytic>
</biblStruct>
<biblStruct></biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Hoffmann, E" uniqKey="Hoffmann E">E. Hoffmann</name>
</author>
<author>
<name sortKey="Stech, J" uniqKey="Stech J">J. Stech</name>
</author>
<author>
<name sortKey="Guan, Y" uniqKey="Guan Y">Y. Guan</name>
</author>
<author>
<name sortKey="Webster, R G" uniqKey="Webster R">R. G. Webster</name>
</author>
<author>
<name sortKey="Perez, D R" uniqKey="Perez D">D. R. Perez</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Mount, D W" uniqKey="Mount D">D. W. Mount</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Altschul, S F" uniqKey="Altschul S">S. F. Altschul</name>
</author>
<author>
<name sortKey="Gish, W" uniqKey="Gish W">W. Gish</name>
</author>
<author>
<name sortKey="Miller, W" uniqKey="Miller W">W. Miller</name>
</author>
<author>
<name sortKey="Myers, E W" uniqKey="Myers E">E. W. Myers</name>
</author>
<author>
<name sortKey="Lipman, D J" uniqKey="Lipman D">D. J. Lipman</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Edgar, R C" uniqKey="Edgar R">R. C. Edgar</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Drummond, A" uniqKey="Drummond A">A. Drummond</name>
</author>
<author>
<name sortKey="Pybus, O G" uniqKey="Pybus O">O. G. Pybus</name>
</author>
<author>
<name sortKey="Rambaut, A" uniqKey="Rambaut A">A. Rambaut</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Smith, G J" uniqKey="Smith G">G. J. Smith</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Drummond, A J" uniqKey="Drummond A">A. J. Drummond</name>
</author>
<author>
<name sortKey="Rambaut, A" uniqKey="Rambaut A">A. Rambaut</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Reed, L J" uniqKey="Reed L">L. J. Reed</name>
</author>
<author>
<name sortKey="Muench, H" uniqKey="Muench H">H. Muench</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Chen, G W" uniqKey="Chen G">G. W. Chen</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Herfst, S" uniqKey="Herfst S">S. Herfst</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Tamuri, A U" uniqKey="Tamuri A">A. U. Tamuri</name>
</author>
<author>
<name sortKey="Dos Reis, M" uniqKey="Dos Reis M">M. Dos Reis</name>
</author>
<author>
<name sortKey="Hay, A J" uniqKey="Hay A">A. J. Hay</name>
</author>
<author>
<name sortKey="Goldstein, R A" uniqKey="Goldstein R">R. A. Goldstein</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Yang, H" uniqKey="Yang H">H. Yang</name>
</author>
<author>
<name sortKey="Chen, L M" uniqKey="Chen L">L. M. Chen</name>
</author>
<author>
<name sortKey="Carney, P J" uniqKey="Carney P">P. J. Carney</name>
</author>
<author>
<name sortKey="Donis, R O" uniqKey="Donis R">R. O. Donis</name>
</author>
<author>
<name sortKey="Stevens, J" uniqKey="Stevens J">J. Stevens</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Tumpey, T M" uniqKey="Tumpey T">T. M. Tumpey</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Lycett, S J" uniqKey="Lycett S">S. J. Lycett</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Matsuoka, Y" uniqKey="Matsuoka Y">Y. Matsuoka</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Fan, S" uniqKey="Fan S">S. Fan</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Jiao, P" uniqKey="Jiao P">P. Jiao</name>
</author>
</analytic>
</biblStruct>
</listBibl>
</div1>
</back>
</TEI>
<pmc article-type="research-article">
<pmc-dir>properties open_access</pmc-dir>
<front>
<journal-meta>
<journal-id journal-id-type="nlm-ta">Sci Rep</journal-id>
<journal-id journal-id-type="iso-abbrev">Sci Rep</journal-id>
<journal-title-group>
<journal-title>Scientific Reports</journal-title>
</journal-title-group>
<issn pub-type="epub">2045-2322</issn>
<publisher>
<publisher-name>Nature Publishing Group</publisher-name>
</publisher>
</journal-meta>
<article-meta>
<article-id pub-id-type="pmid">25533850</article-id>
<article-id pub-id-type="pmc">4274511</article-id>
<article-id pub-id-type="pii">srep07588</article-id>
<article-id pub-id-type="doi">10.1038/srep07588</article-id>
<article-categories>
<subj-group subj-group-type="heading">
<subject>Article</subject>
</subj-group>
</article-categories>
<title-group>
<article-title>Characterization of a Novel Reassortant Influenza A Virus (H2N2) from a Domestic Duck in Eastern China</article-title>
</title-group>
<contrib-group>
<contrib contrib-type="author">
<name>
<surname>Ma</surname>
<given-names>Mai-Juan</given-names>
</name>
<xref ref-type="corresp" rid="c2">b</xref>
<xref ref-type="aff" rid="a1">1</xref>
<xref ref-type="author-notes" rid="n1">*</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Yang</surname>
<given-names>Xiao-Xian</given-names>
</name>
<xref ref-type="aff" rid="a1">1</xref>
<xref ref-type="aff" rid="a2">2</xref>
<xref ref-type="author-notes" rid="n1">*</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Qian</surname>
<given-names>Yan-Hua</given-names>
</name>
<xref ref-type="aff" rid="a3">3</xref>
<xref ref-type="author-notes" rid="n1">*</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Zhao</surname>
<given-names>Si-Yan</given-names>
</name>
<xref ref-type="aff" rid="a4">4</xref>
<xref ref-type="author-notes" rid="n1">*</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Hua</surname>
<given-names>Sha</given-names>
</name>
<xref ref-type="aff" rid="a5">5</xref>
<xref ref-type="aff" rid="a6">6</xref>
<xref ref-type="author-notes" rid="n1">*</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Wang</surname>
<given-names>Tie-Cheng</given-names>
</name>
<xref ref-type="aff" rid="a4">4</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Chen</surname>
<given-names>Shan-Hui</given-names>
</name>
<xref ref-type="aff" rid="a3">3</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Ma</surname>
<given-names>Guang-Yuan</given-names>
</name>
<xref ref-type="aff" rid="a3">3</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Sang</surname>
<given-names>Xiao-Yu</given-names>
</name>
<xref ref-type="aff" rid="a7">7</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Liu</surname>
<given-names>Lin-Na</given-names>
</name>
<xref ref-type="aff" rid="a4">4</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Wu</surname>
<given-names>Ai-Ping</given-names>
</name>
<xref ref-type="aff" rid="a5">5</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Jiang</surname>
<given-names>Tai-Jiao</given-names>
</name>
<xref ref-type="aff" rid="a5">5</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Gao</surname>
<given-names>Yu-Wei</given-names>
</name>
<xref ref-type="aff" rid="a4">4</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Gray</surname>
<given-names>Gregory C.</given-names>
</name>
<xref ref-type="aff" rid="a8">8</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Zhao</surname>
<given-names>Teng</given-names>
</name>
<xref ref-type="aff" rid="a1">1</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Ling</surname>
<given-names>Xia</given-names>
</name>
<xref ref-type="aff" rid="a3">3</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Wang</surname>
<given-names>Jing-Lin</given-names>
</name>
<xref ref-type="aff" rid="a1">1</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Lu</surname>
<given-names>Bing</given-names>
</name>
<xref ref-type="aff" rid="a3">3</xref>
<xref ref-type="author-notes" rid="n2"></xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Qian</surname>
<given-names>Jun</given-names>
</name>
<xref ref-type="aff" rid="a4">4</xref>
<xref ref-type="author-notes" rid="n2"></xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Cao</surname>
<given-names>Wu-Chun</given-names>
</name>
<xref ref-type="corresp" rid="c1">a</xref>
<xref ref-type="aff" rid="a1">1</xref>
<xref ref-type="aff" rid="a2">2</xref>
<xref ref-type="author-notes" rid="n2"></xref>
</contrib>
<aff id="a1">
<label>1</label>
<institution>State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology</institution>
, Beijing, 100071, P. R. China</aff>
<aff id="a2">
<label>2</label>
<institution>School of Public Health, Central South University</institution>
, Changsha, Hunan, 410000 P. R. China</aff>
<aff id="a3">
<label>3</label>
<institution>Wuxi Center for Disease Control and Prevention</institution>
, Wuxi, 214023, P. R. China</aff>
<aff id="a4">
<label>4</label>
<institution>Military Veterinary Research Institute, Academy of Military Medical Sciences</institution>
, Changchun, 130122, P. R. China</aff>
<aff id="a5">
<label>5</label>
<institution>Key Laboratory of Protein and Peptide Pharmaceutical, National Laboratory of Biomacromolecules, Institute of Biophysics</institution>
, Chinese Academy of Sciences, Beijing, 100101,
<country>P. R. China</country>
</aff>
<aff id="a6">
<label>6</label>
<institution>University of Chinese Academy of Sciences</institution>
, Beijing, 100049,
<country>China</country>
</aff>
<aff id="a7">
<label>7</label>
<institution>Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences</institution>
, Harbin, 150001,
<country>China</country>
</aff>
<aff id="a8">
<label>8</label>
<institution>Duke Infectious Disease & Duke Health Institute, Duke University Medical Center</institution>
, Durham, NC, 27710,
<country>USA</country>
</aff>
</contrib-group>
<author-notes>
<corresp id="c1">
<label>a</label>
<email>caowc@bmi.ac.cn</email>
</corresp>
<corresp id="c2">
<label>b</label>
<email>mjma@163.com</email>
</corresp>
<fn id="n1">
<label>*</label>
<p>These authors contributed equally to this work.</p>
</fn>
<fn id="n2">
<label></label>
<p>These authors jointly supervised this work.</p>
</fn>
</author-notes>
<pub-date pub-type="epub">
<day>23</day>
<month>12</month>
<year>2014</year>
</pub-date>
<pub-date pub-type="collection">
<year>2014</year>
</pub-date>
<volume>4</volume>
<elocation-id>7588</elocation-id>
<history>
<date date-type="received">
<day>08</day>
<month>08</month>
<year>2014</year>
</date>
<date date-type="accepted">
<day>28</day>
<month>11</month>
<year>2014</year>
</date>
</history>
<permissions>
<copyright-statement>Copyright © 2014, Macmillan Publishers Limited. All rights reserved</copyright-statement>
<copyright-year>2014</copyright-year>
<copyright-holder>Macmillan Publishers Limited. All rights reserved</copyright-holder>
<license license-type="open-access" xlink:href="http://creativecommons.org/licenses/by-nc-nd/4.0/">
<pmc-comment>author-paid</pmc-comment>
<license-p>This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder in order to reproduce the material. To view a copy of this license, visit
<ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by-nc-nd/4.0/">http://creativecommons.org/licenses/by-nc-nd/4.0/</ext-link>
</license-p>
</license>
</permissions>
<abstract>
<p>While H2N2 viruses have been sporadically isolated from wild and domestic birds, H2N2 viruses have not been detected among human populations since 1968. Should H2N2 viruses adapt to domestic poultry they may pose a risk of infection to people, as most anyone born after 1968 would likely be susceptible to their infection. We report the isolation of a novel influenza A virus (H2N2) cultured in 2013 from a healthy domestic duck at a live poultry market in Wuxi City, China. Sequence data revealed that the novel H2N2 virus was similar to Eurasian avian lineage avian influenza viruses, the virus had been circulating for ≥ two years among poultry, had an increase in α2,6 binding affinity, and was not highly pathogenic. Approximately 9% of 100 healthy chickens sampled from the same area had elevated antibodies against the H2 antigen. Fortunately, there was sparse serological evidence that the virus was infecting poultry workers or had adapted to infect other mammals. These findings suggest that a novel H2N2 virus has been circulating among domestic poultry in Wuxi City, China and has some has increased human receptor affinity. It seems wise to conduct better surveillance for novel influenza viruses at Chinese live bird markets.</p>
</abstract>
</article-meta>
</front>
<body>
<p>Influenza virus remains one of the most common and important causes of human respiratory infections. Influenza virus outbreaks can result in high morbidity and mortality
<xref ref-type="bibr" rid="b1">1</xref>
. Outbreaks may be due to minor changes or antigenic drift (e.g. due to point mutations) or major genetic changes or antigenic shift (e.g. due to reassortment). Historically, pandemics have often been due to antigenic shift that corresponds to the emergence of novel zoonotic influenza viruses which have adapted to cause human-to-human transmission and disease
<xref ref-type="bibr" rid="b2">2</xref>
<xref ref-type="bibr" rid="b3">3</xref>
. Among the three types of influenza virus (A, B, and C), the influenza A viruses embody the greatest significant threat of host switching events, which is illustrated by the pandemic 2009 H1N1 virus (pH1N1), highly pathogenic H5N1 viruses, the recent emergence of human infection with novel avian origin influenza A H7N9 virus, and sporadic human infections with avian influenza A H10N8 virus in China
<xref ref-type="bibr" rid="b4">4</xref>
<xref ref-type="bibr" rid="b5">5</xref>
<xref ref-type="bibr" rid="b6">6</xref>
<xref ref-type="bibr" rid="b7">7</xref>
<xref ref-type="bibr" rid="b8">8</xref>
. Although H5, H7, and H10 subtype viruses have yet to cause efficient human-to-human transmission, their pandemic potential remains a serious public health threat.</p>
<p>Among the 18 currently recognized hemagglutinin (HA) subtypes
<xref ref-type="bibr" rid="b9">9</xref>
<xref ref-type="bibr" rid="b10">10</xref>
<xref ref-type="bibr" rid="b11">11</xref>
, only H1, H2, and H3 viruses have recently been successfully established in humans. However, the best genetic data regarding human influenza only date back to the records of the influenza pandemic of 1918. Since then circulating influenza A viruses have had several antigenic shifts, resulting in the emergence of Asian/57 (H2N2, 1957-1968) and Hong Kong/68 (H3N2, 1968–1969) pandemic strains earlier in last century. H2N2 viruses have not circulated in humans since 1968 and persons born after that year have little or no immunity to H2 viruses
<xref ref-type="bibr" rid="b12">12</xref>
. Currently, H2 subtype influenza viruses are detected not only in avian species, also in swine. The oldest human H2 pandemic viruses are closely related to the currently circulating avian strains, suggesting that the pandemic H2N2 virus had an avian origin and that antigenic change has been slow for H2 viruses
<xref ref-type="bibr" rid="b12">12</xref>
. Hence, H2 viruses remain a public health threat
<xref ref-type="bibr" rid="b13">13</xref>
<xref ref-type="bibr" rid="b14">14</xref>
<xref ref-type="bibr" rid="b15">15</xref>
, and there is considerable risk that H2 viruses may reemerge to cause a pandemic similar to the 2009 H1N1 pandemic. It seems prudent then to study the mechanisms of introduction, adaptation, and transmission of avian H2N2 influenza viruses among terrestrial birds and to examine their potential for transmission to mammals.</p>
<p>Here we describe the characterization of a novel influenza A virus (H2N2) isolated in 2013 from a healthy domestic duck sampled in a live poultry market (LPM) in China. We investigated the pathogenicity and transmissibility of this multi-reassortant H2N2 isolate in mammalian hosts and the serological evidence of previous H2N2 virus infections among geographically-linked humans and live poultry.</p>
<sec disp-level="1" sec-type="results">
<title>Results</title>
<sec disp-level="2">
<title>Identification of influenza virus among live poultry</title>
<p>The cloacal/environmental swabs from 325 adult chickens and ducks, environmental swabs of cages, and poultry drinking water from the LPMs were collected in Wuxi City during the period of June to December 2013. A total of 59 samples (18.2%) (Cycle threshold
<italic>C</italic>
<sub>T</sub>
values ranging from 25.4 to 36.9) were found to have molecular evidence of influenza A virus RNA by a real-time RT-PCR assay for the influenza matrix gene. Following the inoculation of these specimens into embryonated hens' eggs, five viruses were successfully cultured (all from specimens with a C
<sub>T</sub>
value of <33). The insolates underwent full genome analysis by nucleotide sequencing and a BLAST search of the Influenza Sequence Database, which determined that one of isolates was a novel H2N2 influenza virus (Gen-Bank sequence accession numbers KM100132 to KM100139). Additionally, another five isolates were determined to be known influenza viruses: one H5N1 virus isolate, two H5N8 virus isolates, and two H9N2 virus isolates (
<ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html">www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html</ext-link>
). The novel H2N2 virus was designated A/duck/Wuxi/2/2013(H2N2) (abbreviated as: A/duck/WX/2/13 (H2N2)).</p>
</sec>
<sec disp-level="2">
<title>Identification of evolutionary relationships and divergence time estimates</title>
<p>The phylogenies and time to most recent common ancestor (TMRCA) were computed for each gene segment of the novel H2N2 viruses and other influenza viruses. Clearly, all gene segments were nested within the influenza viruses from domestic or/and wild ducks, suggesting a pure avian origin of the novel H2N2 virus. When source locations were compared, remarkably all eight gene segments of the novel H2N2 viruses, HA, NA, NP, PA, PB1, PB2, NS, and M segments were very similar to the clades isolated from birds in east Asian (and east China in particular) in recent years (
<xref ref-type="fig" rid="f1">Fig. 1</xref>
). Given the agreement in both the time and location (
<xref ref-type="fig" rid="f2">Fig. 2</xref>
), we posit that the recent viruses circulating in ducks in East Asian around 2010 are the most likely donors of the six PB2, PB1, PA, NP, NA, and NS gene segments. However, we could not identify the immediate ancestors or ancestor-like viruses for the HA and M gene segments as the novel H2N2 virus' HA and M gene segment had considerable divergence from their genetically closest strains, this may reflect a novel virus or gaps in surveillance data.</p>
<p>We further sought to form a complete picture of the origins of the novel H2N2 virus by integrating the evolutionary analysis of its individual gene segments (
<xref ref-type="fig" rid="f3">Fig. 3</xref>
). Although determining the precise evolutionary pathway of the genesis of the novel H2N2 is greatly hindered by the lack of surveillance data, we can conclude that the novel H2N2 virus emerged from continuously reassortant viruses circulating in Eurasian ducks. We first computed the TMRCA for individual gene segments.
<xref ref-type="table" rid="t1">Table 1</xref>
shows proposed evolutionary events and their timing in the generation of the novel H2N2 virus. As illustrated in
<xref ref-type="table" rid="t1">Table 1</xref>
, the TMRCA for the six gene segments (NA, NP, PA, PB1, PB2, and NS) of the novel H2N2 virus ranged from January 2009 (NS) to October 2011 (NA), suggesting that the novel H2N2 virus has been circulating and left undetected for at least two years (10/2011-12/2013) in domestic and/or wild ducks. For HA, although the genetically closest virus A/environment/Guangdong/2/2009(H2N3) (abbreviated as GD (H2N3)) was identified from the existing influenza surveillance data, their TMRCA is September of 2005, showing a long period of time (~ 8 years) divergence between the novel H7N9 HA and GD(H2N3) HA. Similarly, there existed a long period of divergence time (~ 11 years) between the M of the novel H2N2 virus and that of its genetically closest virus A/duck/Eastern-China/29/2004(H6N2). Thus, we argue that the failure of detecting the immediate ancestors or ancestor-like viruses either from China or other countries for M and HA of the novel H2N2 virus is attributed to insufficient influenza samplings.</p>
</sec>
<sec disp-level="2">
<title>Molecular markers of host specificity and pathogenicity</title>
<p>To determine the host specificity and pathogenicity of novel H2N2 virus, we first analyzed the full-length of HA and PB2 gene sequences, focusing on the important residues in the receptor binding domain (S138A, T160A, G186V, Q226L, and G228S in HA gene, as well as E627K in PB2 of H3 numbering), which could contribute the adaption and virulence of avian influenza virus to human mammal host
<xref ref-type="bibr" rid="b16">16</xref>
<xref ref-type="bibr" rid="b17">17</xref>
. The HA of the novel H2N2 virus did not contain the multibasic cleavage site observed in highly pathogenic H5 or H7 AIVs, nor did the genome contain other major mammalian adaptations, such as the PB2 E627K substitution, which is known to alter host range and virulence. Infection of embryonated eggs was not pathogenic to the embryo. The receptor binding pocket residues have only one human-like amino acid at position A138 (H3 numbering), with the rest being avian-like with E190, G225, Q226, and G228, suggesting α2,3-linked sialic acid receptor binding preference. Other amino acids in the NS1 and matrix (M1; encoded by the M segment) proteins of the novel viruses are supposed be also associated with increased virulence. These amino acids are found in many avian H2N2 influenza viruses, and therefore, their significance for the biological properties of the novel influenza A(H2N2) viruses is currently unclear (
<xref ref-type="table" rid="t2">table 2</xref>
).</p>
</sec>
<sec disp-level="2">
<title>Receptor-binding properties of novel H2N2 virus</title>
<p>Molecular analysis of HA gene showed a human-like virus amino acid at position A138 (H3 numbering). As previous H2N2 viruses isolated from the wild birds have no human-like amino acid changes, we sought to characterize the receptor-binding properties of the novel H2N2 virus. Hence, we analyzed used solid-phase binding assays with A/Changchun/XD/2009(H1N1) and A/Chicken/Jilin/Hu-3/2006(H9N2) as control viruses to examine the receptor-binding properties of the novel H2N2 virus. The novel H2N2 virus was found to bind with both α2,6-linked sialyglycans (human receptor) and α2,3-linked sialyglycans (avian receptor); whereas, it will preferentially bind with the avian receptor (
<xref ref-type="fig" rid="f4">Fig. 4a</xref>
) compared to the XD/H1N1 virus which had strong ability to bind to the α2,6-linked sialyglycans (
<xref ref-type="fig" rid="f4">Fig. 4b</xref>
). In contrast, the avian H9N2 influenza virus bound only to the α2,3-linked sialyglycans (
<xref ref-type="fig" rid="f4">Fig. 4c</xref>
).</p>
</sec>
<sec disp-level="2">
<title>Pathogenicity in chickens</title>
<p>To evaluate the pathogenicity of the novel H2N2 virus in chickens, we intravenously inoculated groups of ten 6-week-old SPF chickens. The viruses isolated were determined to be low pathogenic avian influenza by this inoculation as no clinical signs were observed by the animal care staff and all birds were scored as normal during the 10 day observation period. However, nine chickens developed elevated serum antibodies by the hemagglutination inhibition (HI) assay against the novel H2N2 virus on day 10 (6 chickens with HI titer ≥1:160, 1 with 1:40, and 2 with 1:20), while the two chickens inoculated with PBS were all negative by the HI assay.</p>
</sec>
<sec disp-level="2">
<title>Pathogenicity and transmissibility in mammals</title>
<p>To assess the potential risk of A/duck/WX/2/13 infection in mammals, we administered the virus to BALB/c mice via intranasal routes. Intranasal administration of A/duck/WX/2/13 resulted in no viral replication, without detection of virus on day 3 and 5 post inoculation (p.i). The mice showed no clinical symptoms, weight loss, pathological changes, and had no detectable H2N2 virus serum antibodies by HI assay on day 14 p.i.</p>
<p>To assess the transmissibility of A/duck/WX/2/13 infection in mammals, we also administered the virus to guinea pigs via intranasal routes. Intranasal administration of A/duck/WX/2/13 resulted in no viral replication on day 2, 4, 6, 8, and 10 p.i, using nose wash specimens. The guinea pigs showed no clinical symptoms or weight loss; however, those inoculated guinea pigs had an elevated HI titer from 1:40 to 1:80 against the H2H2 virus by day 14 p.i. Furthermore, no HI titer was detected in any of the guinea pigs from the contact groups.</p>
</sec>
<sec disp-level="2">
<title>Serological analysis among human population and live poultry</title>
<p>Next we sought to assess the evidence of previous novel H2N2 infections among poultry workers and live poultry. Sera collected in the Wuxi City area during 2013 from 100 apparently healthy chickens/ducks and 1984 humans (including 560 poultry-exposed, 617 swine-exposed, and 817 study volunteers with no avian and swine-exposures) were analyzed using HI assays against the novel H2N2 virus. Nine percent of the poultry studied had HI titers ≥ 1:40 (
<xref ref-type="fig" rid="f5">Fig. 5a</xref>
), among which one had a titer of 1:80 and two had an HI titer of 1:160. A total of 98 sera (4.9%) from human population had elevated antibodies against the novel H2N2 virus (HI titer ≥1:40). There was no statistically significant difference in antibody levels between the three exposure groups (
<xref ref-type="fig" rid="f5">Fig. 5b</xref>
). Although, almost all of positive sera for the human population were from people at least 45 years of age (
<xref ref-type="fig" rid="f5">Fig. 5c</xref>
), which likely indicates confounding by cross-reacting antibodies against human H2N2 pandemic virus.</p>
</sec>
</sec>
<sec disp-level="1" sec-type="discussion">
<title>Discussion</title>
<p>China has been implicated as the site of the origin of the 1957 and 1968 pandemic viruses
<xref ref-type="bibr" rid="b18">18</xref>
. Although, the H2N2 virus has not appeared to circulate among humans for several decades, avian H2N2 viruses are still circulating among birds and continue to pose a public health threat. China has been identified as an epicenter
<xref ref-type="bibr" rid="b19">19</xref>
for the generation of novel influenza virus strains; because, it has dense populations of humans and poultry
<xref ref-type="bibr" rid="b20">20</xref>
that often live in close proximity, some of the largest confined animal farm operations in the world, and a variety of enzootic swine and avian influenza viruses
<xref ref-type="bibr" rid="b21">21</xref>
<xref ref-type="bibr" rid="b22">22</xref>
<xref ref-type="bibr" rid="b23">23</xref>
. To better understand the epidemiology of zoonotic influenza viruses, we conducted a surveillance study of avian influenza virus in LPMs in China, and isolated a novel reassortant avian-origin H2N2 virus from a domestic duck in Wuxi City. To our knowledge, this is first time a H2N2 virus has been isolated from a domestic duck in mainland China.</p>
<p>According to data from NCBI
<xref ref-type="bibr" rid="b24">24</xref>
, during the past decades, only a small number avian H2N2 viruses have been detected or isolated from wild birds or domestic poultry in North America, the Netherlands, Sweden, Japan, and Hong Kong. Based upon molecular and phylogenetic analyses, the H2N2 virus evaluated in our present study was a novel recombinant influenza virus strains, demonstrating divergence over time of PB1, PB2, PA, and NP gene segments. The most similar H2N2 strain was identified around 2010, suggesting that novel virus gene segments may be from the same influenza virus strain. Among the other three gene segments (HA, M, and NS) the most similar strains were identified long ago and hence these gene segments may represent independent recombination events. The novel H2N2 virus' NA gene segment is most similar with a H5N2 influenza virus strain isolated in 2012 in Jiangsu Province, which again suggests a recombinant origin of the novel H2N2 virus or this may support gaps in surveillance data due to insufficient influenza sampling between January 2009 and October 2011. However, it remains clear that in general the novel H2N2 virus is quite similar to the 1957 H2N2 human pandemic strain that disappeared in 1968.</p>
<p>The genetic characteristics of HA and PB2 gene segments are frequently associated with zoonotic transmission success or failure among avian influenza strains
<xref ref-type="bibr" rid="b16">16</xref>
<xref ref-type="bibr" rid="b17">17</xref>
. In avian H2 and H3 influenza viruses, HA receptor-binding-site residues corresponding to codon positions 138, 190, 194, 225, 226, and 228 (using the H3 numbering system) are highly conserved
<xref ref-type="bibr" rid="b17">17</xref>
<xref ref-type="bibr" rid="b25">25</xref>
. In human H2 and H3 viruses, leucine and serine substitutions at residues 226 and 228, respectively, have been shown to accompany their adaptation from avian to human hosts
<xref ref-type="bibr" rid="b26">26</xref>
. Our novel H2N2 virus demonstrated no such Q226L and G228L substitution in HA and no E627K substitutions in PB2, which is typical of AIVs (
<xref ref-type="table" rid="t2">Table 2</xref>
). However, position 138 of HA did have substitution, which could increase the binding affinity of human receptor. The receptor binding affinity assay revealed that the novel H2N2 virus had increased α2,6-binding compared with a previously isolated H2N2 avian virus. However, our experimental data suggested that the virus manifested only low replication in guinea pigs, and no replication occurred in direct or indirect guinea pig contacts or in BALB/c mice. This may suggest that the novel H2N2 virus currently has low risk of infection and transmission in mammals, and would have to further adapt to mammalian hosts to become a public health threat. It is interesting to note that Jones
<italic>et al.</italic>
<xref ref-type="bibr" rid="b27">27</xref>
evaluated avian H2N2 influenza viruses and concluded that all H2N2 viruses they tested had almost no human receptor binding affinity using solid-phase enzyme-linked immunosorbent assays, whereas some H2N2 virus could replicate in DBA2/J mice and ferrets. It seems that receptor-binding changes are required for the successful adaptation of avian viruses to humans; however, this trait alone is not sufficient for human-to-human transmission. For example, the classical swine H1N1 influenza viruses have a preference for α2,6-linked glycans sialic acid viral receptors
<xref ref-type="bibr" rid="b28">28</xref>
and a number of self-limiting human transmission infections do occur; however, these viruses have not successfully established themselves in human populations. An examination of the other viral proteins shows that a number of substitutions are present when the novel H2N2 isolate was compared with other H2N2 influenza viruses isolated from swine or mallard ducks, although their significance is unclear.</p>
<p>We examined the pathogenicity of the virus in chickens and also looked for evidence of novel H2N2 virus transmission in geographically-linked poultry and human population. The results revealed that the novel H2N2 virus is a low-pathogenic avian influenza virus (LPAI), whereas 9% of examined poultry had elevated antibodies to the novel virus. However, the serological finding might be explained by antibodies against other H2 viruses such as H2N9
<xref ref-type="bibr" rid="b29">29</xref>
, H2N7
<xref ref-type="bibr" rid="b30">30</xref>
, and H2N3 which have also been rarely found to circulate among ducks in Asia. No matter the H2 type, our data suggest that the H2 viruses continue to circulate within LPMs. Our study found that 4.9% of 1984 human participants had elevated antibody to the novel H2N2 virus, of which, people under age of 45 have little or no immunity, and resistance dramatically increased for those older than 45 (
<xref ref-type="fig" rid="f5">figure 5</xref>
). This was also the case for the 2009 H1N1. The low mutation rate for H2N2 and evidence of waning human immunity make it likely that an H2N2 pandemic could arise from a zoonotic H2N2 strain
<xref ref-type="bibr" rid="b13">13</xref>
<xref ref-type="bibr" rid="b15">15</xref>
<xref ref-type="bibr" rid="b31">31</xref>
.</p>
<p>Avian influenza viruses are often thought to be transmitted from wild birds to domestic birds and then to people. However, influenza viruses from wild birds seldom adapt to and replicate well within domestic birds
<xref ref-type="bibr" rid="b32">32</xref>
, preventing people from being infected by the wild bird avian influenza virus strains. However, when a rare wild bird avian influenza adaption event occurs, such viruses may further adapt for transmission to humans. Several subtypes of avian influenza viruses found in poultry are capable of infecting humans, which include high-pathogenic avian influenza virus (H5N1, H7N2, and H7N7), low-pathogenic avian influenza virus (H9N2, H7N9, and H10N8), and potential other unknown avian influenza viruses
<xref ref-type="bibr" rid="b32">32</xref>
.</p>
<p>In summary, while the immediate potential for this virus to infect humans and to be transmitted efficiently from human-to-human seems unlikely, several characteristics of the virus are quite troubling: the low viral evolution, the increased human receptor binding affinity, and the lack of human immunity for H2N2 virus suggest this and other H2 progeny viruses circulating in China may be a cryptic threat to man. Hence, it seems wise to increase surveillance for novel viruses among healthy poultry in China's LPMs. Such active surveillance and subsequent viral characterization of isolates seem extremely important in evaluating emerging viral threats, in following antiviral susceptibility, and in designing both poultry and human influenza vaccines, all of which are critical components in the public health response to influenza.</p>
</sec>
<sec disp-level="1" sec-type="methods">
<title>Methods</title>
<sec disp-level="2">
<title>Ethics statement</title>
<p>The protocol of the study was conducted in accordance with guidelines of animal welfare of World Organization for Animal Health for animal experiments and Declaration of Helsinki for human experiments, and approved by Animal Welfare Ethics Review of Academy of Military Medical Sciences and Institutional of Review Board of Wuxi Center for Disease Control and Prevention.</p>
</sec>
<sec disp-level="2">
<title>Samples collection</title>
<p>We collected cloacal/environmental swabs from chickens and ducks in LPMs for avian influenza virus surveillance during the period July 2013 to December 2013 in Wuxi City of Jiangsu Province, China. Swabs were placed in transport medium, consisting of phosphate-buffered saline (PBS) containing 50% glycerol, penicillin (2,000 U/ml), gentamicin (250 µg/ml), polymixin B (2,000 U/ml), nystatin (500 U/ml), ofloxacin HCl (60 µg/ml), and sulfamethoxazole (200 µg/ml). Swab specimens were kept on ice for up to 4 h before their preservation −80°C.</p>
</sec>
<sec disp-level="2">
<title>RT-PCR and virus Isolation</title>
<p>The viral RNA of each swab sample were extracted using QIAamp MinElute Virus Spin Kit (Cat.No.57704, Qiagen) as directed by the manufacturer. All samples were tested by a one-step real-time RT-PCR assay targeting the influenza matrix genome segment
<xref ref-type="bibr" rid="b33">33</xref>
, using a one-step RT-PCR kit (Cat. No. 56046, TaKaRa), on an Applied Biosystems 7500 real-time PCR system (Life Technologies, NY, USA). Then, 200 µl RT-PCR-positive samples (C
<sub>T</sub>
values ≤ 38) with the antibiotics were inoculated into 9-10-day-old specific pathogen-free (SPF) embryonated chicken eggs (Beijing Merial Vital Laboratory Animal Technology Co., Ltd., Beijing, China) for virus isolation. Allantoic fluid were harvested after 3 days, and influenza virus were detected by hemagglutination assays with turkey erythrocytes.</p>
</sec>
<sec disp-level="2">
<title>Sequencing analysis and subtype determination</title>
<p>Cultured influenza viruses were analyzed further by a one-step conventional RT-PCR using universal primers targeting each of the eight influenza segments
<xref ref-type="bibr" rid="b34">34</xref>
, using a one-step RT-PCR Kit (Cat. No. 055A, TaKaRa). Amplified RT-PCR products were purified using a QIAquick Gel Extraction Kit (Cat. No. 28704, Qiagen), subcloned into a pGEM®-T Vector System I (Cat. No. A3600, Promega) and sequenced using standard techniques on an Applied Biosystems ABI 3730 DNA genetic analyzer. Sequences were then assembled and edited using Lasergene version 7.0 (DNASTAR, Madison, WI, USA), these sequences were then compared with similar virus sequence from GenBank to determine viral subtype.</p>
</sec>
<sec disp-level="2">
<title>Molecular clock and temporal phylogenetic analysis</title>
<p>To infer the evolutionary histories for each gene segment (PB2, PB1, PA, HA, NP, NA, MP, and NS), we used BLAST
<xref ref-type="bibr" rid="b35">35</xref>
<xref ref-type="bibr" rid="b36">36</xref>
with the default parameters to find the 200 most similar viral sequences to the novel H2N2 virus. These sequences were downloaded from NCBI
<xref ref-type="bibr" rid="b24">24</xref>
and GISAID (
<ext-link ext-link-type="uri" xlink:href="http://gisaid.org">http://gisaid.org</ext-link>
), then aligned with Muscle v3.8.31
<xref ref-type="bibr" rid="b37">37</xref>
followed with manual alignment to codon position in the coding regions. For segments NS and MP, the aligned sequences were edited such that all of the codons in first open reading frame (ORF) were followed by the remaining codons in the second ORF. Genetic distances were calculated using the program “dnadist” and phylogenies were estimated using the program “neighbor” with the default parameters in Phylip 3.68 (version 3.2), with rooting chosen to maximize the regression fit between the isolation date of each influenza sequence and the genetic distance from that sequence to the root of the phylogeny
<xref ref-type="bibr" rid="b38">38</xref>
<xref ref-type="bibr" rid="b39">39</xref>
. Residual analysis was performed to identify and remove significant outliers, which most likely result from isolation data annotation errors in the sequence database. The high correlation between the isolation date of each influenza sequence and its genetic distance to the root suggest a “molecular clock” for the closely related viral sequences. The temporal phylogenies and rates of evolution were inferred using the software BEAST v1.75
<xref ref-type="bibr" rid="b40">40</xref>
. BEAST was used to sample phylogenies and the dates of divergences between viruses from their joint posterior distribution, in which the sequences are constrained by their known date of sampling. The codon-position-specific HKY and Gamma +4 nucleotide substitution model and the uncorrelated lognormal molecular clock model were used in the analysis. The results were similar when both the SRD06 and Yang96 substitution models were used. For the analyses using Bayesian MCMC sampling, in all cases chain lengths were run for sufficient time to achieve convergence both by manual checking and by the effective sample size method, which was recommended to be greater than 200 in BEAST. Furthermore, at least two independent runs of each gene segment were performed and compared to ensure stable results.</p>
</sec>
<sec disp-level="2">
<title>Receptor binding assays</title>
<p>Receptor binding specificity was analyzed by a solid-phase direct binding assay biotinylated sialylglycopolymers: 3′-sialyllactose-PAA-biotin (3′SL-PAA, 3′ Neu5Acα2-3Galβ1-4Glc) and 6′-sialyllactosamine-PAA-biotin (6′SLN-PAA, 6′ Neu5Acα2-6Galβ1-4Glcβ) (Cat. No. 01-038, 01-039, Glycotech, Gaithersburg, MD) as previously described
<xref ref-type="bibr" rid="b27">27</xref>
. Briefly, serial dilutions (0.001875 μm/ml, 0.00375 μm/ml, 0.0075 μm/ml, 0.015 μm/ml, 0.03 μm/ml, 0.06 μm/ml, 0.12 μm/ml, and 0.24 μm/ml) of 3′-sialyllactose-PAA-biotin and 6′-sialyllactosamine-PAA-biotin were prepared in PBS; 100 μl of this dilution was added to each well of the 96-well microtiter plates (Cat. No. costar 30500, Thermo Fisher, MA, USA) and allowed to attach overnight at 4°C. After removal of the glycopolymer solution, the plates were blocked with 0.1 ml of PBS containing 2% bovine serum albumin (BSA) at room temperature for 1 hour. After washing with ice-cold PBS containing 0.1% Tween 20 (PBST) and cold PBS, the plates were incubated in a solution containing influenza virus (64 HA units in PBS containing 2% BSA) at 4°C overnight. After washing with cold PBST and cold PBS, chicken antisera against A/WX/2/2013 (H2N2), A/Changchun/XD/2009 (H1N1), and A/Chicken/Jilin/Hu-3/2006 (H9N2) viruses were added to each well and the plates were incubated at 4°C for 2 hours. The wells were washed with ice-cold PBST and cold PBS then incubated with HRP-linked goat-anti-chicken antibody (Sigma-Aldrich,
<ext-link ext-link-type="uri" xlink:href="http://www.sigmaaldrich.com">www.sigmaaldrich.com</ext-link>
) for 2 hours at 4°C. After washing with ice-cold PBST and cold PBS, the plates were incubated 200 μl of TMB substrate (Cat.No.T8665-100Ml, Sigma-Aldrich) for 20 minutes at room temperature. The reaction was then stopped with 100 µl of 50mM H
<sub>2</sub>
SO
<sub>4</sub>
and the optical density was measured at 450 nm.</p>
</sec>
<sec disp-level="2">
<title>Pathogenicity of avian H2N2 virus in chickens</title>
<p>
<italic>In vivo</italic>
pathogenicity studies on the avian H2N2 isolate were performed in accordance with the World Organization for Animal Health (
<ext-link ext-link-type="uri" xlink:href="http://www.oie.int/fileadmin/Home/eng/Health_standards/tahm/2.03.04_AI.pdf">www.oie.int/fileadmin/Home/eng/Health_standards/tahm/2.03.04_AI.pdf</ext-link>
). In brief, ten 6-week-old SPF chickens (Beijing Merial Vital Laboratory Animal Technology Co., Ltd., Beijing, China) were inoculated intravenously with 0.1ml 10
<sup>6</sup>
Egg 50% infective doses (EID
<sub>50</sub>
) of amniotic allantoic fluid. Two chickens were inoculated with PBS to be used as negative controls. The chickens were observed daily for sickness or death for 10 days post inoculation (p.i), which is used to determine the intravenous pathogenicity index (IVPI). At each observation each chicken was scored 0 if normal, 1 if sick, 2 if severely sick, and 3 if dead. Chickens were determined to be sick if one of the following signs were observed, and severely sick of two or more of the signs were observed: respiratory distress, sneezing, coughing, diarrhea, and cyanosis of exposed skin or wattles, edema of the face or head, and nervous signs. Chickens found to be too sick to eat or drink were euthanized humanely and scored dead at the next observation. The IVPI was calculated as the mean score per chicken per observation over the 10-day period. Viruses with an IVPI greater than 1.2 were considered to be highly pathogenic. The serum sample of each chicken was collected to determine the HI titer of the novel H2N2 virus on day 10 p.i.</p>
</sec>
<sec disp-level="2">
<title>Pathogenicity of avian H2N2 virus in mice</title>
<p>Six- to seven-week old SPF BALB/c mice (Vital River Laboratories Technology Co., Ltd., Beijing, China) were used for infectivity experiments. Animals were weighed and anesthetized with diethyl ether before intranasal inoculation with 50 µl of 10
<sup>1</sup>
-10
<sup>6</sup>
EID
<sub>50</sub>
of the novel H2N2 viruses. A total of 66 BALB/c mice were divided into 6 groups (11 mice for each group) and were inoculated intranasally with the novel H2N2 virus, with doses ranging from 10
<sup>6</sup>
EID
<sub>50 </sub>
to 10
<sup>1</sup>
EID
<sub>50</sub>
. The weight of each mouse was recorded once daily, and the general health status of each mouse was observed twice daily. After onset of disease, the general health status of each mouse was observed three times per day. Three mice of each group were killed on day 3 p.i and three killed on day 5 p.i. At necropsy tissue specimens from the brain, lung, kidney, and spleen were cultured for virus and fixed for histological study. Surviving mice were euthanized on day 14 p.i, and the lungs and serum samples preserved at −80°C.</p>
</sec>
<sec disp-level="2">
<title>Transmission of H2N2 virus in guinea pigs</title>
<p>Infection and transmissibility of the novel H2N2 virus was tested in twelve guinea pigs, which were virus antibody free (Vital River Laboratories Technology Co., Ltd., Beijing, China). Blood samples from each guinea pig were collected before intranasal inoculation of the novel H2N2 virus, with doses ranging from 10
<sup>1</sup>
to 10
<sup>6</sup>
EID
<sub>50</sub>
. For the contact transmission study, 3 guinea pigs infected with 300 µl of 10
<sup>6</sup>
EID
<sub>50</sub>
of H2N2 virus and 3 naïve guinea pigs were placed in the same cage at 24 hours p.i. Nasal, oral, and rectal swabs were collected and body temperature measured from each animal on days 2, 4, 6, 8, 10, and 12 p.i. The aerosol transmission study used the same protocol as the contact transmission study, except the 3 infected guinea pigs were placed in an adjacent cage to the 3 naïve guinea pigs (5 cm away). Swabs and tissue cultures were inoculated into SPF embryonated eggs. EID
<sub>50</sub>
values were calculated by the Reed-Muench method
<xref ref-type="bibr" rid="b41">41</xref>
. A serum sample from each guinea pig was collected on day 14 p.i.</p>
</sec>
<sec disp-level="2">
<title>Hemagglutination inhibition (HI) Assay</title>
<p>HI assays were performed on sera from live poultry and humans in Wuxi City, to determine the prevalence of previous H2N2 influenza infection. The sera samples were pre-treated with receptor-destroying enzyme RDE (II) “Seiken”, (Cat. No. 370013, Denka Seiken Co. Ltd., Tokyo, Japan) at 37°C for 18 hours and then heat-inactivated at 56°C for 30 minutes to eliminate nonspecific inhibitors. The sera samples were next adsorbed with 1% horse red blood cells. Similarly, the sera of mice and guinea pigs in the pathogenicity and transmission studies were examined for evidence of H2N2 virus infection. A HI titer ≥ 1:40 was considered as positive.</p>
</sec>
</sec>
<sec disp-level="1">
<title>Author Contributions</title>
<p>M.J.M., J.Q., and W.C.C. conceived and designed the experiments. M.J.M., X.X.Y., Y.H.Q., S.Y.Z., S.H., T.C.W., S.H.C., G.Y.M., X.Y.S., L.N.L., A.P.W., T.J.J., T.Z., J.L.W., X. L., and Y.W.G. performed the experiments and analysed the data. M.J.M., S.H., and W.C.C. wrote the manuscript. B.L., Y.H.Q., A.P.W., T.J.J., and G.C.G. edited the manuscript.</p>
</sec>
<sec sec-type="supplementary-material" id="s1">
<title>Supplementary Material</title>
<supplementary-material id="d33e24" content-type="local-data">
<caption>
<title>Supplementary Information</title>
<p>Supplementary Figure 1</p>
</caption>
<media xlink:href="srep07588-s1.pdf"></media>
</supplementary-material>
</sec>
</body>
<back>
<ack>
<p>This work was supported by the grants from the Program of International S&T Cooperation of the Ministry of Science & Technology of China (2013DFA30800), the National Natural Science Foundation of China (No.81402730), the Program of Jiangsu Provincial Health Department (H201448), and the Wuxi Major Project of Health Bureau (G201201, Z201404).</p>
</ack>
<ref-list>
<ref id="b1">
<mixed-citation publication-type="journal">
<name>
<surname>Medina</surname>
<given-names>R. A.</given-names>
</name>
&
<name>
<surname>Garcia-Sastre</surname>
<given-names>A.</given-names>
</name>
<article-title>Influenza A viruses: new research developments</article-title>
.
<source>Nat Rev Microbiol.</source>
<volume>9</volume>
,
<fpage>590</fpage>
<lpage>603</lpage>
(
<year>2011</year>
).
<pub-id pub-id-type="pmid">21747392</pub-id>
</mixed-citation>
</ref>
<ref id="b2">
<mixed-citation publication-type="journal">
<name>
<surname>Webster</surname>
<given-names>R. G.</given-names>
</name>
,
<name>
<surname>Bean</surname>
<given-names>W. J.</given-names>
</name>
,
<name>
<surname>Gorman</surname>
<given-names>O. T.</given-names>
</name>
,
<name>
<surname>Chambers</surname>
<given-names>T. M.</given-names>
</name>
&
<name>
<surname>Kawaoka</surname>
<given-names>Y.</given-names>
</name>
<article-title>Evolution and ecology of influenza A viruses</article-title>
.
<source>Microbiol Rev.</source>
<volume>56</volume>
,
<fpage>152</fpage>
<lpage>179</lpage>
(
<year>1992</year>
).
<pub-id pub-id-type="pmid">1579108</pub-id>
</mixed-citation>
</ref>
<ref id="b3">
<mixed-citation publication-type="journal">
<name>
<surname>Lipatov</surname>
<given-names>A. S.</given-names>
</name>
<italic>et al.</italic>
<article-title>Influenza: emergence and control</article-title>
.
<source>J Virol.</source>
<volume>78</volume>
,
<fpage>8951</fpage>
<lpage>8959</lpage>
(
<year>2004</year>
).
<pub-id pub-id-type="pmid">15308692</pub-id>
</mixed-citation>
</ref>
<ref id="b4">
<mixed-citation publication-type="journal">
<name>
<surname>Neumann</surname>
<given-names>G.</given-names>
</name>
,
<name>
<surname>Noda</surname>
<given-names>T.</given-names>
</name>
&
<name>
<surname>Kawaoka</surname>
<given-names>Y.</given-names>
</name>
<article-title>Emergence and pandemic potential of swine-origin H1N1 influenza virus</article-title>
.
<source>Nature.</source>
<volume>459</volume>
,
<fpage>931</fpage>
<lpage>939</lpage>
(
<year>2009</year>
).
<pub-id pub-id-type="pmid">19525932</pub-id>
</mixed-citation>
</ref>
<ref id="b5">
<mixed-citation publication-type="journal">
<name>
<surname>Gao</surname>
<given-names>R.</given-names>
</name>
<italic>et al.</italic>
<article-title>Human infection with a novel avian-origin influenza A (H7N9) virus</article-title>
.
<source>N Engl J Med.</source>
<volume>368</volume>
,
<fpage>1888</fpage>
<lpage>1897</lpage>
(
<year>2013</year>
).
<pub-id pub-id-type="pmid">23577628</pub-id>
</mixed-citation>
</ref>
<ref id="b6">
<mixed-citation publication-type="journal">
<name>
<surname>To</surname>
<given-names>K. K.</given-names>
</name>
<italic>et al.</italic>
<article-title>Emergence of avian influenza A(H10N8) in human after A(H5,6,7,9,10 and N1,2,3,7,9): single intrusion or looming epidemic?</article-title>
<source>J Infect.</source>
<volume>68</volume>
,
<fpage>205</fpage>
<lpage>215</lpage>
(
<year>2014</year>
).
<pub-id pub-id-type="pmid">24406432</pub-id>
</mixed-citation>
</ref>
<ref id="b7">
<mixed-citation publication-type="journal">
<name>
<surname>Claas</surname>
<given-names>E. C.</given-names>
</name>
<italic>et al.</italic>
<article-title>Human influenza A H5N1 virus related to a highly pathogenic avian influenza virus</article-title>
.
<source>Lancet.</source>
<volume>351</volume>
,
<fpage>472</fpage>
<lpage>477</lpage>
(
<year>1998</year>
).
<pub-id pub-id-type="pmid">9482438</pub-id>
</mixed-citation>
</ref>
<ref id="b8">
<mixed-citation publication-type="journal">
<name>
<surname>Subbarao</surname>
<given-names>K.</given-names>
</name>
<italic>et al.</italic>
<article-title>Characterization of an avian influenza A (H5N1) virus isolated from a child with a fatal respiratory illness</article-title>
.
<source>Science.</source>
<volume>279</volume>
,
<fpage>393</fpage>
<lpage>396</lpage>
(
<year>1998</year>
).
<pub-id pub-id-type="pmid">9430591</pub-id>
</mixed-citation>
</ref>
<ref id="b9">
<mixed-citation publication-type="journal">
<name>
<surname>Ma</surname>
<given-names>W.</given-names>
</name>
<italic>et al.</italic>
<article-title>Identification of H2N3 influenza A viruses from swine in the United States</article-title>
.
<source>Proc Natl Acad Sci USA.</source>
<volume>104</volume>
,
<fpage>20949</fpage>
<lpage>20954</lpage>
(
<year>2007</year>
).
<pub-id pub-id-type="pmid">18093945</pub-id>
</mixed-citation>
</ref>
<ref id="b10">
<mixed-citation publication-type="journal">
<name>
<surname>Tong</surname>
<given-names>S.</given-names>
</name>
<italic>et al.</italic>
<article-title>A distinct lineage of influenza A virus from bats</article-title>
.
<source>Proc Natl Acad Sci USA.</source>
<volume>109</volume>
,
<fpage>4269</fpage>
<lpage>4274</lpage>
(
<year>2012</year>
).
<pub-id pub-id-type="pmid">22371588</pub-id>
</mixed-citation>
</ref>
<ref id="b11">
<mixed-citation publication-type="journal">
<name>
<surname>Tong</surname>
<given-names>S.</given-names>
</name>
<italic>et al.</italic>
<article-title>New world bats harbor diverse influenza A viruses</article-title>
.
<source>PLoS Pathog.</source>
<volume>9</volume>
,
<fpage>e1003657</fpage>
(
<year>2013</year>
).
<pub-id pub-id-type="pmid">24130481</pub-id>
</mixed-citation>
</ref>
<ref id="b12">
<mixed-citation publication-type="journal">
<name>
<surname>Nabel</surname>
<given-names>G. J.</given-names>
</name>
<name>
<surname>Wei</surname>
<given-names>C. J.</given-names>
</name>
&
<name>
<surname>Ledgerwood</surname>
<given-names>J. E.</given-names>
</name>
<article-title>Vaccinate for the next H2N2 pandemic now</article-title>
.
<source>Nature.</source>
<volume>471</volume>
,
<fpage>157</fpage>
<lpage>158</lpage>
(
<year>2011</year>
).
<pub-id pub-id-type="pmid">21390107</pub-id>
</mixed-citation>
</ref>
<ref id="b13">
<mixed-citation publication-type="journal">
<name>
<surname>Chen</surname>
<given-names>G. L.</given-names>
</name>
<italic>et al.</italic>
<article-title>Evaluation of replication and cross-reactive antibody responses of H2 subtype influenza viruses in mice and ferrets</article-title>
.
<source>J Virol.</source>
<volume>84</volume>
,
<fpage>7695</fpage>
<lpage>7702</lpage>
(
<year>2010</year>
).
<pub-id pub-id-type="pmid">20504935</pub-id>
</mixed-citation>
</ref>
<ref id="b14">
<mixed-citation publication-type="journal">
<name>
<surname>Hilleman</surname>
<given-names>M. R.</given-names>
</name>
<article-title>Realities and enigmas of human viral influenza: pathogenesis, epidemiology and control</article-title>
.
<source>Vaccine.</source>
<volume>20</volume>
,
<fpage>3068</fpage>
<lpage>3087</lpage>
(
<year>2002</year>
).
<pub-id pub-id-type="pmid">12163258</pub-id>
</mixed-citation>
</ref>
<ref id="b15">
<mixed-citation publication-type="journal">
<name>
<surname>Webster</surname>
<given-names>R. G.</given-names>
</name>
<article-title>Predictions for future human influenza pandemics</article-title>
.
<source>J Infect Dis.</source>
<volume>176 Suppl 1</volume>
,
<fpage>S14</fpage>
<lpage>19</lpage>
(
<year>1997</year>
).
<pub-id pub-id-type="pmid">9240688</pub-id>
</mixed-citation>
</ref>
<ref id="b16">
<mixed-citation publication-type="journal">
<name>
<surname>Steel</surname>
<given-names>J.</given-names>
</name>
,
<name>
<surname>Lowen</surname>
<given-names>A. C.</given-names>
</name>
,
<name>
<surname>Mubareka</surname>
<given-names>S.</given-names>
</name>
&
<name>
<surname>Palese</surname>
<given-names>P.</given-names>
</name>
<article-title>Transmission of influenza virus in a mammalian host is increased by PB2 amino acids 627K or 627E/701N</article-title>
.
<source>PLoS Pathog.</source>
<volume>5</volume>
,
<fpage>e1000252</fpage>
(
<year>2009</year>
).
<pub-id pub-id-type="pmid">19119420</pub-id>
</mixed-citation>
</ref>
<ref id="b17">
<mixed-citation publication-type="journal">
<name>
<surname>Matrosovich</surname>
<given-names>M. N.</given-names>
</name>
<italic>et al.</italic>
<article-title>Avian influenza A viruses differ from human viruses by recognition of sialyloligosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site</article-title>
.
<source>Virology.</source>
<volume>233</volume>
,
<fpage>224</fpage>
<lpage>234</lpage>
(
<year>1997</year>
).
<pub-id pub-id-type="pmid">9201232</pub-id>
</mixed-citation>
</ref>
<ref id="b18">
<mixed-citation publication-type="journal">
<name>
<surname>Zhou</surname>
<given-names>N.</given-names>
</name>
<italic>et al.</italic>
<article-title>Influenza infection in humans and pigs in southeastern China</article-title>
.
<source>Arch Virol.</source>
<volume>141</volume>
,
<fpage>649</fpage>
<lpage>661</lpage>
(
<year>1996</year>
).
<pub-id pub-id-type="pmid">8645101</pub-id>
</mixed-citation>
</ref>
<ref id="b19">
<mixed-citation publication-type="journal">
<name>
<surname>Shortridge</surname>
<given-names>K. F.</given-names>
</name>
<article-title>Pandemic influenza: a zoonosis?</article-title>
<source>Semin Respir Infect.</source>
<volume>7</volume>
,
<fpage>11</fpage>
<lpage>25</lpage>
(
<year>1992</year>
).
<pub-id pub-id-type="pmid">1609163</pub-id>
</mixed-citation>
</ref>
<ref id="b20">
<mixed-citation publication-type="journal">
<name>
<surname>Shu</surname>
<given-names>L. L.</given-names>
</name>
<italic>et al.</italic>
<article-title>An epidemiological study of influenza viruses among Chinese farm families with household ducks and pigs</article-title>
.
<source>Epidemiol Infect.</source>
<volume>117</volume>
,
<fpage>179</fpage>
<lpage>188</lpage>
(
<year>1996</year>
).
<pub-id pub-id-type="pmid">8760967</pub-id>
</mixed-citation>
</ref>
<ref id="b21">
<mixed-citation publication-type="journal">
<name>
<surname>Bi</surname>
<given-names>Y.</given-names>
</name>
<italic>et al.</italic>
<article-title>Novel swine influenza virus reassortants in pigs, China</article-title>
.
<source>Emerg Infect Dis.</source>
<volume>16</volume>
,
<fpage>1162</fpage>
<lpage>1164</lpage>
(
<year>2010</year>
).
<pub-id pub-id-type="pmid">20587196</pub-id>
</mixed-citation>
</ref>
<ref id="b22">
<mixed-citation publication-type="journal">
<name>
<surname>Yu</surname>
<given-names>H.</given-names>
</name>
<italic>et al.</italic>
<article-title>Genetic diversity of H9N2 influenza viruses from pigs in China: a potential threat to human health?</article-title>
<source>Vet Microbiol.</source>
<volume>149</volume>
,
<fpage>254</fpage>
<lpage>261</lpage>
(
<year>2011</year>
).
<pub-id pub-id-type="pmid">21115230</pub-id>
</mixed-citation>
</ref>
<ref id="b23">
<mixed-citation publication-type="journal">
<name>
<surname>Zhang</surname>
<given-names>G.</given-names>
</name>
<italic>et al.</italic>
<article-title>Identification of an H6N6 swine influenza virus in southern China</article-title>
.
<source>Infect Genet Evol.</source>
<volume>11</volume>
,
<fpage>1174</fpage>
<lpage>1177</lpage>
(
<year>2011</year>
).
<pub-id pub-id-type="pmid">21382518</pub-id>
</mixed-citation>
</ref>
<ref id="b24">
<mixed-citation publication-type="journal">
<name>
<surname>Bao</surname>
<given-names>Y.</given-names>
</name>
<italic>et al.</italic>
<article-title>The influenza virus resource at the National Center for Biotechnology Information</article-title>
.
<source>J Virol.</source>
<volume>82</volume>
,
<fpage>596</fpage>
<lpage>601</lpage>
(
<year>2008</year>
).
<pub-id pub-id-type="pmid">17942553</pub-id>
</mixed-citation>
</ref>
<ref id="b25">
<mixed-citation publication-type="journal">
<name>
<surname>Vines</surname>
<given-names>A.</given-names>
</name>
<italic>et al.</italic>
<article-title>The role of influenza A virus hemagglutinin residues 226 and 228 in receptor specificity and host range restriction</article-title>
.
<source>J Virol.</source>
<volume>72</volume>
,
<fpage>7626</fpage>
<lpage>7631</lpage>
(
<year>1998</year>
).
<pub-id pub-id-type="pmid">9696865</pub-id>
</mixed-citation>
</ref>
<ref id="b26">
<mixed-citation publication-type="journal">
<name>
<surname>Connor</surname>
<given-names>R. J.</given-names>
</name>
,
<name>
<surname>Kawaoka</surname>
<given-names>Y.</given-names>
</name>
,
<name>
<surname>Webster</surname>
<given-names>R. G.</given-names>
</name>
&
<name>
<surname>Paulson</surname>
<given-names>J. C.</given-names>
</name>
<article-title>Receptor specificity in human, avian, and equine H2 and H3 influenza virus isolates</article-title>
.
<source>Virology.</source>
<volume>205</volume>
,
<fpage>17</fpage>
<lpage>23</lpage>
(
<year>1994</year>
).
<pub-id pub-id-type="pmid">7975212</pub-id>
</mixed-citation>
</ref>
<ref id="b27">
<mixed-citation publication-type="journal">
<name>
<surname>Jones</surname>
<given-names>J. C.</given-names>
</name>
<italic>et al.</italic>
<article-title>Risk Assessment of H2N2 Influenza Viruses from the Avian Reservoir</article-title>
.
<source>J Virol.</source>
<volume>88</volume>
,
<fpage>1175</fpage>
<lpage>1188</lpage>
(
<year>2014</year>
).
<pub-id pub-id-type="pmid">24227848</pub-id>
</mixed-citation>
</ref>
<ref id="b28">
<mixed-citation publication-type="journal">
<name>
<surname>Ito</surname>
<given-names>T.</given-names>
</name>
<italic>et al.</italic>
<article-title>Molecular basis for the generation in pigs of influenza A viruses with pandemic potential</article-title>
.
<source>J Virol.</source>
<volume>72</volume>
,
<fpage>7367</fpage>
<lpage>7373</lpage>
(
<year>1998</year>
).
<pub-id pub-id-type="pmid">9696833</pub-id>
</mixed-citation>
</ref>
<ref id="b29">
<mixed-citation publication-type="journal">
<name>
<surname>Liu</surname>
<given-names>M.</given-names>
</name>
<italic>et al.</italic>
<article-title>The influenza virus gene pool in a poultry market in South central china</article-title>
.
<source>Virology.</source>
<volume>305</volume>
,
<fpage>267</fpage>
<lpage>275</lpage>
(
<year>2003</year>
).
<pub-id pub-id-type="pmid">12573572</pub-id>
</mixed-citation>
</ref>
<ref id="b30">
<mixed-citation publication-type="journal">
<name>
<surname>Peng</surname>
<given-names>X.</given-names>
</name>
<italic>et al.</italic>
<article-title>Sequence and phylogenetic analysis of H2N7 avian influenza viruses isolated from domestic ducks in Zhejiang Province, Eastern China, 2013</article-title>
.
<source>Virus Genes.</source>
<volume>48</volume>
,
<fpage>391</fpage>
<lpage>396</lpage>
(
<year>2014</year>
).
<pub-id pub-id-type="pmid">24442671</pub-id>
</mixed-citation>
</ref>
<ref id="b31">
<mixed-citation publication-type="journal">
<name>
<surname>Hilleman</surname>
<given-names>M. R.</given-names>
</name>
<article-title>Vaccines in historic evolution and perspective: a narrative of vaccine discoveries</article-title>
.
<source>Vaccine.</source>
<volume>18</volume>
,
<fpage>1436</fpage>
<lpage>1447</lpage>
(
<year>2000</year>
).
<pub-id pub-id-type="pmid">10618541</pub-id>
</mixed-citation>
</ref>
<ref id="b32">
<mixed-citation publication-type="journal">
<name>
<surname>Liu</surname>
<given-names>D.</given-names>
</name>
,
<name>
<surname>Shi</surname>
<given-names>W.</given-names>
</name>
&
<name>
<surname>Gao</surname>
<given-names>G. F.</given-names>
</name>
<article-title>Poultry carrying H9N2 act as incubators for novel human avian influenza viruses</article-title>
.
<source>Lancet.</source>
<volume>383</volume>
,
<fpage>869</fpage>
(
<year>2014</year>
).
<pub-id pub-id-type="pmid">24581684</pub-id>
</mixed-citation>
</ref>
<ref id="b33">
<mixed-citation publication-type="other">World Health Organization. Real-time RT-PCR Protocol for the Detection of A(H7N9) Influenza Virus <
<ext-link ext-link-type="uri" xlink:href="http://www.who.int/influenza/gisrs_laboratory/cnic_realtime_rt_pcr_protocol_a_h7n9.pdf?ua=1">http://www.who.int/influenza/gisrs_laboratory/cnic_realtime_rt_pcr_protocol_a_h7n9.pdf?ua=1</ext-link>
> Date of access 15/06/2013 (
<year>2013</year>
).</mixed-citation>
</ref>
<ref id="b34">
<mixed-citation publication-type="journal">
<name>
<surname>Hoffmann</surname>
<given-names>E.</given-names>
</name>
,
<name>
<surname>Stech</surname>
<given-names>J.</given-names>
</name>
,
<name>
<surname>Guan</surname>
<given-names>Y.</given-names>
</name>
,
<name>
<surname>Webster</surname>
<given-names>R. G.</given-names>
</name>
&
<name>
<surname>Perez</surname>
<given-names>D. R.</given-names>
</name>
<article-title>Universal primer set for the full-length amplification of all influenza A viruses</article-title>
.
<source>Arch Virol.</source>
<volume>146</volume>
,
<fpage>2275</fpage>
<lpage>2289</lpage>
(
<year>2001</year>
).
<pub-id pub-id-type="pmid">11811679</pub-id>
</mixed-citation>
</ref>
<ref id="b35">
<mixed-citation publication-type="journal">
<name>
<surname>Mount</surname>
<given-names>D. W.</given-names>
</name>
<article-title>Using the Basic Local Alignment Search Tool (BLAST)</article-title>
.
<source>CSH Protoc.</source>
<volume>2007</volume>
, pdb top17 (
<year>2007</year>
).</mixed-citation>
</ref>
<ref id="b36">
<mixed-citation publication-type="journal">
<name>
<surname>Altschul</surname>
<given-names>S. F.</given-names>
</name>
,
<name>
<surname>Gish</surname>
<given-names>W.</given-names>
</name>
,
<name>
<surname>Miller</surname>
<given-names>W.</given-names>
</name>
,
<name>
<surname>Myers</surname>
<given-names>E. W.</given-names>
</name>
&
<name>
<surname>Lipman</surname>
<given-names>D. J.</given-names>
</name>
<article-title>Basic local alignment search tool</article-title>
.
<source>J Mol Biol.</source>
<volume>215</volume>
,
<fpage>403</fpage>
<lpage>410</lpage>
(
<year>1990</year>
).
<pub-id pub-id-type="pmid">2231712</pub-id>
</mixed-citation>
</ref>
<ref id="b37">
<mixed-citation publication-type="journal">
<name>
<surname>Edgar</surname>
<given-names>R. C.</given-names>
</name>
<article-title>MUSCLE: multiple sequence alignment with high accuracy and high throughput</article-title>
.
<source>Nucleic Acids Res.</source>
<volume>32</volume>
,
<fpage>1792</fpage>
<lpage>1797</lpage>
(
<year>2004</year>
).
<pub-id pub-id-type="pmid">15034147</pub-id>
</mixed-citation>
</ref>
<ref id="b38">
<mixed-citation publication-type="journal">
<name>
<surname>Drummond</surname>
<given-names>A.</given-names>
</name>
,
<name>
<surname>Pybus</surname>
<given-names>O. G.</given-names>
</name>
&
<name>
<surname>Rambaut</surname>
<given-names>A.</given-names>
</name>
<article-title>Inference of viral evolutionary rates from molecular sequences</article-title>
.
<source>Adv Parasitol.</source>
<volume>54</volume>
,
<fpage>331</fpage>
<lpage>358</lpage>
(
<year>2003</year>
).
<pub-id pub-id-type="pmid">14711090</pub-id>
</mixed-citation>
</ref>
<ref id="b39">
<mixed-citation publication-type="journal">
<name>
<surname>Smith</surname>
<given-names>G. J.</given-names>
</name>
<italic>et al.</italic>
<article-title>Origins and evolutionary genomics of the 2009 swine-origin H1N1 influenza A epidemic</article-title>
.
<source>Nature.</source>
<volume>459</volume>
,
<fpage>1122</fpage>
<lpage>1125</lpage>
(
<year>2009</year>
).
<pub-id pub-id-type="pmid">19516283</pub-id>
</mixed-citation>
</ref>
<ref id="b40">
<mixed-citation publication-type="journal">
<name>
<surname>Drummond</surname>
<given-names>A. J.</given-names>
</name>
&
<name>
<surname>Rambaut</surname>
<given-names>A.</given-names>
</name>
<article-title>BEAST: Bayesian evolutionary analysis by sampling trees</article-title>
.
<source>BMC Evol Biol.</source>
<volume>7</volume>
,
<fpage>214</fpage>
(
<year>2007</year>
).
<pub-id pub-id-type="pmid">17996036</pub-id>
</mixed-citation>
</ref>
<ref id="b41">
<mixed-citation publication-type="journal">
<name>
<surname>Reed</surname>
<given-names>L. J.</given-names>
</name>
&
<name>
<surname>Muench</surname>
<given-names>H.</given-names>
</name>
<article-title>A simple method of estimating fifty per cent endpoint</article-title>
.
<source>Am J Hyg.</source>
<volume>27</volume>
,
<fpage>493</fpage>
<lpage>497</lpage>
(
<year>1938</year>
).</mixed-citation>
</ref>
<ref id="b42">
<mixed-citation publication-type="journal">
<name>
<surname>Chen</surname>
<given-names>G. W.</given-names>
</name>
<italic>et al.</italic>
<article-title>Genomic signatures of human versus avian influenza A viruses</article-title>
.
<source>Emerg Infect Dis.</source>
<volume>12</volume>
,
<fpage>1353</fpage>
<lpage>1360</lpage>
(
<year>2006</year>
).
<pub-id pub-id-type="pmid">17073083</pub-id>
</mixed-citation>
</ref>
<ref id="b43">
<mixed-citation publication-type="journal">
<name>
<surname>Herfst</surname>
<given-names>S.</given-names>
</name>
<italic>et al.</italic>
<article-title>Airborne transmission of influenza A/H5N1 virus between ferrets</article-title>
.
<source>Science.</source>
<volume>336</volume>
,
<fpage>1534</fpage>
<lpage>1541</lpage>
(
<year>2012</year>
).
<pub-id pub-id-type="pmid">22723413</pub-id>
</mixed-citation>
</ref>
<ref id="b44">
<mixed-citation publication-type="journal">
<name>
<surname>Tamuri</surname>
<given-names>A. U.</given-names>
</name>
,
<name>
<surname>Dos Reis</surname>
<given-names>M.</given-names>
</name>
,
<name>
<surname>Hay</surname>
<given-names>A. J.</given-names>
</name>
&
<name>
<surname>Goldstein</surname>
<given-names>R. A.</given-names>
</name>
<article-title>Identifying changes in selective constraints: host shifts in influenza</article-title>
.
<source>PLoS Comput Biol.</source>
<volume>5</volume>
,
<fpage>e1000564</fpage>
(
<year>2009</year>
).
<pub-id pub-id-type="pmid">19911053</pub-id>
</mixed-citation>
</ref>
<ref id="b45">
<mixed-citation publication-type="journal">
<name>
<surname>Yang</surname>
<given-names>H.</given-names>
</name>
,
<name>
<surname>Chen</surname>
<given-names>L. M.</given-names>
</name>
,
<name>
<surname>Carney</surname>
<given-names>P. J.</given-names>
</name>
,
<name>
<surname>Donis</surname>
<given-names>R. O.</given-names>
</name>
&
<name>
<surname>Stevens</surname>
<given-names>J.</given-names>
</name>
<article-title>Structures of receptor complexes of a North American H7N2 influenza hemagglutinin with a loop deletion in the receptor binding site</article-title>
.
<source>PLoS Pathog.</source>
<volume>6</volume>
,
<fpage>e1001081</fpage>
(
<year>2010</year>
).
<pub-id pub-id-type="pmid">20824086</pub-id>
</mixed-citation>
</ref>
<ref id="b46">
<mixed-citation publication-type="journal">
<name>
<surname>Tumpey</surname>
<given-names>T. M.</given-names>
</name>
<italic>et al.</italic>
<article-title>A two-amino acid change in the hemagglutinin of the 1918 influenza virus abolishes transmission</article-title>
.
<source>Science.</source>
<volume>315</volume>
,
<fpage>655</fpage>
<lpage>659</lpage>
(
<year>2007</year>
).
<pub-id pub-id-type="pmid">17272724</pub-id>
</mixed-citation>
</ref>
<ref id="b47">
<mixed-citation publication-type="journal">
<name>
<surname>Lycett</surname>
<given-names>S. J.</given-names>
</name>
<italic>et al.</italic>
<article-title>Detection of mammalian virulence determinants in highly pathogenic avian influenza H5N1 viruses: multivariate analysis of published data</article-title>
.
<source>J Virol.</source>
<volume>83</volume>
,
<fpage>9901</fpage>
<lpage>9910</lpage>
(
<year>2009</year>
).
<pub-id pub-id-type="pmid">19625397</pub-id>
</mixed-citation>
</ref>
<ref id="b48">
<mixed-citation publication-type="journal">
<name>
<surname>Matsuoka</surname>
<given-names>Y.</given-names>
</name>
<italic>et al.</italic>
<article-title>Neuraminidase stalk length and additional glycosylation of the hemagglutinin influence the virulence of influenza H5N1 viruses for mice</article-title>
.
<source>J Virol.</source>
<volume>83</volume>
,
<fpage>4704</fpage>
<lpage>4708</lpage>
(
<year>2009</year>
).
<pub-id pub-id-type="pmid">19225004</pub-id>
</mixed-citation>
</ref>
<ref id="b49">
<mixed-citation publication-type="journal">
<name>
<surname>Fan</surname>
<given-names>S.</given-names>
</name>
<italic>et al.</italic>
<article-title>Two amino acid residues in the matrix protein M1 contribute to the virulence difference of H5N1 avian influenza viruses in mice</article-title>
.
<source>Virology.</source>
<volume>384</volume>
,
<fpage>28</fpage>
<lpage>32</lpage>
(
<year>2009</year>
).
<pub-id pub-id-type="pmid">19117585</pub-id>
</mixed-citation>
</ref>
<ref id="b50">
<mixed-citation publication-type="journal">
<name>
<surname>Jiao</surname>
<given-names>P.</given-names>
</name>
<italic>et al.</italic>
<article-title>A single-amino-acid substitution in the NS1 protein changes the pathogenicity of H5N1 avian influenza viruses in mice</article-title>
.
<source>J Virol.</source>
<volume>82</volume>
,
<fpage>1146</fpage>
<lpage>1154</lpage>
(
<year>2008</year>
).
<pub-id pub-id-type="pmid">18032512</pub-id>
</mixed-citation>
</ref>
</ref-list>
</back>
<floats-group>
<fig id="f1">
<label>Figure 1</label>
<caption>
<title>Phylogenetic relationships of each gene of novel H2N2 strain and closely related viruses.</title>
<p>All branch lengths are drawn to a scale of nucleotide substitutions per site. The novel H2N2 virus is highlighted in red. Detailed phylogenetic trees with virus designations can be found in
<xref ref-type="supplementary-material" rid="s1">Supplemental Figure 1</xref>
.</p>
</caption>
<graphic xlink:href="srep07588-f1"></graphic>
</fig>
<fig id="f2">
<label>Figure 2</label>
<caption>
<title>Temporal reconstruction of the phylogenetic relationships of each gene segment.</title>
<p>The novel H2N2 strain is highlighted by red. Internal nodes are reconstructed common ancestors with 95% highest posterior density (HPD) intervals on their dates given by the light red bars. Detailed temporal reconstruction of phylogenetic trees with virus designations can be found in
<xref ref-type="supplementary-material" rid="s1">Supplemental Figure 1</xref>
.</p>
</caption>
<graphic xlink:href="srep07588-f2"></graphic>
</fig>
<fig id="f3">
<label>Figure 3</label>
<caption>
<title>Reassortment model for the origins of the novel H2N2.</title>
<p>The colors of the novel H2N2 gene segments indicate their origins. The picture of duck was drawn by the author Mai-Juan Ma.</p>
</caption>
<graphic xlink:href="srep07588-f3"></graphic>
</fig>
<fig id="f4">
<label>Figure 4</label>
<caption>
<title>Characterization of the receptor-binding properties of isolated H2N2 virus.</title>
<p>Binding of virus to a2,3-linked (3′SL-PAA) or a2,6-linked (6′SL-PAA) sialylglycan receptors was determined by solid-phase binding assays. (a) DK13-H2N2 (A/duck/Wuxi/2/13 (H2N2)); (b) XD09-H1N1 (A/Changchun/XD/2009 (H1N1)); (c) CK06-H9N2 (A/Chicken/Jilin/Hu-3/2006(H9N2)). Blue line, binding to (3′SL-PAA); red line, binding to (6′SL-PAA).</p>
</caption>
<graphic xlink:href="srep07588-f4"></graphic>
</fig>
<fig id="f5">
<label>Figure 5</label>
<caption>
<title>Hemagglutination inhibition (HI) Antibody against H2N2 virus among human population and live poultry (mean antibody counts shown with 95% confidence intervals).</title>
<p>(a) HI antibody of H2N2 virus among poultry. (b) HI antibody of H2N2 virus among general health population, poultry workers, and swine workers. (c) HI antibody of H2N2 virus among different age population.</p>
</caption>
<graphic xlink:href="srep07588-f5"></graphic>
</fig>
<table-wrap position="float" id="t1">
<label>Table 1</label>
<caption>
<title>Time of most recent common ancestors (tMRCA) for the novel H2N2 virus. Observed closest strains for the H2N2 segments, time of divergence, and the mean evolutionary rate are listed</title>
</caption>
<table frame="hsides" rules="groups" border="1">
<colgroup>
<col align="left"></col>
<col align="center"></col>
<col align="center"></col>
<col align="center"></col>
</colgroup>
<thead valign="bottom">
<tr>
<th align="justify" valign="top" charoff="50">Gene</th>
<th align="center" valign="top" charoff="50">Divergence time</th>
<th align="center" valign="top" charoff="50">Observed closest strain</th>
<th align="center" valign="top" charoff="50">Mean evolutionary rate (10
<sup>−3</sup>
)</th>
</tr>
</thead>
<tbody valign="top">
<tr>
<td align="center" valign="top" charoff="50">PB2</td>
<td align="char" valign="top" char=" " charoff="50">May 2010 (Dec 2009, Oct 2010)</td>
<td align="char" valign="top" char="/" charoff="50">A/mallard/Korea/1203/2010_H10N8</td>
<td align="center" valign="top" charoff="50">2.133 (1.849, 2.421)</td>
</tr>
<tr>
<td align="center" valign="top" charoff="50">PB1</td>
<td align="char" valign="top" char=" " charoff="50">Jul 2010 (Sep 2009, Mar 2011)</td>
<td align="char" valign="top" char="/" charoff="50">A/duck/Hunan/S11200/2012_H4N6</td>
<td align="center" valign="top" charoff="50">2.591 (2.344, 2.846)</td>
</tr>
<tr>
<td align="center" valign="top" charoff="50">PA</td>
<td align="char" valign="top" char=" " charoff="50">Jun 2010 (Apr 2009, Jul 2011)</td>
<td align="char" valign="top" char="/" charoff="50">A/duck/Vietnam/LBM185/2012_H6N6</td>
<td align="center" valign="top" charoff="50">1.864 (1.405, 2.351)</td>
</tr>
<tr>
<td align="center" valign="top" charoff="50">HA</td>
<td align="char" valign="top" char=" " charoff="50">May 2006 (Jan 2005, Aug 2007)</td>
<td align="char" valign="top" char="/" charoff="50">A/environment/Guangdong/2/2009_H2N3</td>
<td align="center" valign="top" charoff="50">3.859 (3.462, 4.278)</td>
</tr>
<tr>
<td align="center" valign="top" charoff="50">NP</td>
<td align="char" valign="top" char=" " charoff="50">Jun 2010 (Oct 2008, Sep 2011)</td>
<td align="char" valign="top" char="/" charoff="50">A/duck/Vietnam/LBM81/2012_H11N9</td>
<td align="center" valign="top" charoff="50">1.562 (1.305, 1.816)</td>
</tr>
<tr>
<td align="center" valign="top" charoff="50">NA</td>
<td align="char" valign="top" char=" " charoff="50">Jun 2011 (Jul 2010, Jan 2012)</td>
<td align="char" valign="top" char="/" charoff="50">A/duck/Jiangsu/m234/2012_H5N2</td>
<td align="center" valign="top" charoff="50">3.049 (2.697, 3.398)</td>
</tr>
<tr>
<td align="center" valign="top" charoff="50">M</td>
<td align="char" valign="top" char=" " charoff="50">Oct 2003 (May 2002, Sep 2004)</td>
<td align="char" valign="top" char="/" charoff="50">A/duck/Eastern-China/29/2004_H6N2</td>
<td align="center" valign="top" charoff="50">1.696 (1.400, 2.011)</td>
</tr>
<tr>
<td align="center" valign="top" charoff="50">NS</td>
<td align="char" valign="top" char=" " charoff="50">Sep 2009 (Nov 2008, Jun 2010)</td>
<td align="char" valign="top" char="/" charoff="50">A/duck/Nanjing/1102/2010_H4N8</td>
<td align="center" valign="top" charoff="50">2.357 (1.977, 2.773)</td>
</tr>
</tbody>
</table>
<table-wrap-foot>
<fn id="t1-fn1">
<p>The mean evolutionary rate is represented as the number of substitutions per site per year. The values in parentheses represent the 95% highest posterior density (HPD) intervals.</p>
</fn>
</table-wrap-foot>
</table-wrap>
<table-wrap position="float" id="t2">
<label>Table 2</label>
<caption>
<title>Selected characteristic amino acids of the novel influenza A(H2N2) virus</title>
</caption>
<table frame="hsides" rules="groups" border="1">
<colgroup>
<col align="left"></col>
<col align="center"></col>
<col align="center"></col>
<col align="center"></col>
</colgroup>
<thead valign="bottom">
<tr>
<th align="justify" valign="top" charoff="50">Viral protein</th>
<th align="center" valign="top" charoff="50">Amino acid position</th>
<th align="center" valign="top" charoff="50">A/duck/WX/2/13</th>
<th align="center" valign="top" charoff="50">Comments</th>
</tr>
</thead>
<tbody valign="top">
<tr>
<td align="center" valign="top" charoff="50">PB2</td>
<td align="char" valign="top" char="." charoff="50">627</td>
<td align="center" valign="top" charoff="50">E</td>
<td align="left" valign="top" charoff="50">E627K: Mammalian host adaptation
<xref ref-type="bibr" rid="b42">42</xref>
<xref ref-type="bibr" rid="b43">43</xref>
</td>
</tr>
<tr>
<td align="center" valign="top" charoff="50">HA</td>
<td align="char" valign="top" char="." charoff="50">138</td>
<td align="center" valign="top" charoff="50">A</td>
<td align="left" valign="top" charoff="50">S138A: Increased virus binding to human-type receptors
<xref ref-type="bibr" rid="b44">44</xref>
</td>
</tr>
<tr>
<td align="left" valign="top" charoff="50"> </td>
<td align="char" valign="top" char="." charoff="50">186</td>
<td align="center" valign="top" charoff="50">N</td>
<td align="left" valign="top" charoff="50">G186V: Increased virus binding to human-type receptors
<xref ref-type="bibr" rid="b45">45</xref>
</td>
</tr>
<tr>
<td align="left" valign="top" charoff="50"> </td>
<td align="char" valign="top" char="." charoff="50">190</td>
<td align="center" valign="top" charoff="50">E</td>
<td align="left" valign="top" charoff="50">E190D: Increased virus binding to human-type receptors
<xref ref-type="bibr" rid="b46">46</xref>
</td>
</tr>
<tr>
<td align="left" valign="top" charoff="50"> </td>
<td align="char" valign="top" char="." charoff="50">225</td>
<td align="center" valign="top" charoff="50">G</td>
<td align="left" valign="top" charoff="50">G225D: Increased virus binding to human-type receptors
<xref ref-type="bibr" rid="b46">46</xref>
</td>
</tr>
<tr>
<td align="left" valign="top" charoff="50"> </td>
<td align="char" valign="top" char="." charoff="50">226</td>
<td align="center" valign="top" charoff="50">Q</td>
<td align="left" valign="top" charoff="50">Q226L: Increased virus binding to human-type receptors
<xref ref-type="bibr" rid="b26">26</xref>
</td>
</tr>
<tr>
<td align="left" valign="top" charoff="50"> </td>
<td align="char" valign="top" char="." charoff="50">228</td>
<td align="center" valign="top" charoff="50">G</td>
<td align="left" valign="top" charoff="50">H2N2 Human: S
<xref ref-type="bibr" rid="b47">47</xref>
</td>
</tr>
<tr>
<td align="center" valign="top" charoff="50">NA</td>
<td align="center" valign="top" charoff="50">65-71</td>
<td align="center" valign="top" charoff="50">No deletion</td>
<td align="left" valign="top" charoff="50">Deletion of 65–71 increased virulence in mice
<xref ref-type="bibr" rid="b48">48</xref>
</td>
</tr>
<tr>
<td align="center" valign="top" charoff="50">M1</td>
<td align="char" valign="top" char="." charoff="50">30</td>
<td align="center" valign="top" charoff="50">D</td>
<td align="left" valign="top" charoff="50">N30D: Increased virulence in mice (most influenza A viruses encode 30D)
<xref ref-type="bibr" rid="b49">49</xref>
</td>
</tr>
<tr>
<td align="left" valign="top" charoff="50"> </td>
<td align="char" valign="top" char="." charoff="50">174</td>
<td align="center" valign="top" charoff="50">R</td>
<td align="left" valign="top" charoff="50">K174K(R): Host marker of influenza A
<xref ref-type="bibr" rid="b44">44</xref>
</td>
</tr>
<tr>
<td align="center" valign="top" charoff="50">NS1</td>
<td align="char" valign="top" char="." charoff="50">42</td>
<td align="center" valign="top" charoff="50">S</td>
<td align="left" valign="top" charoff="50">P42S: Increased virulence in mice (most avian influenza A viruses encode 42S)
<xref ref-type="bibr" rid="b50">50</xref>
</td>
</tr>
<tr>
<td align="left" valign="top" charoff="50"> </td>
<td align="char" valign="top" char="." charoff="50">97</td>
<td align="center" valign="top" charoff="50">E</td>
<td align="left" valign="top" charoff="50">D97E: Increased avian H5N1 viruses' virulence in mammals
<xref ref-type="bibr" rid="b47">47</xref>
</td>
</tr>
<tr>
<td align="left" valign="top" charoff="50"> </td>
<td align="char" valign="top" char="." charoff="50">127</td>
<td align="center" valign="top" charoff="50">N</td>
<td align="left" valign="top" charoff="50">TDRVA127N: Association with H3N2 virulence
<xref ref-type="bibr" rid="b47">47</xref>
</td>
</tr>
</tbody>
</table>
<table-wrap-foot>
<fn id="t2-fn1">
<p>HA amino acid residues have H3 numbering; NA amino acid residues have N2 numbering.</p>
</fn>
</table-wrap-foot>
</table-wrap>
</floats-group>
</pmc>
</record>

Pour manipuler ce document sous Unix (Dilib)

EXPLOR_STEP=$WICRI_ROOT/Sante/explor/H2N2V1/Data/Pmc/Corpus
HfdSelect -h $EXPLOR_STEP/biblio.hfd -nk 000008 | SxmlIndent | more

Ou

HfdSelect -h $EXPLOR_AREA/Data/Pmc/Corpus/biblio.hfd -nk 000008 | SxmlIndent | more

Pour mettre un lien sur cette page dans le réseau Wicri

{{Explor lien
   |wiki=    Sante
   |area=    H2N2V1
   |flux=    Pmc
   |étape=   Corpus
   |type=    RBID
   |clé=     PMC:4274511
   |texte=   Characterization of a Novel Reassortant Influenza A Virus (H2N2) from a Domestic Duck in Eastern China
}}

Pour générer des pages wiki

HfdIndexSelect -h $EXPLOR_AREA/Data/Pmc/Corpus/RBID.i   -Sk "pubmed:25533850" \
       | HfdSelect -Kh $EXPLOR_AREA/Data/Pmc/Corpus/biblio.hfd   \
       | NlmPubMed2Wicri -a H2N2V1 

Wicri

This area was generated with Dilib version V0.6.33.
Data generation: Tue Apr 14 19:59:40 2020. Site generation: Thu Mar 25 15:38:26 2021