{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,28]],"date-time":"2026-07-28T21:12:39Z","timestamp":1785273159857,"version":"3.55.0"},"reference-count":44,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2021,1,26]],"date-time":"2021-01-26T00:00:00Z","timestamp":1611619200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["U1501212"],"award-info":[{"award-number":["U1501212"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["31872497"],"award-info":[{"award-number":["31872497"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100003453","name":"Natural Science Foundation of Guangdong Province","doi-asserted-by":"publisher","award":["2016A030308001"],"award-info":[{"award-number":["2016A030308001"]}],"id":[{"id":"10.13039\/501100003453","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100012166","name":"National Key Research and Development Program of China","doi-asserted-by":"publisher","award":["2016YFD0500207"],"award-info":[{"award-number":["2016YFD0500207"]}],"id":[{"id":"10.13039\/501100012166","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100009997","name":"Earmarked Fund for Modern Agro-Industry Technology Research System","doi-asserted-by":"publisher","award":["CARS-41-G16"],"award-info":[{"award-number":["CARS-41-G16"]}],"id":[{"id":"10.13039\/501100009997","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Microbiol."],"abstract":"<jats:p>Clade 2.3.4.4 H5Nx highly pathogenic avian influenza viruses (HPAIVs) have caused outbreaks in poultry in the world. Some of these viruses acquired internal genes from other subtype avian influenza viruses (AIVs) such as H9 and H6 for the generation of novel reassortant viruses and continually circulated in poultry. Here, we applied a duck-origin virus DK87 and a chicken-origin virus CK66 to assess the biological characteristics of novel reassortant H5N6 HPAIVs and its pathogenesis in ducks. A genetic analysis indicated that the HA genes of the two H5N6 HPAIVs were closely related to the H5 viruses of clade 2.3.4.4 circulating in Eastern Asia and classified into H5 AIV\/Eastern Asia (EA)-like lineage. Their NA genes fell into Eurasian lineage had close relationship with those of H5N6 viruses circulating in China, Laos, Vietnam, Japan, and Korea. All internal genes of DK87 were aggregated closely with H5 AIV\/EA-like viruses. The internal genes (PB1, PA, NP, M, and NS) of CK66 were derived from H9N2 AIV\/SH98-like viruses and the PB2 were derived from H5 AIV\/EA-like viruses. These results indicate that clade 2.3.4.4 H5N6 AIVs have continually evolved and recombined with the H9N2 viruses circulating in Southern China. Pathogenicity test showed that the two viruses displayed a broader tissue distribution in ducks and caused no clinical signs. These results indicated that ducks were permissive for the replication of the chicken-origin reassortant virus CK66 without prior adaptation, but the duck-origin virus DK87-inoculated ducks showed significantly higher viral titers in some organs than the CK66-inoculated ducks at 5 day post-inoculated (DPI). The recovery of viruses from oropharyngea and cloacal swabs of contacted ducks indicated that they transmitted in native ducks by direct contact. Quantitative reverse transcription PCR (qRT-PCR) results revealed that the immune-relative genes (PRRs, IFNs, Mx-1, IL-6, and IL-8) in the lungs of inoculated ducks were expressed regardless of virus origin, but the expression of these genes was significantly higher in response to infection with the DK87 virus compared to the CK66 virus at 3 DPI. Overall, we should provide further insights into how clade 2.3.4.4 H5N6 AIVs undergo genetic and pathogenic variations to prevent outbreaks of this disease.<\/jats:p>","DOI":"10.3389\/fmicb.2021.628545","type":"journal-article","created":{"date-parts":[[2021,1,26]],"date-time":"2021-01-26T08:55:50Z","timestamp":1611651350000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":12,"title":["The Biological Characteristics of Novel H5N6 Highly Pathogenic Avian Influenza Virus and Its Pathogenesis in Ducks"],"prefix":"10.3389","volume":"12","author":[{"given":"Jianni","family":"Huang","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Siyu","family":"Wu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wenbo","family":"Wu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yiwen","family":"Liang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Haibin","family":"Zhuang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhiyu","family":"Ye","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaoyun","family":"Qu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ming","family":"Liao","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Peirong","family":"Jiao","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2021,1,26]]},"reference":[{"key":"ref1","doi-asserted-by":"publisher","first-page":"5913","DOI":"10.1073\/pnas.1001755107","article-title":"Association of RIG-I with innate immunity of ducks to influenza","volume":"107","author":"Barber","year":"2010","journal-title":"Proc. Natl. Acad. Sci. U. S. A."},{"key":"ref2","doi-asserted-by":"publisher","first-page":"1806","DOI":"10.3201\/eid2311.170672","article-title":"Airborne transmission of highly pathogenic influenza virus during processing of infected poultry","volume":"23","author":"Bertran","year":"2017","journal-title":"Emerg. Infect. Dis."},{"key":"ref3","doi-asserted-by":"publisher","first-page":"810","DOI":"10.1016\/j.chom.2016.10.022","article-title":"Genesis, evolution and prevalence of H5N6 avian influenza viruses in China","volume":"20","author":"Bi","year":"2016","journal-title":"Cell Host Microbe"},{"key":"ref4","doi-asserted-by":"publisher","first-page":"23","DOI":"10.1016\/j.virusres.2014.03.012","article-title":"H5N1 infection causes rapid mortality and high cytokine levels in chickens compared to ducks","volume":"185","author":"Burggraaf","year":"2014","journal-title":"Virus Res."},{"key":"ref5","doi-asserted-by":"publisher","first-page":"10452","DOI":"10.1073\/pnas.0403212101","article-title":"The evolution of H5N1 influenza viruses in ducks in southern China","volume":"101","author":"Chen","year":"2004","journal-title":"Proc. Natl. Acad. Sci. U. S. A."},{"key":"ref6","doi-asserted-by":"publisher","first-page":"1626","DOI":"10.1086\/504723","article-title":"Distribution of amantadine-resistant H5N1 avian influenza variants in Asia","volume":"193","author":"Cheung","year":"2006","journal-title":"J. Infect. Dis."},{"key":"ref7","doi-asserted-by":"publisher","first-page":"101","DOI":"10.1016\/j.vetmic.2018.11.018","article-title":"Duck innate immune responses to high and low pathogenicity H5 avian influenza viruses","volume":"228","author":"Fleming-Canepa","year":"2019","journal-title":"Vet. Microbiol."},{"key":"ref8","doi-asserted-by":"publisher","first-page":"53","DOI":"10.1016\/j.virusres.2013.05.010","article-title":"Adaptive mutations in the H5N1 polymerase complex","volume":"178","author":"Gabriel","year":"2013","journal-title":"Virus Res."},{"key":"ref9","doi-asserted-by":"publisher","first-page":"324","DOI":"10.1006\/viro.1998.9508","article-title":"Influenza A virus lacking the NS1 gene replicates in interferon-deficient systems","volume":"252","author":"Garc\u00eda-Sastre","year":"1998","journal-title":"Virology"},{"key":"ref10","doi-asserted-by":"publisher","first-page":"2021","DOI":"10.3201\/eid1912.130340","article-title":"Novel variants of clade 2.3.4 highly pathogenic avian influenza A(H5N1) viruses, China","volume":"19","author":"Gu","year":"2013","journal-title":"Emerg. Infect. Dis."},{"key":"ref11","doi-asserted-by":"publisher","first-page":"4433","DOI":"10.1128\/AAC.00334-09","article-title":"In vitro generation of neuraminidase inhibitor resistance in A(H5N1) influenza viruses","volume":"53","author":"Hurt","year":"2009","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref500","doi-asserted-by":"publisher","first-page":"315","DOI":"10.1038\/nri3665","article-title":"Innate immunity to influenza virus infection","volume":"14","author":"Iwasaki","year":"2014","journal-title":"Nat. Rev. Immunol."},{"key":"ref12","doi-asserted-by":"publisher","first-page":"789","DOI":"10.3389\/fimmu.2018.00789","article-title":"H7N9 avian influenza virus is efficiently transmissible and induces an antibody response in chickens","volume":"9","author":"Jiao","year":"2018","journal-title":"Front. Immunol."},{"key":"ref13","doi-asserted-by":"publisher","first-page":"1146","DOI":"10.1128\/JVI.01698-07","article-title":"A single-amino-acid substitution in the NS1 protein changes the pathogenicity of H5N1 avian influenza viruses in mice","volume":"82","author":"Jiao","year":"2008","journal-title":"J. Virol."},{"key":"ref14","doi-asserted-by":"publisher","first-page":"393","DOI":"10.1089\/jir.2010.0069","article-title":"Highly pathogenic (H5N1) avian influenza induces an inflammatory T helper type 1 cytokine response in the chicken","volume":"31","author":"Karpala","year":"2011","journal-title":"J. Interf. Cytokine Res."},{"key":"ref15","doi-asserted-by":"publisher","first-page":"121","DOI":"10.1111\/j.1750-2659.2009.00084.x","article-title":"Ducks: the \u201cTrojan Horses\u201d of H5N1 influenza","volume":"3","author":"Kim","year":"2009","journal-title":"Influenza Other Respir. Viruses"},{"key":"ref16","doi-asserted-by":"publisher","first-page":"118","DOI":"10.1186\/s13567-014-0118-3","article-title":"Highly pathogenic avian influenza virus infection in chickens but not ducks is associated with elevated host immune and pro-inflammatory responses","volume":"45","author":"Kuchipudi","year":"2014","journal-title":"Vet. Res."},{"key":"ref17","doi-asserted-by":"publisher","first-page":"822","DOI":"10.3201\/eid2305.161905","article-title":"Reassortant clade 2.3.4.4 avian influenza A(H5N6) virus in a wild mandarin duck, South Korea, 2016","volume":"23","author":"Kwon","year":"2017","journal-title":"Emerg. Infect. Dis."},{"key":"ref18","doi-asserted-by":"publisher","first-page":"12058","DOI":"10.1128\/JVI.79.18.12058-12064.2005","article-title":"Molecular basis of replication of duck H5N1 influenza viruses in a mammalian mouse model","volume":"79","author":"Li","year":"2005","journal-title":"J. Virol."},{"key":"ref19","doi-asserted-by":"publisher","first-page":"e95539","DOI":"10.1371\/journal.pone.0095539","article-title":"A 20-amino-acid deletion in the neuraminidase stalk and a five-amino-acid deletion in the NS1 protein both contribute to the pathogenicity of H5N1 avian influenza viruses in mallard ducks","volume":"9","author":"Li","year":"2014","journal-title":"PLoS One"},{"key":"ref20","doi-asserted-by":"publisher","first-page":"e137989","DOI":"10.1371\/journal.pone.0137989","article-title":"A single amino acid in the m1 protein responsible for the different pathogenic potentials of H5N1 highly pathogenic avian influenza virus strains","volume":"10","author":"Nao","year":"2015","journal-title":"PLoS One"},{"key":"ref21","year":"2017"},{"key":"ref22","doi-asserted-by":"crossref","first-page":"337","DOI":"10.1002\/9781118924341.ch13","article-title":"Pathobiology of avian influenza in domestic ducks","volume-title":"Animal influenza.","author":"Pantin-Jackwood","year":"2016"},{"key":"ref23","doi-asserted-by":"publisher","first-page":"113","DOI":"10.20506\/rst.28.1.1869","article-title":"Pathogenesis and pathobiology of avian influenza virus infection in birds","volume":"28","author":"Pantin-Jackwood","year":"2009","journal-title":"Rev. Sci. Tech."},{"key":"ref1500","doi-asserted-by":"publisher","first-page":"673","DOI":"10.1186\/s13104-018-3779-6","article-title":"Effects of mutations in the effector domain of influenza A virus NS1 protein","volume":"11","author":"Pereira","year":"2018","journal-title":"BMC Res. Notes"},{"key":"ref25","doi-asserted-by":"publisher","first-page":"e1003059","DOI":"10.1371\/journal.ppat.1003059","article-title":"Species-specific inhibition of RIG-I ubiquitination and IFN induction by the influenza A virus NS1 protein","volume":"8","author":"Rajsbaum","year":"2012","journal-title":"PLoS Pathog."},{"key":"ref26","doi-asserted-by":"publisher","first-page":"464","DOI":"10.1099\/jgv.0.001015","article-title":"IFN and cytokine responses in ducks to genetically similar H5N1 influenza A viruses of varying pathogenicity","volume":"99","author":"Saito","year":"2018","journal-title":"J. Gen. Virol."},{"key":"ref27","doi-asserted-by":"publisher","first-page":"2180","DOI":"10.1128\/JVI.01975-10","article-title":"The PA protein directly contributes to the virulence of H5N1 avian influenza viruses in domestic ducks","volume":"85","author":"Song","year":"2011","journal-title":"J. Virol."},{"key":"ref28","doi-asserted-by":"publisher","first-page":"200","DOI":"10.1016\/j.vetmic.2018.04.023","article-title":"Genetic analysis and biological characteristics of different internal gene origin H5N6 reassortment avian influenza virus in China in 2016","volume":"219","author":"Sun","year":"2018","journal-title":"Vet. Microbiol."},{"key":"ref29","doi-asserted-by":"publisher","first-page":"116","DOI":"10.1016\/j.vetmic.2015.11.001","article-title":"Characterization of clade 2.3.4.4 highly pathogenic H5 avian influenza viruses in ducks and chickens","volume":"182","author":"Sun","year":"2016","journal-title":"Vet. Microbiol."},{"key":"ref30","doi-asserted-by":"publisher","first-page":"463","DOI":"10.20506\/rst.19.2.1230","article-title":"Highly pathogenic avian influenza","volume":"19","author":"Swayne","year":"2000","journal-title":"Rev. Sci. Tech."},{"key":"ref31","doi-asserted-by":"publisher","first-page":"1834","DOI":"10.1128\/JVI.01648-10","article-title":"NP body domain and PB2 contribute to increased virulence of H5N1 highly pathogenic avian influenza viruses in chickens","volume":"85","author":"Tada","year":"2011","journal-title":"J. Virol."},{"key":"ref32","doi-asserted-by":"publisher","first-page":"8","DOI":"10.1016\/j.virol.2017.08.035","article-title":"Five distinct reassortants of H5N6 highly pathogenic avian influenza A viruses affected Japan during the winter of 2016\u20132017","volume":"512","author":"Takemae","year":"2017","journal-title":"Virology"},{"key":"ref33","doi-asserted-by":"publisher","first-page":"297","DOI":"10.3109\/01480548109018136","article-title":"A computer program for estimating LD50 and its confidence limits using modified Behrens-Reed-Muench cumulant method","volume":"4","author":"Thakur","year":"1981","journal-title":"Drug Chem. Toxicol."},{"key":"ref34","doi-asserted-by":"publisher","first-page":"8112","DOI":"10.1128\/JVI.00006-07","article-title":"The H5N1 influenza virus NS genes selected after 1998 enhance virus replication in mammalian cells","volume":"81","author":"Twu","year":"2007","journal-title":"J. Virol."},{"key":"ref35","doi-asserted-by":"publisher","first-page":"1227","DOI":"10.1111\/tbed.13141","article-title":"Comparative pathogenicity of H5N6 subtype highly pathogenic avian influenza viruses in chicken, Pekin duck and Muscovy duck","volume":"66","author":"Uchida","year":"2019","journal-title":"Transbound. Emerg. Dis."},{"key":"ref36","first-page":"365","article-title":"Cytokine storm in avian influenza","volume":"42","author":"Us","year":"2008","journal-title":"Mikrobiyol. Bul."},{"key":"ref37","doi-asserted-by":"publisher","first-page":"223","DOI":"10.4149\/av_2014_03_223","article-title":"Different outcomes of infection of chickens and ducks with a duck-origin H9N2 influenza A virus","volume":"58","author":"Wang","year":"2014","journal-title":"Acta Virol."},{"key":"ref38","doi-asserted-by":"publisher","first-page":"66","DOI":"10.1186\/1297-9716-45-66","article-title":"Duck MDA5 functions in innate immunity against H5N1 highly pathogenic avian influenza virus infections","volume":"45","author":"Wei","year":"2014","journal-title":"Vet. Res."},{"key":"ref39","doi-asserted-by":"publisher","first-page":"386","DOI":"10.1016\/j.vetmic.2013.06.019","article-title":"Host immune responses of ducks infected with H5N1 highly pathogenic avian influenza viruses of different pathogenicities","volume":"166","author":"Wei","year":"2013","journal-title":"Vet. Microbiol."},{"key":"ref40","doi-asserted-by":"publisher","first-page":"511","DOI":"10.3201\/eid2103.141488","article-title":"Reassortant highly pathogenic influenza A(H5N6) virus in Laos","volume":"21","author":"Wong","year":"2015","journal-title":"Emerg. Infect. Dis."},{"key":"ref41","doi-asserted-by":"publisher","first-page":"e2009","DOI":"10.1002\/rmv.2099","article-title":"Pandemic potential of highly pathogenic avian influenza clade 2.3.4.4 A(H5) viruses","volume":"30","author":"Yamaji","year":"2020","journal-title":"Rev. Med. Virol."},{"key":"ref42","doi-asserted-by":"publisher","first-page":"600","DOI":"10.1186\/s12879-016-1932-1","article-title":"The complexity of human infected AIV H5N6 isolated from China","volume":"16","author":"Zhang","year":"2016","journal-title":"BMC Infect. Dis."},{"key":"ref43","doi-asserted-by":"publisher","first-page":"e6277","DOI":"10.1371\/journal.pone.0006277","article-title":"The special neuraminidase stalk-motif responsible for increased virulence and pathogenesis of H5N1 influenza A virus","volume":"4","author":"Zhou","year":"2009","journal-title":"PLoS One"}],"container-title":["Frontiers in Microbiology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fmicb.2021.628545\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2021,1,26]],"date-time":"2021-01-26T08:55:54Z","timestamp":1611651354000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fmicb.2021.628545\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,1,26]]},"references-count":44,"alternative-id":["10.3389\/fmicb.2021.628545"],"URL":"https:\/\/doi.org\/10.3389\/fmicb.2021.628545","relation":{},"ISSN":["1664-302X"],"issn-type":[{"value":"1664-302X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,1,26]]},"article-number":"628545"}}