References:
Alarcon, P., Brouwer, A., Venkatesh, D., Duncan, D., Dovas, C. I., Georgiades, G.,… Brown, I. H. (2018). Comparison of 2016–17 and Previous Epizootics of Highly Pathogenic Avian Influenza H5 Guangdong Lineage in Europe. Emerging Infectious Diseases, 24(12),2270-2283.doi:10.3201/eid2412.171860
Arai, Y., Ibrahim, M. S., Elgendy, E. M., Daidoji, T., Ono, T., Suzuki, Y.,… Watanabe, Y. (2019). Genetic Compatibility of Reassortants between Avian H5N1 and H9N2 Influenza Viruses with Higher Pathogenicity in Mammals.Journal of Virology, 93(4).doi:10.1128/JVI.01969-18
Beato, M. S., Mancin, M., Yang, J., Buratin, A., Ruffa, M., Maniero, S.,… Capua, I. (2013). Antigenic characterization of recent H5N1 highly pathogenic avian influenza viruses circulating in Egyptian poultry.Virology, 435(2),350-356.doi:10.1016/j.virol.2012.09.016
Bi, Y., Chen, J., Zhang, Z., Li, M., Cai, T., Sharshov, K.,… Gao, G. F. (2016). Highly pathogenic avian influenza H5N1 Clade 2.3.2.1c virus in migratory birds, 2014-2015. Virologica Sinica, 31(4),300-305.doi:10.1007/s12250-016-3750-4
Bi, Y., Chen, J., Zhang, Z., Li, M., Cai, T., Sharshov, K.,… Gao, G. F. (2016). Highly pathogenic avian influenza H5N1 Clade 2.3.2.1c virus in migratory birds, 2014–2015. Virologica Sinica, 31(4),300-305.doi:10.1007/s12250-016-3750-4
Bi, Y., Chen, Q., Wang, Q., Chen, J., Jin, T., Wong, G.,… Gao, G. F. (2016). Genesis, Evolution and Prevalence of H5N6 Avian Influenza Viruses in China. Cell Host & Microbe, 20(6),810-821.doi:10.1016/j.chom.2016.10.022
Dong Zhang, F. G. I. J., & Wang, W. X. L. G. (2019). PhyloSuite: An integrated and scalable desktop platform for streamlined molecular sequence data management and evolutionary phylogenetics studies.doi:10.1111/1755
Duan, L., Bahl, J., Smith, G. J. D., Wang, J., Vijaykrishna, D., Zhang, L. J.,… Guan, Y. (2008). The development and genetic diversity of H5N1 influenza virus in China, 1996–2006. Virology, 380(2),243-254.doi:10.1016/j.virol.2008.07.038
Fouchier, R. A. M., Munster, V., Wallensten, A., Bestebroer, T. M., Herfst, S., Smith, D.,… Infektionssjukdomar. (2005). Characterization of a novel influenza A virus hemagglutinin subtype (H16) obtained from black-headed gulls. Journal of Virology, 79(5),2814.doi:10.1128/JVI.79.5.2814
Gao, G. F. (2014). Influenza and the Live Poultry Trade. Science,344(6181), 235.doi:10.1126/science.1254664
Group, W. O. F. H. (2012). Continued evolution of highly pathogenic avian influenza A (H5N1): updated nomenclature. Influenza and Other Respiratory Viruses, 6(1),1-5.doi:10.1111/j.1750-2659.2011.00298.x
Jin, X., Zha, Y., Hu, J., Li, X., Chen, J., Xie, S.,… Jia, W. (2020). New molecular evolutionary characteristics of H9N2 avian influenza virus in Guangdong Province, China. Infection, Genetics and Evolution,77, 104064.doi:10.1016/j.meegid.2019.104064
Jombart, T., Devillard, S., & Balloux, F. (2010). Discriminant analysis of principal components: a new method for the analysis of genetically structured populations. Bmc Genetics, 11,94.doi:10.1186/1471-2156-11-94
Kalyaanamoorthy, S., Minh, B. Q., Wong, T. K. F., von Haeseler, A., & Jermiin, L. S. (2017). ModelFinder: fast model selection for accurate phylogenetic estimates. Nature Methods, 14(6),587-589.doi:10.1038/nmeth.4285
Karo-karo, D., Bodewes, R., Wibawa, H., Artika, M., Pribadi, E. S., Diyantoro, D.,… Koch, G. (2019). Reassortments among Avian Influenza A(H5N1) Viruses Circulating in Indonesia, 2015–2016. Emerging Infectious Diseases, 25(3), 465-472.doi:10.3201/eid2503.180167
Katoh, K., Misawa, K., Kuma, K., & Miyata, T. (2002). MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform.Nucleic Acids Research, 30(14),3059-3066.doi:10.1093/nar/gkf436
Kim, Y., Biswas, P. K., Giasuddin, M., Hasan, M., Mahmud, R., Chang, Y. M.,… Fournie, G. (2018). Prevalence of Avian Influenza A(H5) and A(H9) Viruses in Live Bird Markets, Bangladesh. Emerging Infectious Diseases, 24(12), 2309-2316.doi:10.3201/eid2412.180879
Lee, D. H., Bertran, K., Kwon, J. H., & Swayne, D. E. (2017). Evolution, global spread, and pathogenicity of highly pathogenic avian influenza H5Nx clade 2.3.4.4. Journal of Veterinary Science,18(S1), 269-280.doi:10.4142/jvs.2017.18.S1.269
Li, X., Chen, J., Jin, X., Hu, J., Xie, S., Dai, Y.,… Jia, W. (2019). Characterization of three H3N2 and one new reassortant H3N8 avian influenza virus in South China. Infection, Genetics and Evolution, 75, 104016.doi:10.1016/j.meegid.2019.104016
Nguyen, L., Schmidt, H. A., von Haeseler, A., & Minh, B. Q. (2015). IQ-TREE: A Fast and Effective Stochastic Algorithm for Estimating Maximum-Likelihood Phylogenies. Molecular Biology and Evolution,32(1), 268-274.doi:10.1093/molbev/msu300
Qi, W., Jia, W., Liu, D., Li, J., Bi, Y., Xie, S.,… Liao, M. (2018). Emergence and Adaptation of a Novel Highly Pathogenic H7N9 Influenza Virus in Birds and Humans from a 2013 Human-Infecting Low-Pathogenic Ancestor. Journal of Virology, 92(2
).doi:10.1128/JVI.00921-17
Rambaut, A., Drummond, A. J., Xie, D., Baele, G., & Suchard, M. A. (2018). Posterior Summarization in Bayesian Phylogenetics Using Tracer 1.7.Systematic Biology, 67(5),901-904.doi:10.1093/sysbio/syy032
Shi, J., Deng, G., Ma, S., Zeng, X., Yin, X., Li, M.,… Chen, H. (2018). Rapid Evolution of H7N9 Highly Pathogenic Viruses that Emerged in China in 2017. Cell Host & Microbe, 24(4),558-568.doi:10.1016/j.chom.2018.08.006
Shi, J., Deng, G., Ma, S., Zeng, X., Yin, X., Li, M.,… Chen, H. (2018). Rapid Evolution of H7N9 Highly Pathogenic Viruses that Emerged in China in 2017. Cell Host & Microbe, 24(4),558-568.doi:10.1016/j.chom.2018.08.006
Shortridge, K. F., Zhou, N. N., Guan, Y., Gao, P., Ito, T., Kawaoka, Y.,… Webster, R. G. (1998). Characterization of Avian H5N1 Influenza Viruses from Poultry in Hong Kong. Virology, 252(2),331-342.doi:https://doi.org/10.1006/viro.1998.9488
Suchard, M. A., Lemey, P., Baele, G., Ayres, D. L., Drummond, A. J., & Rambaut, A. (2018). Bayesian phylogenetic and phylodynamic data integration using BEAST 1.10. Virus Evol, 4(1), y16.doi:10.1093/ve/vey016
Sun, H., Pu, J., Hu, J., Liu, L., Xu, G., Gao, G. F.,… Liu, J. (2016). Characterization of clade 2.3.4.4 highly pathogenic H5 avian influenza viruses in ducks and chickens. Veterinary Microbiology,182, 116-122.doi:10.1016/j.vetmic.2015.11.001
Tong, S., Li, Y., Rivailler, P., Conrardy, C., Castillo, D. A. A., Chen, L. M.,… Donis, R. O. (2012). A distinct lineage of influenza A virus from bats. Proceedings of the National Academy of Sciences,109(11), 4269-4274.doi:10.1073/pnas.1116200109
Velkov, T., Ong, C., Baker, M. A., Kim, H., Li, J., Nation, R. L.,… Rockman, S. (2013). The antigenic architecture of the hemagglutinin of influenza H5N1 viruses. Molecular Immunology, 56(4),705-719.doi:10.1016/j.molimm.2013.07.010
Webster, R. G., & Govorkova, E. A. (2014). Continuing challenges in influenza.Annals of the New York Academy of Sciences, 1323(1),115-139.doi:10.1111/nyas.12462
Wille, M., & Holmes, E. C. (2019). The Ecology and Evolution of Influenza Viruses. Cold Spring Harbor Perspectives in Medicine ,a38489.doi:10.1101/cshperspect.a038489
Xu, X., Subbarao, Cox, N. J., & Guo, Y. (1999). Genetic characterization of the pathogenic influenza A/Goose/Guangdong/1/96 (H5N1) virus: similarity of its hemagglutinin gene to those of H5N1 viruses from the 1997 outbreaks in Hong Kong. Virology, 261(1),15-19.doi:10.1006/viro.1999.9820
Figure 1. Phylogenetic analysis of H5N6 Influenza Viruses.
  1. Phylogenetic tree of hemagglutinin genes of H5N6 viruses sequenced in this study; the clade origins of each gene segment are indicated by different colored bars.
  2. Phylogenetic tree of neuraminidase genes of H5N6 viruses sequenced in this study.
Figure 2. Reassortment of the H5N6 AIV.
The eight gene segments are PB2, PB1, PA, HA, NP, NA, M, and NS (horizontal bars starting from top to bottom of the virion). Different colors represent different virus lineages.
Figure 3. Scatterplots resulting from the DAPC.
Individual isolates from the same AVIs season are depicted as unique color shapes and surrounded by 95% inertia ellipses. The PCA and DA eigenvalues inset panels show the overall variability among individuals and the relative capture of variance for each discriminant function, respectively. The y- and x-axes indicate the first and second discriminant principal components, respectively, which best summarize the differences between clusters while neglecting within-cluster variation. (A) and (B) represent HA and NA genes, respectively.
Figure 4. SeqLogo analysis of amino acid substitutions in H5N6 viruses.
, (B) HA and NA (H5 numbering) are shown.(A) The first three sites (9, 12, 16) in HA signal peptide,The last site (169) in HA2,others in HA1.
Figure 5. Replication and virulence of the H5N6 viruses in mice.
Weight changes in mice after challenge, (B) Survival rate of mice after challenge. (C) The virus content in different organs in the mice on the third day of the challenge. (D) The virus content in various organs in mice on the fifth day of the challenge.
Figure 6. Genotype of H5N6 viruses in different hosts.
Schematic for the genetic Source of human H5N6 isolates. (B) Genotypes of H5N6 viruses isolated in humans, domestic ducks, domestic chickens, domestic geese, environment, and wild birds. The eight gene segments are PB2, PB1, PA, HA, NP, NA, M, and NS (horizontal bars starting from top to bottom of the virion). Different colors represent different virus lineages.