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Genetic and antigenic evolution of H9N2 subtype avian influenza virus in domestic chickens in southwestern China, 2013–2016

<div><p>H9N2 avian influenza virus (AIV) has caused significant losses in chicken flocks throughout china in recent years. There is a limited understanding of the genetic and antigenic characteristics of the H9N2 virus isolated in chickens in southwestern China. In this study a total of 12 field strains were isolated from tissue samples from diseased chickens between 2013 and 2016. Phylogenetic analysis of the Hemagglutinin (HA) and Neuraminidase (NA) nucleotide sequences from the 12 field isolates and other reference strains showed that most of the isolates in the past four years could be clustered into a major branch (HA-branch A and NA-branch I) in the Clade h9.4.2 lineages. These sequences are accompanied by nine and seven new amino acids mutations in the HA and NA proteins, respectively, when compared with those previous to 2013. In addition, four new isolates were grouped into a minor branch (HA-branch B) in the Clade h9.4.2 lineages and two potential N-glycosylation sites were observed due to amino acid mutations in the HA protein. Three antigenic groups (1–3), which had low antigenic relatedness with two commonly used vaccines in China, were identified among the 12 isolates by antigenMap analysis. Immunoprotection testing showed that those two vaccines could efficiently prevent the shedding of branch A viruses but not branch B viruses. In conclusion, these results indicate the genotype of branch B may become epidemic in the next few years and that a new vaccine should be developed for the prevention of H9N2 AIV.</p></div

Secondary structure of the HA protein with mutations.

<p>The reference strain was A/chicken/Sichuan/LFY/2016. Mutations were shown as spheres representing the amino acid location. The mutations from branch A and B were shown as blue spheres and those from branch B were shown as red spheres.</p

Reciprocal HI titer of 12 H9N2 field isolates.

<p>Reciprocal HI titer of 12 H9N2 field isolates.</p

The clinical signs and virus re-isolation from chickens at different times post challenge.

<p>The clinical signs and virus re-isolation from chickens at different times post challenge.</p

Information for the 12 H9N2 subtype AIVs in this study.

<p>Information for the 12 H9N2 subtype AIVs in this study.</p

Antigen map of the 12 H9N2 AIV field isolates.

<p>Antigenic cartography representations of the HI data generated by using a panel of chicken antisera. One unit (grid) represents a 2-fold change in the HI assay results. Viruses in the same HI group were encircled in an oval. Virus designations were abbreviated as follows: YNKM/15 = A/chicken/Yunnan/YNKM/2015, SZQ60/15 = A/chicken/Sichuan/SZQ60/2015, YX/14 = A/chicken/Sichuan/YX/2014, XJ/14 = A/chicken/Sichuan/XJ/2014, CQ/16 = A/chicken/Chongqing/CQ/2016, LP/15 = A/chicken/Chongqing/LP/2015, A615/15 = A/chicken/Yunnan/A615/2015, LFY/16 = A/chicken/Sichuan/LFY/2016, QZ/15 = A/chicken/Guizhou/QZ/2015, ZYL/16 = A/chicken/Sichuan/ZYL/2016, LMC/16 = A/chicken/Sichuan/LMC/2016 and LB/13 = A/chicken/Sichuan/LB/2013.</p