Genetic analysis and biological characteristics of novel clade 2.3.4.4 reassortment H5N6 avian influenza viruses from poultry in eastern China in 2016

•The isolation rate of AIVs from swab samples was 11.03%•H5N6 circulated continuously and exhibited slight-to-moderate virulence in mice•Multiple mutations potentially increase infectivity, transmissibility, and affinity The continuous evolution of highly pathogenic H5N6 avian influenza viruses (AIVs) causes outbreaks in wildfowl and poultry, and occasional human infections. The aim of this study was to better understand the genetic relationships between these H5N6 AIVs from eastern China and other AIVs. In 2016, 1623 cloacal swabs were sampled from poultry in 18 LPMs in eastern China, and subsequently characterized systematically using gene sequencing, phylogenetic studies, and antigenic analysis. In addition, their pathogenicity in mammals was studied in BALB/c mice, which were inoculated with viruses, with survival rate and body weight recorded daily for 14 days. In total, 56 H5N6 AIVs were isolated in eastern China and five representative isolates were selected for further study. In our study, the H5N6 AIVs clustered into clade 2.3.4.4, Group C, and their six internal segments were derived from H6N6 and H9N2 viruses, or both, suggesting extensive reassortant among H5N6 AIVs with other subtypes. These H5N6 viruses could replicate in the lungs without prior adaptation, and exhibited slight-to-moderate virulence in mice. The continuous circulation of these novel H5N6 viruses suggests the importance of persistent surveillance of H5N6 AIVs in poultry.