Objectives: The continuous evolution of highly pathogenic H5N6 avian influenza viruses (AIVs) causes outbreaks in wildfowls and poultry and occasional human infections. To better understand the genetic relationships between these H5N6 AIVs from eastern China and other AIVs.

Methods: In 2016, a total of 1623 cloacal swabs were sampled from poultry in 18 LPMs in eastern China, and subsequently characterized systematically using gene sequencing, phylogenetic study, and antigenic analysis. And their pathogenicity in mammals were studied in BALB/c mice, which were inoculated with viruses and survival rate and body weight were recorded daily for 14 days.

Results: A total of 56 H5N6 AIVs were isolated in Eastern China and five representative isolates were selected for further study. In this 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 reassortment 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.

Conclusions: The continuous circulation of these novel H5N6 viruses suggests the importance of persistent surveillance of the H5N6 AIVs in poultry.