Novel reassortment 2.3.4.4b H5N8 highly pathogenic avian influenza viruses circulating in Xinjiang, China

In 2016, H5N8 avian influenza viruses of clade 2.3.4.4b were detected at Qinghai Lake, China. Afterwards, the viruses of this clade rapidly spread to Asia, Europe, and Africa via migratory birds, and caused massive deaths in poultry and wild birds globally. In this study, four H5N8 isolates (abbreviated as 001, 002, 003, and 004) were isolated from the live poultry market in Xinjiang in 2017. Phylogenetic analysis showed that the hemagglutinin genes of the four isolates belonged to clade 2.3.4.4b, while the viral gene segments were from multiple geographic origins. For 002, the polymerase acidic gene had the highest sequence homology (99.55 %) with H5N8 virus identified from green-winged teal in Egypt in 2016, and the remaining genes exhibited the highest sequence homologies (99.18-100 %) with those of H5N8 viruses isolated from domestic duck sampled in Siberia in 2016. The polymerase basic 1 gene clustered together with H5N8 virus identified from painted stork of India in 2016, and the remaining genes had relatively close genetic relationships with H5N8 viruses identified from the duck of Siberia in 2016 and turkey in Italy in 2017. For the other three isolates, the nucleoprotein gene of 001 had the highest sequence homology (98.82 %) and relatively close genetic relationship with H9N2 viruses identified from poultry in Vietnam and Cambodia in 2015-2017, and all the remaining genes had the highest sequence homologies (99.18 %-99.58 %) and relatively close genetic relationships with H5N8 viruses identified from poultry and waterfowl sampled in African countries in 2017 and swan sampled in China in 2016. Multiple basic amino acids were observed at cleavage sites in the hemagglutinin proteins of the H5N8 isolates, indicating high pathogenicity. In addition, the L89V, G309D, R477G, I495V, A676T and I504V mutations in the polymerase basic 2 protein, N30D and T215A mutations in the matrix 1 protein, P42S mutation, and 80-84 amino acid deletion in the nonstructural 1 protein were detected in all isolates. These mutations were associated with increased virulence and polymerase activity in mammals. Therefore, our results indicate that the H5N8 isolates involved multiple introductions of reassorted viruses, and also revealed that the wetlands of Northern Tianshan Mountain may play a key role in H5N8 AIVs disseminating among Central China, the Eurasian continent, and East African Countries.