Isolation, identification and phylogenetic analysis of six strains of H5N6 highly pathogenic avian influenza virus / 中华微生物学和免疫学杂志

ABSTRACT

Objective:To analyze phylogenetic structure and molecular characteristics of H5N6 avian influenza virus (AIVs) isolated from live poultry market (LPM).Methods:Oropharyngeal and cloacal swabs from poultry, and environmental samples were collected from LPM in Urumqi in December 2018, AIVs were isolated and identified by inoculation of chicken embryo, hemagglutination test and RT-PCR, the viral whole genome was amplified with the universal primers of influenza A virus, and then sequenced, pairwise sequence alignments, phylogenetic and molecular characteristics analysis were performed by BLAST, Clustal W, MEGA-X and DNAStar software.Results:Six strains of H5N6 AIVs were isolated from poultry samples, the identity between the viral genes was high (99.4%-100.0%), so the isolates were the same source. BLAST analysis revealed that the viral NP sequence had the highest identity (99.7%) with H5N6 AIVs isolated from poultry in Suzhou, while the sequence of the remaining 7 viral genes had the highest identity (99.0%-100.0%) with H5N6 AIVs isolated from environment in Guangdong during 2017 to 2018. Phylogenetic analysis showed that the viral HA belonged to Clade 2.3.4.4C, and the viral HA, NA, PB1, PA, NP, and MP were all clustered together with H5N6 AIVs isolated from mink in Eastern China in 2018, while the PB2 and NS were clustered together with H5N6 AIVs isolated from environment in Guangdong from 2017 to 2018. The HA cleavage site contained multiple basic amino acid residues, which was highly pathogenic AIVs (HPAIVs). S137A and T160A mutations of HA could increase binding to human-type receptor SAα2, 6-Gal. Additionally, the viral multiple mutations, including 59-69 deletion in NA, the L89V, G309D, R477G, I495V, I504V, D391E, and A661E in PB2, as well as the P42S, D92E, and 80-84 deletion in NS1, could enhance the viral virulence and pathogenicity to mammals. Conclusions:The 6 strains of H5N6 HPAIVs isolated from LPM have relatively close genetic relationship with H5N6 AIVs isolated from mink in Eastern China and environment in Guangdong during 2017 to 2018, the viral multiple mutations could increase its pathogenicity to mammals, which could pose a potential risk to public health.