Surveillance And Phylogenetic Of Avian Influenza Virus H10-H12 Subtypes Isolated From Wild Birds In Shanghai,China From 2016 To 2019

Wild birds are natural hosts of avian influenza virus(AIVs).Because of their migration habits,they carry the virus for transmission,play the role of "active carriers" for the preservation,transmission and variation of AIVs.At present,16 HA and 9 NA subtypes of AIVs have been isolated from wild birds,and these HA subtypes in wild birds have different degrees of isolation rate and host restriction.H1-H12 subtypes are most frequently detected in Anseriformes,of which H3,H4 and H6 subtypes are often detected,while H10-H12 subtypes have a lower detection rate,and even many of their HA-NA subtype combinations have not been detected so far.At present,there is little research on the H10-H12 subtypes isolated from wild birds,and the viral ecology and molecular genetic evolution of these subtypes are inadequate understood.Therefore,in order to better assess the prevalence patterns and elucidate genetic variation condition of H10-H12 subtypes in wild birds,and to further understand the role of wild birds in the global spread of these subtypes,it is necessary to increase the surveillance of H10-H12 subtypes in wild birds.Shanghai is an important stopover and wintering place on the East Asian-Australasian Flyway,where millions of migratory birds pass through or winterize every year.In recent years,with the development of economy and wetland restoration projects being strengthened,the suitable habitat in Shanghai attracts wild birds.In view of the importance of H10-H12 subtypes to the ecology of AIVs,we monitored AIVs in wild birds in Shanghai from 2016 to 2019.In this study,the epidemiology,genome sequence and molecular evolution analysis of H10-H12 subtypes were studied.The main results are as follows:(1)The most common subtypes were H4N2 and H6N2 accounting for about 40% of the total positive samples.The H10-H12 subtypes were infrequently detected,accounting for about 2.8%.By statistical analysis of hosts and source areas of H10-H12 subtypes in the influenza virus database,we found that these subtypes were mainly detected in North America,Europe and Asia(South Korea,Japan and China)and more than 80% of the isolates were from wild birds.(2)Sequence analysis of 12 H10-H12 subtypes with complete genomes were analyzed,the 12 isolates shared a low degree of sequence identity.In the phylogenetic tree,some internal gene segments of 4 H12N2 strains belonged to North American lineage,all the other genes belonged to the Eurasian lineage and shared a high degree of sequence identity and relatedness with those from domestic ducks and wild birds in countries in the East Asian-Australasian Flyway,including Russia,Mongolia,Japan,Korea and China.It has been shown that wild bird migration plays an important role in the transmission of AIVs and promotes gene exchange between Eurasian and North American.(3)Analysis of key amino acid sites showed that 12 H10-H12 subtypes were characterized as low-pathogenic AIVs and would preferentially bind to avian receptors.Some key amino acid residues,such as amantadine resistance and increased pathogenicity of mammals found no mutations.(4)Reassortment analysis suggested that the internal genes segments of these isolates were recombined to varying degrees.Among the 12 H10-H12 subtypes,except for 2H11 subtypes,the internal genes of all the other subtypes had partial or completely different phylogenetic positions on the evolutionary tree.4 H12N2 subtypes showed a higher degree of reassortment,which may have been occurred intercontinental gene exchange in the Eurasian and North American.In summary,the H10-H12 subtypes carried by wild birds in Shanghai have great genetic diversity and the genetic evolution of the complicated relationship.To better understand the viral ecological characteristics and assess the risk of arise in the area of these subtypes in wild birds,it is necessary to increase long-term surveillance of these subtypes in wild birds.