Surveillance And Evolution Analysis Of Avian Influenza Virus In Wild Birds In Khanka Lake,Heilnongjiang,2013-2018

Wild birds are natural hosts of avian influenza virus(AIV),and almost all subtypes of avian influenza virus(H1-H16,N1-N9)can be detected in wild birds.Migratory birds,especially migratory waterfowls,can carry and spread AIVs across countries and even the mainland during migration,and they can obtain AIV genes from poultry on the migration routes.This accelerates the spread and rearrangement of AIV and enriches the genetic diversity of AIV.Epidemiological surveillance on wild bird AIV in Khanka Lake can in-depth the understanding of the "abundance" of AIV gene bank in migratory birds,and it can explore the rules of AIV transmission in migratory birds.It is of great significance for the prevention and control of AIV.Molecular epidemiological surveillance on AIV in wild birds was carried out in Khanka Lake from September 2013 to October 2018.On one hand,the distribution of AIV subtypes in wild birds in Khanka Lake was investigated.On the other hand,the genetic evolution of avian influenza isolates was analyzed.From September 2013 to October 2018,3767 wild waterfowl samples were collected in Khanka Lake during the spring and autumn wild birds migration,including 3309 fresh feces samples,438 oropharyngeal/cloacal swab samples and 20 tissue samples.AIV was isolated from the SPF chicken embryo,allantoic fluid was detected by the hemagglutination test and finally identified by RT-PCR test of AIV.The results showed that 29 samples were positive for AIV and the isolation rate of AIV was 0.77%.1 H3N3,1 H3N5,1 H3N8,2 H4N6,1 H5N2,11 H5N6,1 H5N8,1 H6N6,1 H10N7 and 9 H16N3 were identified.Virus isolation results show that the isolation rate is higher in autumn than spring;and the isolation rate is higher in oropharyngeal/cloacal swabs.The HA cleavage site analysis of 23 isolates showed that 11 H5N6 isolates and 1 H5N8 isolate accorded with the typical molecular characteristics of highly pathogenic AIV.In addition,a G228S amino acid mutation in HA protein of 3 H16N3 isolates,which enhanced the ability of the virus to bind mammal a-2,6 receptors,and suggested that they had the potential to infect human beings.The comparative analysis of the NA key amino acid sites showed that 11 H5N6 isolates had the neck amino acid deletion,which enhanced the pathogenicity to mice.The genetic evolution of the external genes showed that there were some gene rearrangements among different subtypes of AIVs in wild waterfowl in Khanka Lake(H3,N6).The results of genetic evolution of internal genes showed that the internal genes of AIV in wild waterfowls in Heilongjiang region had diverse origin.There is extensive and frequent AIV gene exchange among Asia,Europe and even North America.Most of the gene fragments in the study were closely related to those of viruses isolated from Mongolia,Korea and Japan,which indicated the existence of frequent gene exchange and flow of AIV among these countries.BG/LX-97/14(H3N3)isolate(no NA gene sequence),was a multi-source recombinant strain,its PB2 gene derived from Japan/Korea wild waterfowl-origin strains;the PB1,PA1 and H3 genes was derived from Japanese/Korean avian-origin strains;the NP,M and NS genes were derived from Alaska wild waterfowl-origin strains.It was speculated that wild waterfowl could carry AIVs from North America to Eurasia.The transcontinental transmission of AIVs was also found in the N8 gene of BG/JHM-133/15(H3N8)and the PB2 genes of 3 H16N3 isolates.Alaska and Khanka Lake are probably important sites for the transmission and the gene spread of AIV between North America and Eurasia continents.The results of genetic evolution analysis and phylogenetic analysis showed that the whole genome of H5N6 subtype AIV detected in Khanka Lake had a very close genetic relationship with the H5N6 isolates in South Korea and Japan from 2016 to 2017,which indicated that they had the same evolutionary source.The H5N6 subtype AIVs were origined from Khanka Lake in the autumn of 2016,then they were spread to South Korea and Japan by migratory waterfowl.In addition,H5N6 subtype AIVs were also detected in poultry in Vietnam during the winter of 2017-2018,and some of the strains were originated from Khanka Lake.