Biological Characteristics Of Wild Bird-derived Avian Influenza Virus In China In Recent Years

Wild birds are regarded as the natural hosts of avain influenza virus(AIV),and play an important role in the preservation,transmission and variation AIV.Influenza viruses isolated from poultry and mammals including humans are directly or indirectly derived from wild birds.In order to understand the prevalence and biological characteristics of AIV in wild birds in China,6356 fresh fecal samples were collected from the wild bird habitats in China from 2017 to 2019.25 different subtype of AIVs were isolated,the isolation rate was about 0.39%.Subtype identification showed there were five HA subtypes and four NA subtypes,forming seven different combinations,including three H3N8,three H6N1,three H6N2,one H6N8,twelve H9N2,one H13N8 and two H16N3.Among them,the two H16N3 were the first isolated in China.The key amino acid sites of HA cleavage motif indicated that 25 isolates in this study were low pathogenic,however,one H3N8 was found that it was bearing the molecular marker of PB2 E627 K mutation,which increased the pathogenicity of AIV to mammals.Phylogenic evolution of the HA genes of 15 isolates of H3N8,H6,H9N2(two of twelve),H13N8 and H16N3 were belonged to the Eurasian lineage,and 10 H9N2 isolates were divided into North American lineage;NA genes of the 23 isolates come from the Eurasian lineage,except one H6N8 and one H9N2 from North America.The internal genes,PB2 and PA were divided into eight groups.PB1,NP and M of the 25 strains were divided into seven groups,and the NS genes were divided into five groups.The 25 isolates in the study were divided in 18 genotypes,which indicated that there were multiple ressortment occurring.The pathogenicity of representatives of wild bird-derived H9N2 in poultry showed that they could not cross species barrier to infect SPF chickens,but two viruses induced seroconversion,which suggested that the H9N2 subtype AIV in wild birds in China was non-pathogenic to poultry.The pathogenicity of the isolates in mice showed that WB/TJ/2-300/2018(H3N8)could cross the blood-brain barrier and replicate in the brain of mice with low titer,but other nine isolates couldn't replicate in the brain of mice.Three H6 and three H9N2 could replicate in the turbinate and lung of mice.It showed that most wild bird-derived AIVs could infect mice without pre-adaptation,and possess the potential to cross the species-barrier to infect mammals and even human.Two H16N3 couldn't replicate effectively in mice,which indicated that they were low pathogenicity to mammals.Applying reverse genetics technology,we found that the naturally PB2 E627 K mutation of H3N8 subtype AIV increased the replication titer in lung of mice and enhanced the pathogenicity in mice,it indicated that E627 K of wild bird-derived H3N8 subtype AIV mutation under natural conditions could enhance its pathogenicity in mammals.In summary,the wild bird-derived AIV in this study displayed genetic diversity and multiple ressortant,although they cannot cross species barrier to infect poultry,some isolates can infect mice without pre-adaptation,and possessing the potential to human.Therefore,the continuous strengthening of AIV surveillance in wild birds will be helpful to better understand the prevalence and biological characteristics of AIV,also provide the early warning for avian influenza outbreak and important data for the scientific prevention and control of avian influenza.