Surveillance And Evolution Analysis Of Avian Influenza Virus In Poyang Lake Region,2016-2017

Objectives:To study the epidemic situation of avian influenza viruses(AIVs)in Poyang Lake region during 2016-2017,evaluate effects of migratory birds' activities to the epidemic situation and ecology of local AIVs,analyze the genetic diversity and molecular epidemiological characteristics of the AIVs in Poyang Lake region,and provide the data base for new AIVs' prediction and risk assessment.Method:Three stages of sampling were conducted in this study,poultry-related environmental samples were collected before and after the wild birds migrating to Poyang Lake,and the wild birds-related environmental samples were collected when the migratory birds arrived in Poyang Lake.Types of poultry-related environmental specimens included fecal swabs,cages surface wiping swabs,poultry drinking water,shedding feather,weeds wiping swabs,fresh eggs wiping swabs,and chopping board wiping swabs in live poultry markets(LPMs).The types of wild birds-related environmental samples included fecal swabs,drinking water and shedding feather samples.All samples were inoculated into 9-11 days SPF chicken embryos for AIVs isolation.After detected with influenza A virus test strip,collect allantoic fluid which shows positive results.Complete genome of AIVs obtained by next generation sequencing(NGS)and analyzed by MEGA6.06 combined with GISAID and Influenza Research Database.Results:(1)12 subtypes of AIVs were isolated during the study period.In November 2016(the first sampling)and May 2017(the third sampling),1336 and 1335 poultry-related environmental samples were collected respectively in Poyang Lake region.In total 227 AIVs were isolated,1 strain from poultry farm and 226 from LPMs,including subtype H1N3,H3N1,H3N2,H3N6,H3N8,H4N2,H4N6,H5N6,H6N6,H9N2 and H9N9.In December 2016(the second sampling),1995 environmental samples collected in wild bird habitats.One H6N1 subtype AIVs was isolated.All of them were low pathogenic avian influenza viruses(LPAIVs)except H5 subtypes were highly pathogenic avian influenza viruses(HPAIVs).(2)The positive rate of virus isolation was 9.35%in the first sampling(pre-bird migration),and 7 subtypes were identified.The number of each subtype was H9N2(32),H5N6(31),H6N6(23),H3N2(8),H3N8(8),H4N2(1),H4N6(1).The positive rate of virus isolation was 7.64%in the third sampling(after bird migration).Ten subtypes were detected,the number of each subtype was H9N2(39),H6N6(19),H3N2(14),H3N8(7),H9N9(4),H5N6(2),H4N6(2),H3N1(2),H1N3(2),H3N6(1).There was no significant difference in the positive rate of virus isolation between the two samplings.No new mutations related to increased mammalian virulence were found in the whole genome sequence analysis.(3)Two H3N1 strains was first identified in Poyang Lake region,and this was the second time isolated in China since 2003.The NA segment clustered with H1N1 isolated from wild birds in Korea,China and other countries belong to Eurasian lineage and far from NA of HPA1 H5N1.(4)Evolutionary analysis of H6N1 subtype AIVs isolated from wild birds showed that HA fragment belonged to North-American lineage,NA fragment belonged to the Eurasian lineage and clustered with N1 subtypes AIVs isolated from local poultry,suggesting frequent gene reassortant among AIVs circulating in wild birds and local poultry.(5)The third sampling was conducted in the fourth week after recover of LPM following 3 months' closure.The positive rate of virus isolation in the first and third live poultry market sampling was compared,and results showed that the positive rate in Wannian county was significantly lower than that of the first sampling,while the other districts and counties had reached the level before the closure of markets.Conclusions:(1)Several subtypes of avian influenza virus continue to circulate in Poyang Lake region based on multi-batch samplings,and H3N1 subtype was isolated for the first time in Poyang Lake region.Vast majority of AIVs were detected in LPMs and possess relatively higher risk for human infectetions;AIVs positive rate in environmental samples collected from backyard/poultry farm and wild bird habitats were quite low,with relatively lower risk of human infection caused by exposure to poultry related environment.(2)Although sequence analysis did't find any new mutations relate to increased virulence in mammals,evolutionary analysis suggested that AIVs continued to reassortant in poultry and wild birds,the major subtypes such as H9N2 and H5N6 were in the same phylogenetic clade with viruses isolated from human cases,and H3N1 subtype AIVs was first identified in this area,suggesting the importance of continuing research on avian influenza in the region.(3)The results of sampling before and after migration indicated that the positive rate of avian influenza virus in live poultry market did not change significantly,but the proportion of subtypes of avian influenza virus changed and the number of subtypes increased.Futher studies are needed to scientifically assess the impact of migratory bird movements to ecology of local avian influenza virus.