Molecular Epidemiology And Pathogenicity Of H9N2 Subtype Avian Influenza Virus In Eastern China From 2016 To 2018

H9N2 subtype avian influenza virus(AIV)is low pathogenic and causes respiratory symptoms and drop in egg production in chicken with long-term virus shedding,resulting in great economic losses due to high mortality related to secondary infection with other pathogens.In recent years,H9N2 viruses pose a threat to public health,causing human infection in China.Compared to other subtypes AIVs,there are relatively fewer studies on the pathogenic mechanism of H9N2 virus in mammals.H9N2 subtype AIV has been circulating worldwide in many avian species and transmited with high efficiency between poultry.It can provide internal genes for other subtypes to produce new virus causing a pandemic risk.It is important to carry out a long-term surveillance and pathogenic characteristics for H9N2 virus.In this study,we executed the epidemiological surveillance of avian influenza virus in live poultry markets(LPMs)and poultry farms in Eastern China from October 2016 to September 2018.We performed the viral isolation and identification,and RT-PCR for the genes of H9N2 subtype AIV.Additionally,we analyzed the transmission characteristic and pathogenicity in mice for the representative H9N2 subtype AIV strains.1.Surveillance of H9N2 avian influenza virus;endemic in ChinaBase on the epidemiological investigation in LPMs during 2016-2018,we collected 2183 samples of cloacal/laryngotracheal swabs from poultry in LPMs in Eastern China.A total of 518 strains of AIV and NDV were identified by inoculation in SPF embryonated chicken eggs and HA-HI assay.Among the viruses,there are 252 strains of H9N2 virus with isolation rate 11.54%(252/2183).Additionally,156 H9N2 subtype AIV strains were isolated from clinical samples from commercial chicken flocks.The H9N2 virus has the highest isolation rate(48.65%,252/518)among viruses from LPMs,followed by the H5(21.81%,113/518),H3(14.1%,73/518),H7(4.83%,25/518)and so on.The isolation rate of H9N2 subtype AIV in LPMs has increased significantly since Oct.2016.H9N2 subtype AIV had occasionally been isolated in LPMs at a rate of 0.21%-0.78%during Jul.2011 to Sept.2016(the statistical period is about 12 months).However,the isolation rate increased and reached to 8.49%to 15.12%during Oct.2016 to Sept.2018.The virus has adapted the waterfowl and chicken.The geographic distribution of isolates are mainly in provinces of Jiangsu,Anhui,Shandong in Eastern China.H9N2 subtype AIV was common isolated in summer during 2016-2018,which often isolated in autumn and winter before 2016.It indicated that the virus became well thermal stability,and had no obvious seasonal epidemic characteristics.2.Genetic characterization of the H9N2 avian influenza viruses circulated in poultry in Eastern ChinaTo analyze the genetic evolution of H9N2 virus,selected 394 HA segments,302 NA segments and 69 sets of internal genes segments in this paper.And then,constructed the phylogenetic tree by the neighboring-joining(NJ)method based on the sequences of eight genes of 69 strains of H9N2 virus.The HA and NA gene both belong to Y280-Like lineage,and the internal PB2 and M gene both belong to G1-Like lineage.The other four internal genes all belong to F/98-Like lineage.The H9N2 isolates in this study all belong to the S genotype.It showed that 389 isolates have seven N-glycosylation sites in HA protein(HA1:11,123,280,287,295;HA2:154,213),and lost position 11 and increased position 295 of N-glycosylation site in HA.It showed that 294 H9N2 virus(294/302)have five N-glycosylation sites(69,86,146,200,234)in NA protein which are consistent with the strain WJ57.Among the isolates,11(11/302)strains added 44th and 14(14/302)strains increased the 402th potential N-glycosylation site in NA protein.The amino acid at position 226 in HA protein,which affects the receptor-binding properties of the virus,has mutated from Q to L.indication the ability of binding the receptor of mammals.It is reported that HA 381K and PA 672L are crucial molecular signatures of the airborne transmission of the H9N2 subtype AIVs.In this study,K381R mutation occurred in six strains(6/394),and no mutation occurred at the position 672 in PA protein.PA protein K356R,PB2 protein E627K are two important mutations that affect the pathogenicity of H9N2 subtype AIV in mammals.In this study,all isolates have 627E in PB2 protein and 356R in PA protein.3.Transmission characteristics and pathogenicity of H9N2 subtype avian influenza virusThe isolation rate of H9N2 virus in chicken flocks has increased significantly since 2016,and there have been some reports of human infections.At present,there is no systematic study on whether the transmission ability and pathogenicity of different genotype strains are different.Sixteen of H9N2 subtype AIV isolates in the past twelve years were selected for airborne transmission experiments,including three strains of O genotype(SD07,W4 and Dh0801D11),one strain of T genotype(HeN86),and twelve strains of S genotype.Among the 16 strains,twelve strains are consistent with amino acid of HA 381K and PA 672L,include W4,HeN86,Cl,HeN131,SQ68,WJ57,TM118,SDKD1,AH320,AH463,FJ1802 and 292HZ.All of S genotype H9N2 subtype AIV strains have airborne characteristics,even though GC510 and A6 strain had 381R in the HA protein.However,we only detected a seroconversion but fail to detect virus shedding in C1 group.Other strains of non-S genotype,which possess above molecular markers,have aerosol transmission characteristics except HeN86.Moreover,the airborne transmission ability of the S genotype H9N2 strains in the recent two years increased,the exposed group had earlier and more long-time virus shedding up to 9 days post infection and a higher seroconversion level.Two H9N2 subtype AIV strains of S genotype tested,AH320 and SDKD1,could transmit with airborne droplet from chicken to guinea pig,the latter shed virus up to 14 days post exposure.S genotype H9N2 subtype AIV tested were low pathogenic in mice,with lower weight growth rate between 17.06%-22.89%compared with PBS control group(29.45%).Moreover,the weight growth of the mice in SDKD1 group was the least.S genotype viruses had different pattern of tissue distribution and viral load in mice.They replicated mainly in lung of mice at 3 dpi and 5dpi with no virus detected at 7 dpi,and some strains can also be detected in the hearts,kidneys,livers and spleens.Based on histopathological examination of lung in mice at 3 dpi and 5 dpi,they caused different degree of lung lesion,characterized by blood congestion,hemorrhage,alveolar interstitial proliferation and extensive inflammatory cell infiltration.Therefore,it indicated that the S genotype viruses generally became transmissible via respiratory droplets between chickens,were sufficient for chicken-to-guinea pig transmission through respiratory droplet,only induced slight weight loss in infected mice without death,and replicated in more tissues of mice,suggesting a possible threat to public health.