Epidemiological Analysis And Biological Characterization Of H3 Subtype Avian Influenza Virus In Eastern China From 2017 To 2019

In influenza A viruses,the H3 subtype has a wide range of hosts,infecting not only poultry but also pigs,horses,dogs and other mammals and even humans.Therefore,H3 virus poses an important threat to livestock production and public health,which deserves continuous epidemiological surveillance.In this study,based on the epidemiological investigation of low pathogenicity avian influenza virus(AIV)in live poultry market(LPM)in Eastern China from 2017 to 2019.we conducted genomic evolutionary analysis and HA gene codon-usage-bias analysis of the isolated H3 subtype AIV.Additionally,some biological characteristics of the representative H3 strains were also determined.The results will enrich the basic data resources of low pathogenic AIV in China and provide certain reference for the mechanism study of AIV interspecies transmission.1.Epidemiological surveillance of low pathogenic avian influenza virus in Eastern China during 2017-2019 plus isolation and identification of H3 subtype avian influenza virusFrom September 2017 to June 2019.epidemiological surveillance of low pathogenic AIV was carried out in LPM in Eastern China.A total of 1866 samples including cloacal and laryngeal swabs of poultry from Jiangsu,Anhui,Shandong and other province plus LPM environmental water samples were collected.In particular.346 samples distributing in 109 flocks were identified as AIV positive,with an overall positive rate of 18.54%,and the flock positive rate of 44.67%,respectively.According to the distribution of HA subtypes,the highest positive rate of AIV was H9(72.83%,252/346),followed by H3(26.59%,92/346).Also,one H1 virus and one H10 virus were detected.As divided in different hosts,H9 was most abmundant in chickens with the positive rate of 96.80%(242/250),and the rest were all H3 subtype.In ducks.H3 subtype possessed the highest positive rate of 91.76%(78/85),followed by H9 subtype(5.88%.5/85),and then both the H1 subtype(1.17%,1/85)and H10 subtype(1.17%.1/85).In geese.H3 subtype and H9 subtype shared equal positive rate(50%,5/10).From environmental water samples,only one AIV of H3 subtype was isolated.According to AIV in different months,numbers of low pathogenicity AIV could yet be detected in summer and autumn,without evident seasonality.Furthermore,H3 subtype positive samples scattered in all 28 groups were subjected for virus purification plus PCR identification of HA and NA genes.The results showed that low pathogenicity H9 and H3 subtypes were widely exited in East China,in which H3 subtype had obvious advantage in ducks and was exclusively H3N2 subtype.2.Genetic evolution analysis of H3N2 subtype avian influenza virusAccording to space-time,host differences,19 representatives of H3N2 subtype AIV from chapter one were selected for genome sequencing and genetic evolutionary analysis.As analyzed,the 19 H3N2 strains all belonged to the Eurasian lineage in each of the phylogenetic trees of PB2,PB1,PA,HA,NP,NA,M and NS genes.And,they were all highly homologous with the influenza viruses in wild birds but genetically distant from those in mammals such as pigs,horses,dogs and human,suggesting a low risk of H3N2 viruses that could spread directly from birds to mammals.In addition,some of the PB1 genes of H3N2 subtype AIV clustered with H5N6 viruses,indicating potential reassortment between the subtypes.All the HA gene carried a uniform amino acid motif of PEKQTR?GLF at the cleavage site,in accordance with the molecular characteristics of low pathogenic AIV.HA sites 226 and 228 associated with the receptor-binding preference were all Q and G,respectively,indicating a prior binding affinity to avian receptors.Particularly,one virus of 180138 possessed a G225D mutation,which was worthy of further study.Analysis of key amino acid sites in internal genes showed that site mutations relevant to enhanced pathogenicity in mammals such as E627K and D701N in PB2 genes were not found in any of the investigated H3N2 isolates.In order to explore whether the H3N2 subtype AIV may be related to interspecies transmission,we conducted HA gene analysis from the perspective of codon usage bias.The dataset comprised HA sequences of H3N2 subtype from avian(n=19,our sequenced H3 isolates),and from other different hosts including human(n=60).dog(n=55),pig(n=54).horse(n=21)by retrieving from NCBI database.The analysis of nucleotide composition and relative synonymous codon usage showed that the content of AU was higher than that of GC in HA genes from all the hosts,preferring to use codons ended with A or U.The average value of effective number of codons in HA genes of different hosts ranged from 47.10±1.64?53.22 ±0.33,indicating the existence of certain bias for codon usage but at a low level.Neutral analysis showed that the codon usage bias of H3 virus from each host was more affected by natural selection than mutation pressure,with the corresponding proportions of 95.65%&4.35%in bird,68.75%&31.25%in human,73.17%&26.83%in dog,60.74%&39.26%in pig,63.57%&36.43%in horse,respectively.In correspondence analysis,HA gene showed an overall clustering tendency according to host origin but a relatively obvious difference between avian and mammalian H3 viruses,somewhat suggesting the host-jump potential of H3N2 subtype AIV directly from birds to mammals was low.3.Biological characteristics of 6 H3N2 subtype avian influenza virusAccording to clade distribution in the phylogenetic trees of the 19 H3N2 subtype AIV genomically sequenced in chapter two,6 strains of 171029,180133,180562,181222,190566 and 180138(with HA-G225D)were selected to determine some of the biological characteristics such as infectivity in vivo/vitro,thermal/low-pH stability.The EID50 of the 6 viruses ranged from 107.000?108.500,indicating their good propagation in chicken embryos.The TCID50 on different cells showed that all the tested viruses replicated to higher titers on MDCK(104.769-106.000)than on A549(101.500-102.571)or CEF(103.289-104.500)cells,despite that the number and size of formed plaques on MDCK cells were variant among the 6 viruses.In the thermal stability assay,180133 was determined as thermal instable and 180562 as moderate thermal stable,while the rest four viruses were all heat-stable.Except for 190566,all the other H3N2 viruses lost their infectivity in MDCK cells after heat treatment at 56? for 10 minutes.The low-pH stability test showed that each of the viruses still retained the ability to infect MDCK cells after being treated with buffers at pH 4.0?7.0.In animal experiment,all the infected mice survived in a 14-day observation period and exhibited overall growing tendencies of body weight change when intranasally inoculated 6-week-old BALB/c mice with 106.0EID50 per virus.In addition,only limited virus replication were detected in the lungs of mice challenged with 171029,180133 and 180562 viruses on day 5 post infection,indicating the low pathogenicity to mice of the six H3N2 subtype AIVIn summary,H3N2 subtype AIV currently had a high positive rate in ducks in LPM,and its PB1 gene might participate in the gene reassortment with contemporaneous H5N6 virus.Low potential of cross-species transmission of H3N2 subtype AIV to mammals was inferred from the phylogenetic analysis of each gene and the analysis of HA gene codon usage bias.Avian H3N2 viruses had not shown high pathogenicity to mammalian model mice,but the increase of thermostability might promote the virus spread and variation,which still deserved further continuous monitoring.