Isolation,Identification And Biological Characteristics Analysis Of Swine Influenza Virus In Guangdong Province In 2016～2017

Swine influenza caused by swine influenza virus,is a kind of respiratory infectious disease.Recently,H1N1,H1N2 and H3N2 are circulating in swine herb.There are SAα-2,3 and SAα-2,6 in espiratory tract and swine are considered as“mixing vessel”.Avian-,human-and swine-origin influenza viruses can infect swine and generate novel reassortants which have the ability to cause potential pandemic and pose a threaten to public health.Guangdong area is considered as an influenza ecology due to it’s special geography,feeding condition,human population density and dietary culture.So it’s necessary to detect swine influenza viruses’circulation,evolution and biological characteristics in Guangdong province.By analyzing the biological characteristics of swine influenza virus,circulation,genetic evolution,growth feature and pathogenicity of virus are detected in Guangdong area,this study slected swine influenza in Guangdong province as the research target.1522 nasal swabs were collected in Guangdong province between 2016～2017,of which 287 swabs were collected from pig barn and 1235 swabs were collected from slaughter farm including132 sera were collected from pig barn in total.Total RNA was extracted from nasal swabs and M gene of influenza A virus which was detected by RT-PCR.The results showed that69 samples were positive to IAV M gene,and the nucleic acid rate was 4.53%,of which 15positive samples were collected from pig barn with 5.23%nucleic acid positive rate,and 54samples were collected in slaughter farm with 4.37%nucleic acid positive.Nucleic acid positive samples were inoculated in MDCK cells and 13 swine influenza viruses were isolated,and the viral isolation rate was 0.85%,of which 8 strains were isolated from pig barn with the viral isolation rate of 2.79%,and 5 strains were isolated from slaughter farm withthe viral isolation rate of 0.4%.HI was performed by using of positive serum and subtype of 13 viruses which were identified and the results indicated that 4 viruses were H1subtype and 9 viruses were H3 subtype.Sera were detected by the use of HI assay,and the results showed that 26.5%（35/132）sera were positive to H3 subtype of SIV with titer of2log2～7log2 and 27.3%（36/132）sera were positive to H1 subtype of SIV with titer of1log2～7log2.The positive rate of sera to SIV was 53.8%and none of sera was positive to between H1 and H3 subtype SIV.The whole genome of SIV were sequenced and the results indicated that all isolated viruses posing the feature of y binding toα-2,6 SA preferentiall.HA genes of H1 subtype SIV belongs to Eurasian avian-like lineage and HA genes of H3 subtype SIV belongs to recent human lineage.N1 genes lie in Eurasian avian-like lineage,N2 genes lie in North American triple reassortant swine lineage and recent human lineage.PB2,PB2,PB1,PA and NP genes line in 2009A/H1N1 lineage.M genes belong to 2009A/H1N1 lineage and Eurasian avian like lineage.NS genes lie in 2009A/H1N1 and north American triple reassortant swine lineage.H1 subtype and H3 subtype SIVs were classified into 2 genotype and 5 genotypes,respectively.Mutations,Q591R,A588I and T339K in PB2 protein,N30D and T215A in M1protein,P42S in NS1 protein showed that these viruses have effective infection in mammalian.Amino acid is P at 13 sites in PB1 conserved in human influenza viruses,which indicated that viruses pose the features of human influenza viruses.To detect the pathogenicity of SIVs,6 viruses had been titered over 10⁶TCID₅₀,including 2 H1N2and 4 H3N2 viruses which were selected for mice infection experiment.The results showed that GD/A41 caused mice to lose body weight over 25%and killed 80%mice.The other 5viruses caused a little bit body weight change.6 viruses replicated in respiratory tract and the height viral titer were detected in lung.All mice seroconverted in 14 days post infection.GD/A1720 caused mice seroconverted with 7log2～9log2.The circulation,evolution and pathogenicity of SIVs which circulated in Guangdong area during 2016～2017 were evaluated by virus isolation,serum test assay,sequencing and mice infection experiment.These findings will promote researches for the circulation and biological characteristics of SIVs in Guangdong province,and provide data referenced for the prevention and control of influenza virus.