Isolation And Identification Of Swine Influenza Virus In Yangzhou From 2019 To 2020 And Preparation Of Its Monoclonal Antibody

Swine influenza(SI)is mainly endemic in pig herds.Swine influenza virus(SIV)is an important pathogen,which mainly included H1N1,H1N2 and H3N2 subtypes.The virus also easily recombined with other subtype influenza viruses in pigs that increased susceptibility risk to humans.China have the most complex SIV ecosystem,including Classic swine influenza(CS),Northern american triple reassortant(TR)and Eurasian avian-like(EA),which result complicated SIV popularity and evolution.Therefore,It's has a great significance for early warning,prevention and control of SI by continuous epidemiological surveillance,evolutionary analysis,assessment of virus virulence changes,and accumulation of diagnostic tools.In this study,nasal swab samples were firstly collected from pigs in slaughterhouses in Yangzhou in 2019-2020,then virus isolation,identification,whole-genome sequence and evolution analysis were conducted;Secondly,the pathogenicity of H1 subtype SIV to mouse was evaluated;Finally,monoclonal antibodies against H1N1 subtype SIV were preparated for diagnostic method deveolopment.The specific research content is as follows.1.Isolation,identification and genetic evolution analysis of swine influenza virus in Yangzhou area from 2019 to 2020In 2019-2020,the pigs were continuously sampled at one slaughterhouse in Yangzhou,Jiangsu Province,where a total of 802 nasal swab samples were collected.The virus was isolated by the SPF chicken embryo inoculation method,and the hemagglutination test combined with the RT-PCR method was used to identify the virus subtypes.Two H1 subtype SIVs were successfully isolated and the isolate rate was 0.25%.One of was HIN1 subtype SIV named as A/Swine/Jiangsu/1070/2019(JS1070);the other was H1N2 subtype SIV named as A/Swine/Jiangsu/1281/2019(JS1281).The genetic evolution analysis of the HA gene showed that the two SIVs were belonged to the EA lineages,and the alkaline cleavage sites of HA genes in both SIVs were 339PSIQSR?G347,which was accorded with the molecular characteristics of low pathogenicity.NA genes were belonged to different lineages,JS1070 belonged to EA lineage,and JS1281 belonged to TR lineage.The internal genes PB2,PB1,PA,NP,M and NS genes of the two SIV virus were all belonged to the CS lineages.2.Biological characteristics and mouse pathogenicity of swine influenza isolatesThe SIV isolates were purified by the limited dilution means,and their biological characteristics and pathogenicity to mice were studied.The 50%egg infectious doses(EID50)of JS1070 and JS1281 were 7.7 lg/0.1 mL and 8.5 lg/0.1 mL,respectively,and 50%tissue culture infectious doses(TCID50)of JS1070 virus and JS1281 virus were 5.22 lg/0.1 mL and 6.24 lg/0.1 mL,respectively.Also,the mean death times(MDT)in chicken embryos caused by minimum lethal dose of JS1070 virus and JS1281 virus were 81 h and 69 h,respectively.When 6-week-old BALB/c mice were intranasally infected with 106 EID50 viruses,the body weight of the mice were decreased at about 10%rate and the survival rate of the mice was 66.7%by the JS1070 virus infection,while the body weight of the mice were decreases at about 22.3%and the survival rate of mice was 50%by the JS1281 virus infection.Also,pathological tissue sections results showed that both SIVs caused severe lung tissue lesions.The virus could only be detected at 3th day in the heart,while the virus could be detected up to 9th day in the lungs,which the virus titers of JS1281 was higher than that of JS1070 in the heart and lungs.Compared with the control group,the expression level of cytokines IL-6,IL-12 and TNF-? in the lungs were up-regulated in both SIVs infected mice,but the cytokines induced by JS1281 was higher than that by JS1070.All in all,the above results indicated that both two H1 subtype SIVs caused disease in mice but the JS1281 is more virulent to mice.3.Preparation of monoclonal antibody against H1N1 subtype SIVThe H1N1 subtype virus JS1070 was selected for HA monoclonal antibody preparation.The virus was purified by sucrose concentration gradient centrifugation and inactivated with formaldehyde to prepare an oil emulsion inactivated vaccine.6-week-old BALB/c mice were immunized subcutaneously through the abdomen,and boosted by intraperitoneal injection after three immunizations.The spleen cells were taken 72 hours later to prepare hybridoma cells.The positive clones were screened by hemagglutination inhibition(HI)method,and finally seven monoclonal antibody hybridoma cells that can stably secrete antibodies were obtained and named as 1B7,3C10,3D6,3D7,4F11,5B6 and 5C11,respectively.HI results showed that all 7 monoclonal antibodies had HI titers(7?8log2)against H1N1 subtype SIV(JS1070).The antibody subclass identification results showed that the subclass of 1B7 was IgG3,the subclasses of 3D6 and 5C11 were IgM,the subclass of 3D7 was IgG2a,and the subclasses of 3C10,4F11 and 5B6 were IgG1.The results of HI and indirect immunofluorescence test(IFA)showed that 5 monoclonal antibodies except for 3D6 and 5C11 were reactively with the parent JS1070,which and have no cross-reactivity to H3,H4,H5,H6,H7,H9,and H10 influenza viruses,indicating that the monoclonal antibodies had good specificity.The monoclonal antibody 3C10 had better cross-reactivity with swine H1N2 subtype JS1281 virus,avian H1N1 subtype JY virus,human H1N1 subtype PR8 virus and CA09 virus,indicating that 3C10 had a good broad spectrum.Also,monoclonal antibody 3C10 had better neutralization characteristic and immunoblotted characteristic.In summary,the monoclonal antibody 3C10 has better comprehensive properties and can be applied to develop subsequent immunological diagnosis methods for the H1 subtype influenza virus.