Isolation And Identification Of A Senecavirus And Development Of An Inactivated Vaccine

Senecvirus A(SVA)is the pathogen of Senecvirus disease(SVD).In 2015,the first outbreak of SVA in Guangdong Province in China,followed by reports of SVA epidemic in Heilongjiang,Hubei,Henan and other provinces.The widespread spread of the disease in China seriously jeopardizes the healthy development of pig industry in China.At present,there is no commercial vaccine to control the infection of SVA,and there is no objective evaluation method for vaccine immune response.In this study,a SVA CH-HNCY-2019 was isolated and identified from clinical samples of a suspected SVA infected pig farm in Henan Province,the whole genome sequence was determined,and genetic evolution analysis was performed;established SYBR Green ? real-time PCR nucleic acid detection method and indirect ELISA antibody detection method for SVA;SVD oil emulsion inactivated vaccine based on SVA epidemic strain was prepared,and the immune efficacy of the vaccine was evaluated.The research contents include the following four aspects:1.Isolation,identification and genetic evolution analysis of an epidemic strain of SVASenecavirus A(SVA)is the pathogen of senecvirus disease(SVD).The molecular epidemiological investigation and genetic evolution analysis of SVA are of great significance to the prevention and control of SVD.In this study,a SVA CH-HNCY-2019 was isolated and identified from clinical material collected of a suspected SVA infected pig farm in Henan Province by RT-PCR,virus isolation and identification,and indirect immunofluorescence test.The whole genome sequencing and genetic evolution analysis were carried out.The results showed that CH-HNCY-2019 could be well replicated in BHK-21 cells and produced obvious cell lesions.The titer of virus reached 1×107TCID50·0.1mL-1 60 hours after inoculation.The whole length of CH-HNCY-2019 genome is 7294 nucleotides,95.9%to 99%with Chinese isolates,and the lowest homology with SVV-001.CH-HNCY-2019 is closely related to most of the strains isolated from China in 2017-201 8,and relatively far from the early isolates in the United States.Compared with other domestic and foreign SVA isolates,the 722 amino acids of VP1 protein of CH-HNCY-2019 were changed from L to Q;499 amino acids of VP3 were changed from A to V,528 amino acids changed from P to S,582 amino acids changed from E to K.A new SVA strain was successfully isolated in this study.The results suggested that the Chinese strains of SVA were diverse and evolving.Therefore,we should strengthen the molecular epidemiological investigation,biosafety measures and vaccine development of SVA to prevent the spread of SVA in China.2.Establishment and application of S YBR Green ? real-time PCR for detection of Senecvirus A.In order to establish a method for rapid diagnosis of SVA and objective evaluation of vaccine immune response,this study designed a pair of specific primers based on the conservative region of the SVA VP1 gene to amplify the target fragment of the SVA VP1 gene and construct the pMD18-T-SVA-VP1 standard plasmid product.Using this as a template,by optimizing the reaction conditions,a SYBR Green ? real-time PCR detection method for SVA VP1 gene was established,and its sensitivity,specificity and stability were tested.The results show that the detection limit of this method was 10 copies·?L-1,which is 100 times that of ordinary PCR.In addition,the detection results of TGEV,PEDV,PRRSV and other pathogens are all negative,and the repeat coefficient of variation within and between groups is less than 2%,indicating that this method has good specificity and repeatability.At the same time,this method was used to study the proliferation of SVA CH-HNCY-2019 on BHK-21 cells.In this study,a SYBR Green ? real-time PCR method for SVA VP1 gene was successfully established,which provided technical support for early diagnosis of SVA infection,molecular epidemiological monitoring,and evaluation of immune efficacy of SVA vaccine.3.Establishment and application of indirect ELISA based on SVA epidemic strainsIn order to establish a rapid,accurate and specific method for SVA serological detection and immune efficacy evaluation of SVA vaccines,this study used purified SVA epidemic strains was used as the antigen and the reaction conditions were optimized to establish an indirect ELISA antibody-based detection method.The optimal coating amount of antigen was 0.4 ?g/well,the optimal dilution of the serum to be tested was 1:000,the optimal action time of the antigen primary antibody was 1 h,and the optimal dilution of the enzyme-labeled secondary antibody was 1:2 000,the optimal action time of the secondary antibody was 1 h.Under optimized conditions,when the S/P value of the serum to be tested was greater than or equal to 0.1176,it is judged as positive;when the S/P value is less than 0.0904,it is negative.The test results of specificity,sensitivity,repeatability and coincidence rate show that the established method has no response to pathogen-positive sera such as PEDV,PRV and TGEV,indicating that the specificity of the method was good;in addition,the sensitivity of the method is equivalent to that of the neutralization test,The coefficient of variation within and between groups is less than 5%,indicating that the method has good sensitivity and repeatability.Using this method and the neutralization test to detect 39 serum samples at the same time,the coincidence rate was 89.7%.The positive detection rate of 96 clinical pig serum samples was 5.2%.The indirect ELISA antibody detection method based on SVA epidemic strains established in this study has high repeatability,specificity,sensitivity and coincidence rate,which can be used for clinical detection of SVA and provide technical support for the diagnosis,prevention and control of SVA and the evaluation of immune efficacy of SVA vaccine.4.Development and evaluation of inactivated vaccine based on SVA strainIn view of the fact that there is no commercial vaccine to prevent and control the prevalence of SVD in China,this study used BHK-21 cells to expand SVA CH-HNCY-2019,and then prepared the SVD oil emulsion inactivated vaccine after inactivation with formaldehyde.Two week old female BALB/c mice were immunized with different doses of SVD inactivated vaccine and challenged with SVA CH-HNCY-2019 was challenged 4 weeks after immunization to evaluate the immunogenicity and immune efficacy of the vaccine.The results showed that 107 TCID50 immunized mice produced anti SVA specific antibody 2 weeks after immunization,and the antibody level was the highest 5 weeks after immunization,and the vaccine could induce mice to produce a high level of neutralizing antibody.After challenge,the 107 TCID50 immunized mice did not show the characteristic clinical symptoms and pathological changes of SVD,and SVA was not detected in tissues and organs,which indicated that the SVD oil emulsion inactivated vaccine prepared in this study could effectively resist the infection of SVA,and provided an auxiliary tool for the prevention and control of SVD epidemic in Chinese pigs.