Isolation And Identification Of SVA And Its Genomic Sequence Analysis And Immunogenicity Study

Senecavirus A(SVA)can infect pigs of different ages and cause the death of newborn piglets,posing a great threat to the pig industry in countries around the world.Therefore,the biological characteristics,genetic evolution analysis and immunogenicity of SVA are of great significance for controlling the spread of SVA.In March 2018,this study collected 6 suspected SVA-induced pigs with blisters,vesicular skin and blister fluid from the sputum-producing pigs for RT-PCR.The results showed that 3 of the nucleic acid electrophoresis results were SVA positive.In order to further analyze the biological characteristics of SVA,three positive samples were aseptically treated with Baby hamster syrian kidney cells(BHK-21)and Porcine kidney-15cells(PK-15)for virus isolation.The results showed that the BHK-21 cells inoculated with the disease had a significant cytopathic effect(CPE)after 3 generations of blind passage.The PK-15 cells inoculated with the disease only produced a slight CPE after 3 generations of blind transmission.The cell culture medium was positive for SVA nucleic acid detection.It was indicated that three SVA were successfully isolated from the disease materials,and the three strains were named GD-SVA1-2018,GD-SVA2-2018,and GD-SVA3-2018,respectively.In order to observe the dynamic proliferation of SVA in BHK-21 cells,the pathological changes of BHK-21 cells inoculated with SVA were observed under a light microscope.The results showed that after inoculation of SVA in BHK-21 cells,a small amount of lesions appeared at 4 h,partial lesions appeared at 8 h,complete lesions appeared at 16 h,and the lesions accumulated at 24 h and began to fall off.To investigate the proliferation titer of each SVA isolate on BHK-21,TCID₅₀of GD-SVA1-2018,GD-SVA2-2018,and GD-SVA3-2018 was determined by the TCID₅₀assay.The results showed that the virus titers of the SVA isolates on BHK-21 were 10^7.75TCID₅₀/m L,10^8.25TCID₅₀/m L,and 10^8.0TCID₅₀/m L,respectively.The results of the susceptibility of SVA isolates to chloroform showed that SVA after chloroform treatment caused CPE production in BHK-21 cells,which was consistent with the biological characteristics of SVA non-encapsulated membrane.In this study,the 5'and 3'gene fragments of SVA gene sequence were amplified by RT-PCR,and the successfully identified PCR products were cloned into p MD18-T vector.Three positive recombinant plasmids were sent to the company for sequencing;The remaining gene fragments of the SVA whole gene sequence were amplified by overlapping RT-PCR,and the successfully identified PCR products were sent to the company for sequencing.The sequencing results were spliced by Seqman and other software,and GD-SVA1-2018 and GD-SVA2-2018 were completed.GD-SVA3-2018 whole genome sequencing.The nucleotide sequence similarity analysis of the three isolates showed that the sequence similarity of GD-SVA1-2018,GD-SVA2-2018 and GD-SVA3-2018 was higher than 99.5%,and the similarity of VP1 gene was 100%.Then,for the SVA VP1 gene,the similarity and genetic relationship between the isolates of this study and SVA strains at home and abroad were further analyzed.The similarity analysis found that the isolates of this study and the recent SVA epidemic strains in Guangdong were highly similar.Genetic evolution analysis found that the isolates of this study were on the same branch as the domestic GD03/2017 and GD01/2017 strains.The SVA strain of this branch was closely related to USA/GBI26/2015.In order to establish an SVA animal infection model,3×10^8.25TCID₅₀SVA was inoculated into sows by intranasal route.This experiment was conducted to observe the SVA infection of sows by observing the clinical symptoms of sows and monitoring the SVA viral load in blood and feces.The results showed that sows inoculated with SVA may have elevated body temperature and vesicular symptoms;SVA in SVA inoculated SVA can detect SVA,and all SVA-infected sows detect SVA virus copy number in blood.It is indicated that blood containing SVA copy number can confirm SVA infection.The SVA animal infection model was successfully established.In order to preliminaries to explore the immunological efficacy of SVA inactivated vaccine,we prepared a large number of SVA virus solution,inactivated SVA with?-propiolactone solution,and emulsified the inactivated virus solution with 201 adjuvant to prepare SVA.Inactivate the vaccine.We inoculated 2-month-old sows at a vaccine dose of3 m L/pig,and in the same manner after 3 weeks,a control group inoculated with 3 m L/pig of PBS was working.One week after the second immunization,the experimental pigs were inoculated with 3×10^8.25TCID₅₀SVA.After the basic exemption,the blood of the experimental pigs was collected every other week,and the serum antibody titer after immunization was detected by a neutralization test.The results showed that the serum antibody titer increased after the second week after immunization,and the antibody titer reached 1:512 in the fourth week after immunization.After SVA infection,the blood of the experimental pigs was collected every other day,and the viral load of SVA in the blood was considered by q RT-PCR.The results showed that SVA was successfully infected with the control group inoculated with PBS;the immunized group inoculated with the SVA inactivated.In summary,This study successfully isolated 3 SVA and completed the whole genome sequencing of 3 SVA;Genetic evolution analysis showed that the SVA isolates in this study were recent epidemic strains in Guangdong;Successfully establish SVA sow infection model,lay the foundation for studying SVA infection mechanism;Prepare SVA with good immunogenicity Live vaccines provide a reference for the prevention and control of SVA in China.