Establishment Of Luciferase Immunosorbent Assay For Porcine SVA And Epidemiological Investigation

Senecavirus A(SVA)is an infectious virus that can cause blister disease in pigs and acute death in newborn piglets.In this study,we fused SVA VP1 with Renilla luciferase(Rluc)gene to construct p CDNA3.1(+)eukaryotic expression vector,and prepared soluble crude proteins containing VP1 and Rluc genes.To develop a novel SVA VP1-Rluc LISA method for capturing specific antibodies by lucifinase-labeled antigens,and to use this method to isolate growing-finishing pigs(6-26 weeks of age)and sows(> 26 weeks of age)to conduct seroepidemiological investigation of SVA in order to provide data support for the infection of SVA in Sichuan Province.1 Establishment of porcine Seneca virus VP1-Rluc LISA methodIn this study,Mega7.0 and oligo7.0 software were used to analyze SVA VP1 and Rluc genes and design specific primers.After fusion of amplified fragments,p CDNA3.1(+)eukaryotic expression vector was constructed.In 10 μg plasmid quality,under the condition of transfection time 48 h successfully expresses the size is about 63 KDa protein.The results were verified by Western blot and IFA.The results showed that the recombinant protein VP1-Rluc had luciferase activity and could specifically bind to the murine anti-VP1 polyclonal antibody of SVA.In this study,VP1-Rluc LISA method of SVA was established using the prepared VP1-Rluc recombinant protein.The results show that the method has good specificity and does not cross-react with PRRS,CSF and other virus.High sensitivity,SVA serum dilution up to1:6 400;The coefficient of variation between batches and within batches was less than 10%.Compared with SVA IFA method,Kappa(κ)coefficient was 0.897,showing strong consistency.2 SVA serological survey of large-scale pig farms in some areas of Sichuan ProvinceIn this study,a total of 2298 serum samples were collected from 15 regions including Chengdu,Guang ’an and Nanchong during 2021 to 2022.Among them,1428 serum samples were collected from growing-finishing pigs(6-26 weeks of age)and 1428 serum samples were collected from sows(> 26 weeks of age);According to geographical location,870 serum samples of 26 weeks of age were divided into western Sichuan,northeast Sichuan,central Sichuan and southern Sichuan.In this study,LISA method was established to conduct serological investigation of SVA in Sichuan province and SVA ELISA method was used to verify and supplement the detection data.The results showed that the positive rate of SVA in growing-finishing pigs was 12.8%for individual and 40.3% for population.The individual positive rate was 25.2% and the population positive rate was 70.8%.According to geographical location,the SVA positive rate of growing-finishing pigs in the four regions was similar,while the SVA positive rate of sows ranged from 63.6% to 83.3%,and the highest was found in central Sichuan.The positive rates of SVA antibody in growing-finishing pigs and sows in the fourth quarter were19.7% and 39.0%,respectively.The positive rates of SVA antibody in growing-finishing pigs and sows in the third quarter were the lowest(9.3% and 16.3%,respectively).In general,SVA was widely distributed in Sichuan Province with subclinical transmission.