Pathogenesis Of Senecavirus A And Establishment Of An Indirect ELISA For Detecting The Anti-VP2 Antibody

Senecavirus A(SVA),original named as seneca valley virus(SVV),is an emerging important virus that causes swine vesicular disease to affect world swine industry.SVA can infect pigs of all ages,causing sudden death of piglets and lethargy,anorexia,vesicles,and scabs in the snout and hoofs of fattening pigs,which have a certain impact on the export of the breeding industry.Since the first report of SVA infection in swine farms in Guangdong,China in 2015,vesicular disease cases have been increasing in many provinces in Hubei,Heilongjiang,and Fujian.At present,research on the pathogenesis,clinical symptoms,pathological changes,and virus control of SVA is not comprehensive,and there is no complete retrospective serological investigation in China.In this study,we observed the clinical symptoms after infection with SVA in pigs of different ages,and studied the changes of neutralizing antibodies,viremia,and structural protein antibodies with the infection time.At the same time,we detected indirect ELISA antibodies.The method was optimized and serological surveys were carried out in different provinces and at different times.The specific contents of this study are as follows:1.Pathogenesis of Senecavirus A isolate SW-CH-SD in swineTotal 40 SVV-negative healthy pigs of different ages were selected and randomly divided into 8 groups each with 5 pigs,that is,30-35 days old infection group(oral injection and nasal drip),30-35 days old control group,55-65 days old infection group(oral injection and nasal drip),55-65 days old control group,90-100 days old nose drip challenge group,90-100 days old oral nose challenge group,90-100 days old hoof Intramuscular injection group and 90-100 day-old control group.Each group was inoculated with 10 ml SVV-CH-SD strain on the first day through different challenge routes,and the clinical symptoms were recorded daily.The blood samples were taken from the vena cava at 0,3,7,14,21,and 28 days after challenge,and the viremia and antibodies were detected by real-time PCR and ELISA.All pigs were sacrificed on the 28th day after challenge,the macroscopic pathological changes were observed and the heart,liver,spleen,lung,kidney,oral fluid and lymph nodes of the infected group were collected for determining the viral load of each organ tissues.The results showed that only one pig in the day-old challenge group had fever,but no deaths occurred,and the organs had no specific lesions.SVA could be detected in the heart,liver,spleen,lung,kidney,oral fluid,and lymph nodes of infected pigs by real-time PCR.The viremia in the serum reached its peak on the third day after challenge,and then rapidly,and decreased until it completely disappears at 28 day post challenge(dpc).Serum neutralization test showed that the anti-SVA neutralizing antibody reached a peak at 14 dpc,and then decreased slowly,in the challenge groups.Anti-VP1,VP2,VP3,and VP4 antibodies measured by indirect ELISA reached a peak 14 dpc,and then decreased slowly.It indicated that SVV-CH-SD isolate has can infect all days old pigs,but only has low clinical virulence in 90-100 days-old pigs.It provides helpful to study on the pathogenic mechanism and prevention and control of this disease.2.Development and application of an indirect ELISA for detecting antibody against VP2 of SVA in swineThe VP2 gene was amplified by RT-PCR from SVV-CH-SD strain isolated from a clinically affected pig farm in Shandong Province,and cloned into the pET-3 2a(+)expressed plasmid.After correct confirmation by double-enzyme identification and sequencing,it was transformed into competent cells BL21(DE3)and induced expression was performed.The results show that the recombinant strain can express a fusion protein with a molecular weight of 55 kDa with good antigenicity.A purified VP2 protein was used as the coating antigen,and a series of conditions were optimized to establish an SVA VP2 indirect ELISA antibody detection method.This method has good specificity and does not react with five common swine epidemic virus-positive sera.The sensitivity of this method is 91.6%,the coincidence rate is 91.9%,and the repeatability is good.After using this test method to detect antibodies in 2917 sera from 12 provinces in China from 2016 to 2019,the positive rate was 71%.The results indicated that the indirect ELISA detection method established in this experiment can be used an effective method for rapid detection of SVA antibodies.