Epidemiological Investigation Of Porcine Kobuvirus Disease In Sichuan Province And Establishment Of An Indirect ELISA Method Detecting

Porcine kobuvirus (PKoV) is the member of Kobuvirus genus, Picornaviridae family. PKoV was first detected in a Hungary farm in 2008 in apparently healthy pigs, then in 2009, it was also detected in China. Since that, PKoV has been detected in Thailand, Spain, Japan, Korea, the United States, Brazil, the Netherlands, Czech Republic and recently in Italy, East Africa. PKoV is an emerging virus widely distributed worldwide with high prevalence rate in diarrheic pigs, it was thought to be a potential pathogeny of swine diarrhea. Considering PKoV was also prevalent in non-diarrheal swinery and the lack of a cell culture system to propagate PKoV in vitro, more further study would be needed to clarify the biology properties and pathogenicity of this emerging virus.The aim of this study was to determine the prevalence of PKoV disease in Sichuan Province China, and to analyze the phylogenetic and genetic relationships of the VP1 region between Sichuan PKoV and reference kobuvirus strains. VP1 protein is the most exposed structure protein of kobuviruses, and it is also the the immunodominant part of the picornavirus capsid protein.. PKoV VP1 main antigenic epitope region was expressed in Escherichia coli expression system, purified recombinant protein was used to establish an indirect ELISA method to detect PKoV antibody in serum samples, providing some reference for further serological diagnosis study on PKoV.A total of 163 porcine intestinal and fecal samples were collected during 2011-2012, Sichuan Province, China. The number of non-diarrheal and diarrheal stool was 51 and 112, respectively. Using primers targeting the 3D region,53.4%(87/163) pigs were positive for PKoV. The proportion of asymptomatic pigs and those with diarrhea that were PKoV-positive were 29.4%(15/51) and 64.3%(72/112), respectively. Detection rate of PKoV in suckling pigs, weaned pigs, growing/finishing pigs, and sows were,66.7%, 39.1%,23.5% and 40.0%, respectively. PKoV infection was significantly associated with diarrhea in pigs (χ2=17.126, p=3.5×10-5) using Pearson’s chi-square test. Statistical analysis showed that porcine kobuvirus infection strongly correlated with suckling pigs, which were younger than 4 weeks old (χ2=10.941, p=9.4×10-4). These observations imply that young pigs and pigs with diarrhea might be more susceptible to PKoV infection.RT-PCR was used to amplify PKoV VP1 gene sequence. The obtained 35 PKoV VP1 gene sequences were submitted to GenBank (Accession numbers:KF157917-KF157951), these VP1 sequences were 80.7%-100% similar at the nucleotide level, and 87.2%-100% similar at the amino acid level.The 35 PKoV VP1 gene sequences belonged to four different branches, indicating multiple PKoV strains were circulating in Sichuan Province. Two pigs were found to be co-infected with multiple PKoV strains. This is the first report, with supporting evidence, of multiple strains of PKoV co-infecting a single pig. Possible recombination events were analyzed for sequence A2 by RDP and Simplot software; A2 might have been generated from recombination between Al and A3 in the VP1 region. Recombination events have contributed to the virus genome evolution and genetic diversity within hosts that we observed.Antigen epitope of PKoV VP1 protein was analysed by using BepiPred、Bcepred、 DNAStar Protean. Through comprehensive consideration of all prediction results, 1-462bp region of PKoV VP1 protein was selected, which had higher antigenic index, and the main epitopes concentrated in this region. Selected sequence was sent to Genscript company for codon optimization and gene synthesis. The synthetic sequence was cloned to pET32a(+) by directed cloning. Constructed plasmid pET32-VP1 was transferred into E.coli BL21(DE3) competent cell. Induced expression conditions were optimized. The optimal induced expression conditions were as follows:37℃ 4h,0.2mmol/L IPTG. The recombinant protein was 35.6KDa and existed as insoluble protein. The prokaryotic expression products were purified by using Ni column. Purified recombinant protein had specific immunity response with PKoV antibody positive serum in Western blot detection, indicating the good antigenicity of recombinant protein.Purified recombinant protein was used to act as coating antigen, the indirect ELISA detecting PKoV antibody was established initially, every step was further optimized. Antigen coating optimal concentration was 2μg/mL, and the optimal serum dilution was 1:200. Incubation at 37℃1h plus 4℃ overnight was the optimal recombinant protein antigen coating condition.5% skim milk powder was the ideal blocking buffer, and the determined blocking time was 90min. Optimal serum reaction time was 60min. Optimal working concentration of HRP rabbit anti-pig antibody was 1:6000, optimal reaction time was 60min. The optimal TMB reaction time was 15min. Cut off value for the indirect ELISA was 0.250. The established indirect ELISA had good specificity, cross reactions were not observed when standard positive serum of TGEV, PEDV, PRV, CSFV, PPV, PRRSV were detected.170 porcine serum samples were detected with the established indirect ELISA,65/170 (38.2%) were positive for PKoV antibody. The established indirect ELISA would provide some reference for further serological diagnosis study on PKoV.