Prevalence Of Porcine Circovirus Type 3 In South China And Development Of An Indirect ELISA For Antibody Detection

Porcine circovirus type 3?PCV3?is a novel emerging porcine circovirus of recent years,which is suspected to be associated with Porcine dermatitis nephropathy syndrome?PNDS?and porcine respiratory disease complex?PRDC?.Several reports have described its presence in various countries or regions from North America,Asia,Europe to South America.Currently,a severe epidemic situation exists in China.PCV3 has prevailed in many provinces,the infection rate showing a year by year ascending trend.Studies on the epidemiology and diagnostics of PCV3 are urgently needed.In order to investigate the prevalence of PCV3 in south China,1319 diseased samples were collected from 203 pig farms in Guangdong,Guangxi,Hainan,Fujian,Jiangxi and Hunan provinces through December 2016 to February 2018.PCR assays were was conducted to detect the PCV3 genomic DNA.Positive samples were used to amplify and sequence the complete genome of PCV3.The PCR results demonstrated that the positive rate was 31.61%?417/1319?at sample level and 44.83%?91/203?at pig farm level.Complete genomic sequences were obtained from 22 PCV3 positive samples.The 22 complete genome sequences shared 98.4%to 100%nucleotide identity among each other and 98.4%⁹8.8%nucleotide identity when compared with other PCV3 strains available in GenBank.Phylogenetic analysis based on PCV3 Cap gene showed that PCV3 strains divided into three clusters.PCV3 strains HuN-CS and GD-ZQ-1 belonged to PCV3a clustered within US/MO2016,PCV3 strains GD-SH-4 belonged to PCV3b,whereas the other 19 strains in this study belonged to PCV3c.This study indicated that PCV3 had been widely spread in south China.A truncated PCV3-Cap protein was successfully expressed in a prokaryotic expression system.The purified recombinant Cap protein was used as the coating antigen to develop an enzyme linked immunosorbent assay?ELISA?and the optimal reaction conditions were determined.The optimal coating concentration of the Cap protein was 2?g/mL and coating condition was overnight at 4?.5%skim milk-PBST was used as blocking buffer over 2hours at 37?.The optimal dilution ratio of the tested serum was 1:50,which was allowed to react with the coated plates for 45 min at 25?.Diluted HRP conjugated anti-pig IgG?1:40000?was subsequently added to the reaction for 25 min.TMB was incubated for 10 min at 25?and then 2M H₂SO₄ was used to terminated the reaction.An OD value over 0.213was determined as positive for the indirect ELISA and under 0.197 as negative,while an OD value between 0.197 and 0.213 would be considered as suspicious.The indirect ELISA developed in this study showed high specificity and repeatability for detection of antibody against PCV3.Serology investigation showed that more than 95%of the boars and sows in commercial pig farms were positive for PCV3 antibody,which indicated that the pig herds in South China are suffering from severe PCV3 infection.This study provides rich data for the epidemiological study of PCV3 in Southern China.The indirect ELISA lays foundation for the development of a PCV3 ELISA kit,and is helpful in the prevention and control of PCV3.