Detection Of PCV Infection In Wild Boars In Some Areas Of Jiangxi Province And Establishment And Application Of PCV4 Antibody ELISA Detection Method

Porcine circovirus(PCV)is a small,envelope-free virus belonging to the genus Circovirus in the family Circoviridae,with a ring-shaped,single-stranded,bidirectional DNA genome.To date,four genotypes of the virus are known,PCV1-PCV4.Porcine circovirus type 4(PCV4),a new member of the genus Circovirus,was first reported in Hunan Province,China,in 2019.To further understand the prevalence and genetic situation of this virus in wild boar population,138 tissue samples of wild boars,including lymph nodes,kidney,spleen and other tissue organs,were collected from 5 different mountainous areas in Jiangxi Province in this study,and the tissue samples were tested by fluorescent quantitative PCR(Taq-Man q PCR)using probe method.The positivity rates were 21.74%,22.46%,5.80%and 19.57%,respectively.The whole genome sequences of 19 PCV2 strains and 2 PCV4 strains were successfully amplified by amplifying the full length and sequencing of the positive tissue samples of PCV2 and PCV4 by ordinary PCR method and the sequence analysis showed that the 19 PCV2 strains contained 18 PCV2 b subtypes and 1 PCV2 d subtype,which were more homologous with PCV2 of domestic pig origin;the 2 PCV4 strains were also more homologous with PCV4 of domestic pig origin.In this experiment,PCV4-Cap/Rep genes were ligated to p ET-32 a vector to construct p ET-32a-PCV4-Rep and p ET-32a-PCV4-Cap recombinant expression plasmids,respectively.The recombinant plasmid was transformed into BL21 expression receptor state for expression,induction,and optimization of induction conditions.The purified recombinant protein was obtained,and the purified recombinant protein was emulsified with the Freund’s adjuvant in a certain ratio and injected into mice.According to the serological potency test of mice,the antibody potency could reach a minimum of more than 1:64000,so the murine-derived polyclonal antibody was successfully prepared.Then the amplified target genes were separately ligated to eukaryotic expression vectors(Flag-N)and transfected into cells for expression.The proteins in the wild boar positive samples were also extracted and the target bands were all detected using the obtained mouse-derived polyclonal antibodies as primary antibodies.An indirect ELISA assay for PCV4 was established using purified PCV4-Rep and PCV4-Cap recombinant proteins as encapsulated antigens,respectively.By optimizing various conditions of the assay,the optimal conditions for each of the two recombinant proteins were determined.Rep protein: the antigen was coated overnight at 4°C;the coating concentration was 1.25 μg/ml;the serum dilution was 1:800;the enzyme secondary antibody dilution was 1:4000;5% skimmed milk powder was closed at 37°C for 2 h;the optimal reaction conditions for the established indirect ELISA assay were when both the serum and secondary antibody acted for 1 h.Cap protein: the antigen was coated overnight at 4°C;the coating concentration was 2.5 μg/ml;the serum dilution was 1:400;the enzyme secondary antibody dilution was 1:4000;1% BSA was closed at 37°C for 90 min;the optimal reaction conditions for the established indirect ELISA assay were when both the serum and secondary antibody had an action time of 1 h.By testing positive sera of two other viruses of porcine origin,the results were presented as negative,indicating that the ELISA method established in this test does not cross-react with the above viruses and has good specificity.Since the coefficients of variation obtained between and within batches were within the critical range,it proved that the inter-and intra-batch reproducibility was good.As the coefficients of variation obtained between and within batches were within the critical range,the inter-and intra-batch reproducibility was proved to be good.Serological tests were performed on random samples from pig farms in five different regions of Jiangxi,and the results showed that the positive rate of PCV4-Rep antibodies was highest in Ji’an(11.76%);while the positive rate of PCV4-Cap antibodies was highest in Yichun(5.94%),so this test provides a new way for rapid seroepidemiological detection of PCV4.