Molecular Epidemiological Investigation Of PCV4 Of Inner Mongolia And Preliminary Establishment Of Indirect ELISA Antibody Detection Method

Porcine circovirus type 4(PCV4),a newly discovered Porcine circovirus type 4(PCV4),was first detected in pigs in Hunan Province,China in 2019.However,due to its relatively short time of discovery,information on its prevalence and pathogenicity is still unclear.In this study,PCV4 was tested on 1,683 pig serum samples collected from 51 pig farms in the Inner Mongolia Autonomous Region from 2016 to 2018 using PCR detection methods.The results showed that the total positive rate of PCV4 in the pig herd was 1.6%(27/1683),the positive rate of PCV4 at the farm level was 21.6%(1 1/51).Among them,the total positive rate of PCV4 in the pig herd in 2016 was 0.2%(1/485),in 2017 it was 1.7%(12/686),and in 2018 it was 2.7%(14/512);the positive rate of PCV4 at pig farm level was 6.7%(1/15)in 2016,25%(5/20)in 2017,and 31.3%(5/16)in 2018,indicating that the infection rate of PCV4 was increasing year by year.In addition,the co-infection of PCV2,PCV3,and PCV4 was explored by PCR,and the results showed that PCV4 infection is not related to PCV2 and PCV3 infection.In order to clarify the genetic variation and evolutionary laws of PCV4,a total of 3 PCV4 genome sequences were obtained in this study.The homology analysis showed that PCV4 has high genetic stability.At the same time,the 8 PCV4 sequences that can be downloaded by NCBI are compared with the 23 complete genome sequences of 17 circoviruses.The analysis of nucleotide and deduced amino acid sequence homology shows that PCV4 and mink circle Cycloviruses have the highest homology.Genetic diversity analysis of PCV4 showed that there were 7 amino acid mutations in the Cap protein sequence,and 6 amino acid mutations in the Rep protein sequence.There were 19 nucleotide mutation sites in ORF1 gene and 17 nucleotide mutation sites in ORF2 gene,but there were no insertions or deletions.Phylogenetic analysis of the PCV4 genome sequence,Cap gene sequence,and Rep gene sequence respectively showed that PCV4 is in an independent branch and has the closest evolutionary relationship with mink circovirus.Taking the ORF2 gene sequence of the Inner Mongolia PCV4 strain amplified by our laboratory as a reference,its codons were optimized and sent to the company for plasmid synthesis,and the truncated Cap protein gene was amplified by designing specific primers.The target gene was cloned into the pET-28a vector to construct the pET28a-PCV4-Cap recombinant expression plasmid.The recombinant plasmid was transformed into the expression host strain BL21(DE3)pLysS for induction and expression,and the PCV4 Cap recombinant protein was prepared.By optimizing the expression conditions and analyzing by SDS-PAGE,the Cap recombinant protein can be expressed in a large amount in the precipitate and the maximum expression is at a final concentration of IPTG of 0.2 mmol/L and induction at 37℃ for 6 hours,and its size is about 27 kDa.It was verified by Western blot that the band was single,which was the same as the expected result.The affinity chromatography method was used to purify the His-tagged pET28a-PCV4-Cap recombinant protein through a nickel column.After purification,the highest purity of the target protein could reach 95.16%.Purified and renatured pET28a-PCV4-Cap recombinant protein and Freund’s adjuvant were used in a ratio of 1:1,and then emulsified and injected into Balb/c mice aged 4 to 6 weeks to prepare murine polyclonal antibodies.The potency can reach more than 1:300000.The purified and renatured recombinant protein was used as the coating antigen to establish a PCV4 indirect ELISA detection method.By optimizing the reaction conditions,it was finally determined:the best coating concentration of the antigen is 1μg/ml,and the coating is overnight at 4℃;use 5%BSA,blocking at 37℃ for 1 hour is the best blocking condition;the best dilution of the serum to be tested is 1:400,incubated at 37℃ for 1 h;the best dilution of the enzyme-labeled secondary antibody is 1:5000,incubated at 37℃ for 15 min;The best color development condition of the substrate is 15 min at room temperature.The indirect ELISA detection method established in this study has a coefficient of variation of less than 10%for inter-assay and intra-assay reproducibility,and does not produce serological cross-reactivity with PCV2,PRRSV,PRV,and SVV positive sera,which proves that the method has good reproducibility and specificity.It provides a new way for the rapid detection of PCV4 seroepidemiology.