Molecular Epidemiology Of Porcine Circovirus In Heilongjiang Region And Preparation Of Polyclonal Antibodies Against PCV2 Cap Protein

Porcine Circovirus(PCV)is a single-stranded circular DNA virus of the genus Circovirus of the circoviridae family,divided into 4 genotypes: PCV1,PCV2,PCV3,and PCV4.PCV1 was first discovered in 1974 and is non-pathogenic to pigs.PCV2 is the main pathogen causing ring-related diseases in pigs,such as weaned piglet multisystem failure syndrome,congenital tremor in newborn piglets,porcine dermatitis nephrotic syndrome and sow reproductive disorders,causing serious economic losses to the pig industry.PCV3 infection was first reported in the United States in 2016,which can cause a variety of pig-related diseases clinically.As an emerging virus,PCV4 was first detected in Hunan province,China in 2019,and its pathogenic mechanism and epidemic status are not fully understood.PCV2 is one of the important pathogens threatening the healthy development of pig industry,in order to fully understand the overall situation of circovirus infection in pigs in China,and to find out the situation of circovirus infection in the pig breeding industry in Heilongjiang Province,especially small and medium-sized farms,we applied meta-analysis to evaluate the overall prevalence of PCV2 infection in Chinese pigs,carried out molecular epidemiological investigation of porcine circovirus in Heilongjiang Province,and prepared PCV2 Cap protein polyclonal antibodies.The research carried out provided theoretical support and technical support for the formulation of porcine circovirus prevention and control measures.In this study,CNKI,Wanfang,Weipu and Pubmed databases collected the literature on PCV2 prevalence published from 2001 to 2021,a total of 5934 related literature was retrieved,and the data of 48 literature were extracted for meta-analysis after screening,and the results showed that the positive rate of PCV2 in pigs in China was 41%,the highest positive rate of PCV2 in Northeast China(61%),and the lowest positive rate in Northwest China(17%).The positive rate of PCV2 in clinically symptomed pigs was 42%,and the positive rate of healthy pigs was 36%.The positive rate of PCV2 detected in tissue disease material was 42%,and the positive rate in serum samples was 28%.The positive rate of PCV2 in fattening pigs was 51%,and the positive rate of PCV2 in piglets was 39%.The positive rate of PCV2 in large-scale pig farms is 41%,and the positive rate of retail farmers is 19%.The analysis results show that the overall positive rate of PCV2 in pigs in China is high,and there are obvious differences in the positive rate in different regions and different provinces,and corresponding prevention and control measures should be taken to reduce the spread of PCV2.From 2019 to 2022,we collected 276 clinical samples in some areas of Heilongjiang Province,tested PCV1,PCV2,PCV3,PCV4,and performed molecular genetic evolution analysis of the positive strains obtained.The results showed that the positive rate of :P CV2 was the highest,63.04%(174/276),the positive rate of PCV3 was 34.78%(96/276),and PCV1 and PCV4 were not detected.Genome-wide and ORF2 gene amplification was performed on PCV2-positive samples,resulting in a total of 18 PCV2-wide genome sequences and 43 ORF2 sequences.The nucleotide homology of PCV2 whole genome sequence was 92.9%-99.9%,the nucleotide homology with the reference strain was 90.4%-100%,and the homology of ORF2 gene was 86.0%-99.8%.Amino acid sequence alignment showed that there were 33 amino acid variant sites in Cap protein.Genetic evolution analysis showed that PCV2 a type accounted for 16.7%(3/18),PCV2 b type accounted for 16.7%(3/18),and PCV2 d type accounted for 66.7%(12/18)in PCV2 full-length genome sequence.The proportion of PCV2 a type in PCV2 ORF2 gene sequence was 9.3%(4/43),PCV2 b type was 6.97%(3/43),and PCV2 d type was 83.72%(36/43).Whole genome amplification was performed on the detected PCV3-positive samples,and 12 PCV3 whole genome sequences were obtained,and the PCV3 whole genome sequence glycotide homology was 98.6%-99.9%,and the nucleotide homology with the reference strain was 98.1%-99.6%,and the homology analysis showed that PCV3 had high genetic stability.In this study,we amplified the PCV2 Cap gene,constructed p ET-30a-PCV2-Cap prokaryotic expression plasmid,expressed PCV2 Cap protein,and after verifying the correct expression by SDSPAGE and Western blot,the immune mice prepared polyclonal antibodies,and identified the prepared polyclonal antibodies,and the results showed that the target protein with a size of about34 KDa was successfully expressed in BL21 competent cells.The prepared polyclonal antibody titer reached 1:15000,and Western blot could detect a specific band of 34 KDa size,indicating that the prepared polyclonal antibody had good specificity and laid a foundation for the development of subsequent diagnostic reagents.