Establishment Of Differential Diagnosis And Molecular Epidemiological Investigation Of Porcine Circovirus Type 2 And Type 3

Porcine Circovirus(PCV),a member of the Circovirus genus of the Circovirus family,can cause swine reproductive and respiratory diseases,weaner piglet syndrome,dermatitis and nephritis and other symptoms,and bring great economic losses to the pig industry in China.Porcine circovirus is currently classified into four types:PCV1,PCV2,PCV3 and PCV4.PCV2 and PCV3 were the main subtypes prevalent in pigs in China.In recent years,the phenomenon of mixed infection of these two types gradually appeared.Since it is difficult to distinguish between PCV2 and PCV3 in pigs infected with PCV2 in clinical appearance,it is imperative to establish a differential detection method for PCV of the two types and carry out molecular epidemiological investigation to further understand and reveal the evolution and pathogenic mechanism of the virus.In order to understand the infection of PCV2 and PCV3 in pigs in Shandong Province,Taq Man dual fluorescent quantitative PCR method was first established to distinguish PCV2and PCV3.Two pairs of specific primers and two Taq Man probes were designed according to the registered PCV2 and PCV3 gene sequences in Gen Bank.Primer concentration,probe concentration,annealing temperature and other conditions were optimized by using checkerboard method to determine the best reaction conditions and system.The standard curve was drawn using standard plasmid as template,and the sensitivity,specificity and reproducibility of the method were tested.The results showed that the correlation coefficients(R~2)of the dual fluorescence quantitative PCR method established in this study were all greater than 0.99.The method did not amplify PCV1,porcine parvovirus,porcine pseudorabies virus,etc.The detection limits of this assay for PCV2 and PCV3 were 10~2copies/?Land 1copy/?L,respectively,which was more sensitive than conventional PCR assay.The coefficients of variation for both intra-and inter-group repeated trials were less than 3%.The above results showed that the dual Taq Man fluorescence quantitative PCR method for PCV2 and PCV3 established in this study had good specificity,sensitivity and reproducibility,which provided technical support for subsequent studies.Subsequently,the method was applied to the detection of 77 samples from some pig farms in Shandong Province.The results showed that the positive rate of PCV3 was 14.29%(11/77),the positive rate of PCV2 was 32.47%(25/77),and the mixed rate of PCV3 and PCV2 was 7.79%(6/77)in some pig farms in Shandong province.In order to further understand the molecular biological characteristics and genetic variation of PCV2 and PCV3in Shandong,genome amplification,sequencing and analysis were performed on the isolated PCV2 and PCV3 strains.The results showed that the full genome homology of the 25 strains of PCV2 isolated in this study ranged from 96%to 99.8%,and the main subsets were PCV2b and PCV2d.The sequence homology of the 11 PCV3 strains was 98.4%-99.9%among each other,and distributed in PCV3a,PCV3b and PCV3c subsets.The Cap protein sites and Rep protein sites of PCV2 and PCV3 isolated in this study were mutated.Compared with PCV3,PCV2 had more Cap protein mutation sites.These data provided technical support and molecular biological basis for nucleic acid detection,epidemiological characteristics,genetic variation and prevention of PCV2 and PCV3 in Shandong.In view of the widespread prevalence of PCV3 in the world,it is of great significance to establish a rapid and accurate serological diagnostic method for the prevention and control of PCV3.This study expressed PCV3 Cap protein prokaryotic to establish an indirect ELISA method for rapid detection of PCV3 antibodies.The main antigen region of PCV3-cap gene was amplified by PCR method.The amplified product was ligated to the p ET-28a(+)prokaryotic expression vector and transferred to Ecoli BL21 for prokaryotic expression.The36.4 k Da protein was successfully expressed.Western blot analysis showed that the expressed protein had good reactivity after purification by inclusion bodies.The concentration of antigen coating,serum dilution ratio,secondary antibody dilution ratio and other conditions were explored to establish an indirect ELISA method.The optimal concentration of antigen coating was 2.5?g/m L,the optimal serum dilution ratio was 1:400,and the optimal dilution ratio of goat anti-pig enzyme labeled antibody was 1:30000.In this method,the lowest detection titer of PCV3 positive serum reached 1:25600.The positive sera of PCV2,CSFV,PRRSV,PRV and PEDV were negative.The variation coefficients of intra-and inter-batch repeated experiments were less than 3%.According to the optimized ELISA method,1299sera were detected,and the positive rate of PCV3 was 11%.The results showed that the established ELISA method had good sensitivity,specificity,repeatability and clinical applicability.In addition,we also immunized 6 week-old BALB/c mice with purified PCV3Cap protein to produce polyclonal antibodies.Blood samples were collected and serum samples were separated within two weeks after triple immunization.The prepared polyclonal antibody was confirmed to specifically recognize the eukaryotic expression protein of PCV3Cap.PCV3 strain was isolated and cultured with PK-15 cells,and the challenge test was carried out with the abrasive solution of PCV3 positive disease material in mice.The results showed that the PCV3 virus isolated in this study could not infect PK-15 cells and BALB/c mice.Subsequently,in order to further understand the pathogenic mechanism of PCV3,we preconstructed a dual-copy infectious clone plasmid of PCV3 and conducted a preliminary study on its pathogenicity.First,PCV2 was used as a template,and primers were designed to amplify the whole genome of PCV2.Dual copies of the full-length PCV2 gene sequence were successively inserted into Blunt plasmid by means of the mutation induced by the restriction site.The results showed that the transfected recombinant plasmid could produce infectious progeny virus,indicating that the infectious clone plasmid of PCV2 was successfully constructed.Reference to construct PCV2 dual copy infectious clone the whole genome amplification PCV3 plasmid construction method,the dual copies of the PCV3 full-length genome sequence by Eco R I/Bam H I and Bam H I/Sac I progressive inserted into a Blunt plasmid,build dual copy PCV3 infectious clone plasmid.The constructed plasmid was transfected into PK-15 cells and identified by IFA with mouse anti-PCV3 cap-polyclonal antibody.The obvious green fluorescence was visible,which proved that PCV3 dual-copy plasmid could successfully express viral protein.However,no virus particles were observed under electron microscopy,indicating that the dual-copy PCV3 plasmid constructed in this experiment could not save the virus particles,but could only express the virus protein instantaneously.In this study,a dual-copy PCV3 genome-wide plasmid and an infectious dual-copy PCV2 genome-wide plasmid were successfully constructed,providing a reference for the study of the molecular characteristics and pathogenesis of PCV2 and PCV3.