| Porcine parvovirus(PPV)is a collective term for a variety of parvoviruses that infect pigs in the Parvoviridae family,including the classic porcine parvovirus 1(PPV1),and the porcine parvovirus 2 to 7(PPV2-PPV7).Seven PPVs are divided into 4 different virus genera: PPV1 is Protoparvovirus,PPV2~PPV3 is Tetraparvovirus,PPV4~PPV6 is Copiparvovirus,and PPV7 is Chapparvovirus.To understand the infection status of PPV1-PPV7,this study established a multiplex PCR method to detect and analyse clinical samples from pigs in Henan Province;gene sequencing was performed on PPV2 and PPV7,which have a high infection rate,to analyze their genetic and evolutionary characteristics;the classical prokaryotic expression system E.coli BL21(DE3)is used to express the PPV7 Cap protein,and an indirect ELISA assay was established.This study supports the serological detection of PPV7.Details of the study are as follows:1.Establishment of PPV1/2/5 triple PCR and PPV3/6/7 triple PCR methodsIn this study,nucleic acids were extracted from PPV1/2/3/5/6/7 positive tissue samples kept in our laboratory,and standard positive plasmids were prepared to establish triple PCR assays for PPV1/2/5 and PPV3/6/7 respectively.The optimal primer concentrations for PPV1/2/5 triple PCR were: 0.8,0.2,0.4 μmol/L,PPV3/6/7 triple PCR were:0.4,0.4,0.2μmol/L.The specificity test showed that PPV1/2/5 or PPV3/6/7 did not cross-react with other PPVs,porcine reproductive and respiratory syndrome virus(PRRSV),porcine circovirus type 2(PCV2)and pseudorabies virus(PRV),with a sensitivity of 103 copies/μL for PPV1/2/5 triple PCR and 104 copies/μL for PPV3/6/7 triple PCR,which were consistent with single PCR;the clinical samples tested by the established multiplex PCR method were compared with the single PCR method and the compliance rate was 100%.The results indicate that the multiplex PCR method developed in this study is highly specific and has a high compliance rate,which can be used for the detection of clinical samples and provides a new technical tool for the rapid identification of PPV1/2/3/5/6/7 infections.2.Epidemiological survey of PPV in Henan Province from 2021 to 2022In this study,748 samples were selected from samples sent to pig farms in Henan Province in 2021-2022,and clinical samples were tested using the assay established in Trial1.The results showed that PPV2(16.04%)had the highest infection rate,followed by PPV7(15.11%).PPV1/2/3/5/6/7 were widely prevalent in all regions of Henan Province throughout the year,with the peak season being autumn and winter;PPV1/3/5 were not detected in farrowing piglets,PPV2/6/7 could be detected in pigs at all growth stages,nursery and fattening pigs with respiratory diseases,and PPV2/6/7 were detected in pigs with respiratory diseases.Among the different types of samples,PPV2 and PPV7 were more frequently detected in oral fluid,lung and spleen lymph nodes and less frequently in blood samples,PPV3 was often found in serum and tissues,PPV5 in oral fluid and PPV6 in blood samples;Mixed infections of PPV are serious and complex.PPV2 and PPV7 can be mixed with other PPV,PRRSV and PCV2,and PPV7 is the most common mixed infection with PCV2.This study provides a basis for further assessment of PPV infection and prevalence characteristics and clinical hazards..3.Molecular epidemiological investigation of PPV2 and PPV7In this experiment,4 PPV2 and 11 PPV7 positive samples were selected from different areas of Henan Province.And the PPV2 complete genome and PPV7 Cap gene were sequenced,and perform homology analysis,phylogenetic analysis and recombination analysis with domestic and foreign reference sequences to understand the prevalence and genetic variation of PPV2 and PPV7 in Henan Province.The results showed that the complete genome nucleotide homology of PPV2 prevalent strains ranged from 95.0% to99.7%,and they were in different evolutionary branches and were distantly related.The PPV2ZMD-20/2021,PPV2ZK-9/2021 and PPV2SQ-16/2021 strains were probably recombinant strains.Nucleotide homology of 11 PPV7 endemic strains of Cap gene ranged from 90.2% to 99.7%,amino acid homology ranged from 90.0% to 99.8%,and both nucleotide and amino acid homology to 45 reference sequences ranged from 84.7% to99.4%.The 11 PPV7 endemic strains were distributed in branches B,D,E and G.There were three gene lengths of 1 425,1 422 and 1 410 bp,and 15 bp were easily missing at 542-556 bp.It was concluded that PPV2 had large genetic evolutionary differences and recombination existed,and the location of recombination was not unique.PPV7 Cap gene had large differences,and both missing and non-deleted strains were widely prevalent in Henan Province.4.Establishment and application of the PPV7 indirect ELISA methodIn this study,the classical prokaryotic expression system E.coli BL21(DE3)expressed PPV7 Cap protein as an encapsulated antigen and an indirect ELISA method was established for the detection of PPV7 antibody.The optimal TMB development time was 15 min,and the test was positive when the S/P value was ≥ 0.3414.The specificity test showed no cross-reactivity with antibodies against CSFV,PRRSV,PCV2 and PPV1.The sensitivity was 1:600,and the intra-and inter-batch coefficients of variation were less than 7%.The method was applied to test 154 serums from pigs in Henan Province.The positive rate was42.86% for primiparous sows,57.50% for fattening pigs,50.00% for nursery pigs and46.88% for piglets.The results showed that the indirect ELISA method established by using p ET28a-PPV7 Cap protein as an encapsulated antigen has high specificity and sensitivity,good reproducibility and can be used for clinical PPV7 antibody detection.This study lays the foundation for the epidemiological study of the clinical novel porcine parvovirus and supports the analysis of the genetic evolution of PPV2 and PPV7.The established indirect ELISA assay has laid the foundation for the study of serological detection methods for PPV7. |
