Detection And Genetic Variation Analysis Of Porcine Parvovirus 7 In Partial Areas Of Hunan Province

Porcine parvovirus（PPV）is one of the most common pathogens,causing reproductive disorders of sows with many genotypes,which will cause serious losses to the swine industry.In 2016,PPV7 was first identified in USA,and has since been reported in China,Poland,South Korea,Sweden,and Brazil,it has been distributed widely in several areas of China.PPV7 is a novel porcine parvovirus,the present study of the epidemiology and pathogenesis are still in the initial stage.Therefore,it is urgent to accelerate the in-depth study of PPV7,especially the etiological investigation,the molecular characteristic and the expression of VP2protein.1.Detection of Porcine Parvovirus 7 in some areas of Hunan ProvinceIn this study,PCR was used to detect 422 samples of pig tissue and semen DNA,and the results showed that the number of PPV7 positive samples was 70（the positive rate was 16.59%,70/422）,among which the positive rate of PPV7 in pig tissue samples was 19.58%（65/332）,and in pig semen samples was 5.56%（5/90）.The complete genome sequence of one strain of PPV7 and the full-length VP2 gene of nine PPV7 strains were obtained,homology analysis showed that nucleotide and amino acid homologies of nine strains of PPV7 VP2 genes were88.7-99.85%and 88.8-99.8%,respectively,and no homologous recombination was found.The co-infection of PPV7 and PCV3 was further detected by RT-PCR.The positive rate of PPV7in PCV3 positive samples was 31.4%,while the positive rate of PPV7 in PCV3 negative samples was 14.4%.These results showed that the positive rate of PPV7 in PCV3 positive samples was significantly higher than that in PCV3 negative samples.The RT-PCR Ct values of PCV3 and PPV7 were analyzed statistically,the results showed that the Ct value of PCV3in PPV7 positive samples was significantly lower than that in PPV7 negative samples,suggesting that PPV7 is an important cofactor in PCVAD and PPV7 may stimulate PCV3replication,thus enhancing PCV3 viremia.In addition,the PPV7 positive rate in PCV3positive samples from sows with reproductive disorders（45.9%）was also significantly lower than that in PCV3 positive samples without reproductive disorders.2.Genetic variation analysis of PPV7 genome and VP2 gene sequenceThe complete genome sequences of 45 strains of PPV7 and 59 gene sequences of NS1and VP2 from Gen Bank were collected for genetic evolution analysis and antigenic epitope prediction.Reconstruction of Maximum clade credibility（MCC）tree from 45 complete PPV7genomes,evolutionary analysis found that Chinese PPV7 strain（2004）was the most likely PPV7 ancestor strains.Evolutionary rate analysis found that VP2 gene has a more rapid evolutionary rate(2.19×10^-3/site/year)than NS1 gene(8.01×10^-4/site/year),which is comparable to the rates of most RNA viruses.The antigenic profiles of PPV7 Cap were revealed and there were indications that PPV7 used antigenic shift to escape from the host’s immune surveillance.We predicted 6 linear B-cell epitopes（A-F）for PPV7 VP2.According to the results of bioinformatics analysis,the homology of VP2 amino acids between PPV7 and PPV1 was only 11.6%,and no homology sequences were found in the comparison of the epitope sequences of them,suggesting that PPV7 and PPV1 have very low cross-immune protection.The B cell epitopes identified in this study were expected to be used in the design of PPV7 vaccine or the study of serological diagnostic.3.Expression,purification and identification of PPV7 VP2 proteinAs the main structural protein of PPV,VP2 protein has good immunogenicity and can induce the production of specific neutralizing antibodies.In order to study the expression characteristics of PPV7-YY-25 Hunan strain,this study used the insect baculovirus expression system to express the VP2 protein of PPV7-YY-25 Hunan strain.The results showed that the recombinant plasmid pFast Bac-1-PPV7-VP2 was successfully constructed,IFA and Western blot analysis showed that the recombinant protein could be expressed correctly in cells and secreted into supernatant.PPV7 VP2 protein can be used for the preparation of polyclonal antibodies and the establishment of PPV7 ELISA serological detection method,but also for VP2 protein assembling into virus-like particles to lay the experimental foundation,and can be helpful to the development of new PPV7 vaccine.Through the epidemiological investigation of PPV7 in some areas of Hunan Province,this study enriched the epidemiological data of PPV7 infection and laid a scientific foundation for the monitoring,diagnosis and prevention of the disease.The genetic evolution and epitope prediction of PPV7 revealed the evolutionary rate of PPV7 and predicted the linear B cell epitope of PPV7,which provided a certain reference for the development of vaccine and the establishment of serological diagnosis method of PPV7.Finally,the VP2 protein of PPV7strain was studied,which laid the foundation for the preparation of PPV7 polyclonal antibody and the establishment of ELISA detection method.