| Porcine parvovirus(PPV)is a non-enveloped,single-stranded,negative-strand DNA virus belonging to the Parvoviridae family.PPV genotype 1(PPV1),a major cause of SMEDI syndrome(stillbirth,mummied fetus,embryonic death,and infertility),was first isolated in Germany in 1965.Six new PPV genotypes(PPV2-PPV7)have been identified by methods including high-throughput sequencing.In China,PPV was first identified in 1983,and the prevalence of different genotypes of PPV in China has become a serious threat to Chinese swine industry.Co-infection of 7 PPV genotypes(PPV1-PPV7)may have a synergistic effect on SMEDI syndrome caused by PPV1.Since the emergence of new PPV in China,most studies have focused on the genetic characteristics of a single isolate or the epidemiology of PPV in a certain area,but there is a lack of systematic investigation on the epidemiology of PPV1-PPV7 in China.In addition,there is no commercial vaccine against PPV2-7 strains.Porcine reproductive and respiratory syndrome(PRRS)is a highly contagious swine disease resulting in huge economic losses to the global swine industry.The major clinical symptoms of PRRS include reproductive disorders in pregnant sows and respiratory symptoms in piglets.Since PRRSV was first identified in China in 1995,PRRSV has been spread throughout China,and the virus is constantly evolving to generate multiple genotypes and multiple lineages of PRRSV.Both PPV and PRRSV can cause reproductive disorders in sows.How to effectively prevent and control the diseases caused by PPV and PRRSV infections in China is two major problems.In this thesis,we carried out the following research works.At first,a series of PCR assays were established to detect and distinguish PPV1 to PPV7.A total of 435 clinical samples were collected from 8 provinces from 2016 to 2020,and the epidemiology of PPV1-PPV7 was systemically analyzed.Secondly,based on the PRRSV infectious cloning platform,two PRRSV chimeric viruses were constructed to evaluate its cross-protection efficacy.At the same time,the PRRSV infectious clone was used as a live vaccine vector to construct a chimeric virus recombinant expressing PPV2 Cap protein as a PPV live vector vaccine candidate strain.Study 1.Prevalence,co-infection and genetic evolution of porcine parvovirus types 1-7 from 2016 to 2020In this study,we developed a set of PCR differential detection methods for PPV1-PPV7 with satisfied specificity,sensitivity and reproducibility.These methods were applied to detect PPV1-PPV7 in 435 clinical samples collected from 8 provinces in China from 2016 to 2020.A total of 55.40%samples were PPV positive.PPV2 and PPV3 genotypes have the highest positive rate(both 22.53%).Co-infections of distinct PPVs were detected in all eight provinces,and 27.36%(119/435)of the samples were co-infected with at least 2 to 5 PPV genotypes.In addition,the co-infection rate of PPVs with PCV2 was 22.30%(97/435).In this study,20 representative PPV1-PPV7 complete genomes were determined and genome-based phylogenetic analysis was performed.The results showed that the genetic diversity of PPV was not increased significantly in the past five years.One HBTS20180519-152 was detected as recombinant with JX15 and JX38 serve as the parental strains.Both JX15 and JX38 were from wild boar while HBTS20180519-152 was from domestic pig,suggesting the transmission of PPV from wild boar to domestic pig.Selection pressure analysis based on VP2 sequences of PPV 1-PPV7 revealed that they were mainly under negative selection,with only a small number of positive selection sites detected in VP2 of PPV7.Overall,this study systematically investigated the prevalence,co-infection,and evolution of PPV1-PPV7 in pig farms in China from 2016 to 2020.Study 2.Construction of PPV recombinant PRRSV live vector vaccine candidate strainsBased on the epidemiological survey of porcine parvovirus in China,PPV2 is the predominant genotype circulating at present.At first,the PRRSV infectious clone rJSTZ1712-12(a natural attenuated strain)previously established in our laboratory was used as a backbone to construct two chimeric PRRSV strains including consensus sequences of genes encoding minor envelope proteins and major envelope proteins.The cross-protection efficacies of these two chimeric strains(rJS-ORF2-4-CON and rJS-ORF5-6-CON)were evaluated by pig inoculation and challenged with s heterologous NADC30-like isolate.The results indicated that minor and major envelope proteins of PRRSV play synergistic roles in inducing heterologous neutralizing antibodies and conferring cross protection.Secondly,we further used the rJSTZ1712-12 infectious clone as a live viral vector,and the Cap protein coding gene of PPV2 was cloned from clinical samples.The Cap coding gene of PPV2 was inserted between the nonstructural protein and the structural protein encoding genes of PRRSV by restriction enzyme ligation and homologous recombination.Overall,two PRRSV chimeric viruses were successfully constructed and their replication characteristics and cross protection efficacies were evaluated in this study.Meanwhile,a chimeric PRRSV strain expressing PPV2 Cap was constructed and rescued.The chimeric virus may be used as a candidate to develpe a genetically engineered vaccine against PPV and PRRSV prevalent strains,which could help to achieve the purpose of double protections with one injection. |
