Study On Genetic Variability And DNA Vaccine Of Porcine Reproductive And Respiratory Syndrome Virus (PRRSV)

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of porcine reproductive and respiratory syndrome (PRRS), causing heavy economic losses to the swine industry all over the world.In this study, we designed three pairs of PRRSV specific primers to amplify ORF5, ORF7, and Nsp2genes of4PRRSV strains. We used pMD18-T vector to construct plasmids, and verified gene sequences by gene sequencing. Molecular biology software was used to analyze the sequences we got and the sequences of other PRRSV strains from NCBI. We analyzed the genetic homology and drew phytogenetic trees of PRRSV ORF5, ORF7, and Nsp2, respectively. The results indicated that the identity percent of the ORF5gene sequences was ranged from89.4%to99.5%; the identity percent of the ORF7gene sequences was ranged from94.1%to100%; the identity percent of the Nsp2gene sequences was ranged from83.3%to99.7%. The PRRSV strain HB was highly homologous with the strain of the vaccine strain of CH-la; DL-US strain was highly homologous with VR-2332; JX strain was highly homologous with highly pathogenic PRRSV (HP-PRRSV) strains which have30amino acids deleted in the Nsp2genes. The Nsp2gene of DL-TB strain had high homology with the vaccine strain of CH-la, but the ORF5and ORF7genes of this strain had high homology with HP-PRRSV strains. Therefore, we speculated that the DL-TB strain was the recombinant PRRSV strain of the traditional PRRSV and HP-PRRSV. Currently, HP-PRRSV was the overwhelming strain in our country, and it was different with the traditional PRRSV in pathogenicity. HP-PRRS was characterized by high temperature, high pathogenicity, and high mortality. How to prevent and control the HP-PRRS is becoming an urgent issue.Immunization is a main strategy to solve the problem. There are two types of commercial vaccines for PRRSV:killed vaccine and modified live vaccine. However, both of them have inherent drawbacks. Killed vaccine is weak in immunogenicity and it can’t always provide protective immunity against PRRSV infection. The modified live vaccine has a risk of reverting to virulence although it can confer protection against clinical diseases. As both of them can only confer limit and incomplete protection against PRRSV infection, a safer and more efficient PRRSV vaccine is in need. We used three strategies to enhance the immunogenicity of DNA vaccine of PRRSV. First, we constructed a plasmid expressing the fusion protein of GP4and GP5of PRRSV. Secondly, a fusion expression plasmid of cytotoxic-T-lymphocyte-associated protein4(CTLA4) and GP5of PRRSV was constructed. This targeted DNA vaccine could target antigen to antigen presenting cells (APCs). PRRSV-specific antibodies, neutralizing antibodies (NAs), cytokines of IFNy and IL4, and the Lymphocyte proliferation activity were evaluated. Both of the fusion expression plasmids could induce a significant enhancement of humoral and cellular immune responses. Thirdly, this effect could be further augmented when we boosted the mice with a killed PRRSV vaccine after DNA vaccine priming. Both the fusion expression and heterologous "prime-boost" strategies will be useful to improve the immune efficacy of vaccines against PRRSV in pigs in the future.