Molecular Epidemiology Surveillance Of Viral Pathogens Related To Swine Reproductive Failure In Gansu Province And Primary Study Of DNA Vaccine Co-expressing GP5 And M Of HP-PRRSV

Porcine reproductive and respiratory syndrome (PRRS) is characterized by reproductive failure in pregnant sows and respiratory disease in all pigs. Since 2006, a highly pathogenic PRRS (HP-PRRS) has been found in China, which caused an immense economic loss in China. The pathogen was highly pathogenic PRRSV (HP-PRRSV), which was found a dis-continuous deletion of 30 aa in nsp2 protein. The HP-PRRSVs are still prevalent in swine herd in China and become a main cause of reproductive failure of sows and death in young pigs.In order to investigate the pathogens causing the reproductive failure of sows in Gansu province in 2007-2010, the specific primer pairs against PRRSV nsp2 and ORF5 gene were designed, according to the conserved sequences of PRRSV genome. The PCR detection showed that most of the clinical samples were PRRSV-positive. In order to well understand the genetic variation of PRRSV in China, we selected 7 strains sequenced in this study and some other Chinese reference strains to analyze the phylogenetic relationship and amino acid aligment. The phylogenetic analysis shows that Chinese PRRSV strains could be divided into 4 sub-groups. The classical strains mainly located in sub-group I,ⅡandⅢ, while the HP-PRRSVs located in sub-group IV. The HP-PRRSV was in far relation to traditional vaccine strains RespPRRS MLV and CH-1R, which indicated that these vaccines might be of malfunction in prevention of HP-PRRSV infection. The nsp2 protein of sub-group IV had some different types of deletion, among which most of the HP-PRRSVs were characterized by a dis-continuous deletion of 30 aa in nsp2 protein. Viruses of this type were the dominant strains in China. Our PRRSV strains 07N, 128, TS, XB and XIN belonged to sub-group IV and were of HP-PRRSV; strain XJ belonged to sub-groupⅢ, which was classical strain; Interestingly, QD strain belonged to sub-groupⅠin whole genome tree and nsp2 protein tree, while belonged to sub-group IV in GP5, M, and N protein trees. It may be concluded that QD strain was a recombinant strain between classical strain and HP-PRRSV strain. From the amino acid alignment, all sub-groups were characterized by different amino acids. The HP-PRRSV had the same characterization with virulent strains in residues responsible for virulence in GP5 and M protein. Moreover, amino acid mutation appeared in neutralizing epitope. The glycosylation sites began to accumulate and were close to the downstream neutralizing epitope. Hence, the glycosylation sites could cover up the neutralizing epitope, detain the production of neutralizing antibody, and enable the virus to escape from the immune response of host. In all, the HP-PRRSV infection in Gansu province is very common and it needs further reseaches for molecular epidemiology of PRRSV and development of new vaccine against PRRSV in China.In order to investigate the other viral pathogens related to swine reproductive failure in Gansu province, several primer pairs were designed to detect the viral pathogens, e.g. porcine parvovirus (PPV), porcine circovirus typeⅡ(PCV2), pseudorabies virus (PRV), and classical swine fever virus (CSFV). The detection results showed that two samples were PPV-positive, while five samples were PCV2-positive. No PRV- or CSFV-positive sample was detected in all samples. To further understand the genetic characterization of PPV and PCV2 isolated in Gansu province, the genomes of PPV and PCV2 were amplified with specific primers, respectively. The phylogenetic analysis of PPV showed that the PPV strains could be divided into 4 sub-groups. LZ strains isolated in this study belonged to II sub-group, which was common PPV strains in China; while JY strains belongs to III sub-group, which was composed of German PPV strains. This was the first isolation of Germany-like PPV in China. Phylogenetic analysis of PCV2 showed that the PCV2 strains could be divided into 5 sub-groups. The strains 124 and XIN isolated in this study belonged to sub-group 2b; strains XJ and XB belonged to sub-group 2d; while strain TS belonged to sub-group 2e. It can be concluded that the viral pathogens related to swine reproductive failure in Gansu Province are complicate. Molecular epidemiology should be conducted in order to well understand these epizootics in Gansu province.Vaccine prevention is a key measure to prevent the epizootics in domestic animals. With the outbreak of HP-PRRS in China, it is badly in need of an effective vaccine to prevent the prevalence of HP-PRRSV in China. However, the traditional inactivated vaccines usually did not provide enough immunologic potency to swine herds. In this study, DNA vaccine co-expressing GP5/M or modifiedΔGP5/M was constructed using pcDNA3.1 vector. The experiment in mouse model showed that both kinds of vaccines could induce high level of ELISA antibodies in mouse, as well as the virus-specific neutralizing antibodies. Moreover, the level of neutralizing antibody of modified GP5 group was higher than that of ordinary GP5 group. Cellular immune detections, e.g., lymphocyte proliferation assay, IFN-γdetection, and T-lymphocyte subsets detection, showed that both kinds of vaccines had good immune effect, compared with the control. The DNA vaccines co-expressing GP5 and M of HP-PRRSV would provide a new approach to develop the HP-PRRSV vaccine.