The Changes Of The Lymphocyte Subpopulations In Peripheral Blood Of The Pigs Inoculated With Highly Pathogenic PRRSV GD Wild And Vaccine Type And The Duplex Real-time RT-PCR Differential Detection

Porcine reproductive and respiratory syndrome (PRRS), the causative agent of which is PRRSV, is considered to be one of the most important infectious diseases causing reproductive failure in sows and respiratory disease in pigs of all ages. PRRS, one of the OIE listed diseases has caused enormous economic losses in porcine industry, and Chinese government defined it as a B disease. It will play an important role in the prevention and control of HP-PRRSV in China.But there were few report about the immune mechanism of the HP-PRRSV. In the study, the changes of the lymphocyte subpopulations in peripheral blood of the SPF pigs inoculated with highly pathogenic PRRSV GD wild strain and GD vaccine strain was analyzed, which was in a cellular way to discuss the effect between the lymphocyte subpopulations in peripheral blood of the SPF pigs and highly pathogenic PRRSV GD wild and vaccine strain.According to the aligment of complete genome among the39PRRSV isolates in Genbank and PRRSV GDr180vaccine virus, the conservative regions were found in Nsp2and Orf7which can be designed MGB probes for discriminating the GDr180vaccine virus and other PRRSV strains, then a duplex real-time RT-PCR was developed. Two sets of specific primers and fluorescence dye labeled MGB probes which were expected to differentiate PRRSV GDr180vaccine strain from wild type were designed based on the nucleotide sequence of Nsp2and ORF7respectively. Both of those two MGB probes were probed that they were able to hybridize to the specific sequence of PRRSV GDr180and the wild strain. The reliability of this method was confirmed by detecting the virus culture, serum and tissue samples from PRRSV infected pig. The sensitivity reached to thel9.4copies/μL of GDr180vaccine virus and34.2copies/μL of GD wild virus. Meanwhile, there were no cross-reactions among the8different PRRSV strains, Classical Swine Fever Virus (CSFV), Pseudo rabies Virus (PRV), Porcine Circovirus2(PCV2), and Porcine Parvovirus (PPV) which verify the specificity of this method. To sum up, the specificity, sensitivity and reliability of this duplex real-time RT-PCR method make it feasible for the differential detection of PRRSV GDr180vaccine strain from its wild type.