Development And Application Of Two Indirect ELISAs For Detection Of Antibodies Against Porcine Reproductive And Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome virus(PRRSV) is the causative agent of PRRS, a disease characterized by reproductive failure in sows, including early farrowing with stillborn piglets and late-term abortions, as well as respiratory distress in young pigs and all influenza-like disease in grow-finish swine. This disease brings large economic losses to the global swine industry. Therefore, it is extremely important to build available PRRSV diagnostic assays.The nucleocapsid protein(N) is the most abundant and immunogenic component of the virion. Among the nonstructural genes of PRRSV, Nsp9 contained is the most conserved region of arterivirus(and nidovirus) genomes and encodes the key enzymes for RNAdependent RNA polymerase(RdRP). These make them a suitable candidate for the detection of virus-specific antibodies and diagnosis of the disease. In this study, the N and Nsp9 protein of PRRSV was successfully expressed and used as the antigen to develop indirect ELISAs for detection of antibody against PRRSV. The established assays have a certain value in clinical practice of the immune surveillance and disease purification by diagnosing and differentiating inactivated vaccine from infected PRRSV by wild virus or attenuated vaccine.Firstly, to develop the N-iELISA and Nsp9-iELISA. The N and Nsp9 were expression by prokaryotic expression strain. The expression of the protein N and Nsp9 was induced by treating the culture with isopropyl-β-D-thiogalactoside(IPTG), and then were expressed as the denatured and soluble form, respectively, and then the two indirect ELISAs were optimized, and then the cut-off values were determined as 0.172 and 0.32.Secondly, to analysis the specificity and the sensitivity of the two developed iElISA. A total of 62 experimental pig serum samples which were picked and confirmed by Western blot. the overall agreements between the N-iELISA and Nsp9-iELISA with Western blot were 95.16% and 87.10%, respectively. Another 37 clinical serum samples were selected and tested by the two iELISAs and IDEXX-ELISA, and confirmed by Western blot. The result of IDEXX ELISA and the agreements between the N-iELISA and Nsp9-iELISA were 91.89% and 81.08%, respectively.Finally, to apply the two developed iELISA by testing experimental and field serum samples. Using the two iELISAs, swine sera collected sequentially from pigs experimentally infected with PRRSV different strain were used to react with N and Nsp9 protein in NiELISA and Nsp9-iELISA, respectively. We observed the kinetics of the anti-N and anti-Nsp9 antibody responses from pigs experimentally infected with PRRSV. The result was that the all serum samples were postive after 7 days postinoculation and 10 days postinoculation(infected with PRRSV CH-1R, or 21 days postinoculation infected with PRRSV SD-16) for the NiELISA and Nsp9-iELISA. There was a decrease slightly of the antibody titer to the N protein, while the antibody titers of Nsp9 continued to be high during the first 30 days.104 for inactivated PRRSV vaccine of pigs serum samples from different herds in different cities in Shanxi, China were tested, the positive rate of 93.27% and 2.88% respectively; 552 attenuated PRRSV vaccine pigs serum samples were also tested, the positive rate of 89.85% and 81.34% respectively.Therefore, the N-iELISA and Nsp9-iELISA that was developed successfully in this study can be used for detection antibodies against PRRSV. The established assays can be used successfully to diagnose and differentiate infected PRRSV from inactivated vaccine and wild virus or attenuated vaccine.