Development Of ELISA Differentiation Diagnosis For A Dual-marker Vaccine Of Porcine Reproductive And Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome (PRRS) which characterized by the productivefailure in pregnant sows or respiratory problems in piglet is caused by porcine reproductive andrespiratory syndrome virus（PRRSV）, and become one of the most important diseases to swine industryworldwide. In China, the first PRRSV strain was isolated in1996, and in2006the highly pathogenicporcine reproductive and respiratory syndrome virus (HP-PRRSV) emerged and resulted in signigicanteconomic loss to swine industry.Vaccination is wildly used as the most effective strategy of control PRRS, both attenuated andinactivated vaccines have been developed for PRRSV, attenuated live vaccines led to better results inimmune efficiencies against PRRSV and used in most farms. Because the use of attenuated vaccinesCH-1R, JXA1-R, HuN4-F112and TJM, there are four types of PRRSVs in swine herds in China:classical PRRSV strain, vaccine strain derived from classic PRRSV strain, HP-PRRSV strain andvaccine stain derived from HP-PRRSV strain. However, there is no serological method to differentiatethe field infected animals from the vaccinated, and make it extremely difficult of PRRS eradication.Based on the infectious clone of PRRSV HuN4-F112strain, we developed a dual-marker vaccine forHP-PRRS (rHN4-Δ25+NP49), proving that the vaccine has good immune protection, and it hasunique genetic markers, which can be used to develop differential diagnosis of the vaccined pigs and thePRRSV infected.In this study we first investigate a RT-PCR method for differential diagnosis of rHN4-Δ25+NP49and other PRRSV strain. On the other hand, we focus on the ELISA for detection of the antibodyto NP49and25AA.1.Development of a RT-PCR method for detection and differentiation of rHN4-Δ25+NP49andother PRRSV strain. According to the Nsp2gene sequences of PRRSV in GenBank, one pair ofspecific primer was designed to amplify the partial Nsp2gene, then a RT-PCR method for differentialdiagnosis of rHN4-Δ25+NP49and other PRRSV strain was established. As a result, the amplifyfragment of the HP-PRRSV strains, classical PRRSV strains and rHN4-Δ25+NP49strain will be234bp,321bp and306bp, respectively. The method was used to detect120samples collected before, thereare65PRRSV-positive serum samples and39samples were rHN4-Δ25+NP49-positive, the result wasconsistent with the background of the samples. The result indicated that the method could differentiaterHN4-Δ25+NP49strain from other PRRSV strains.2.Development of two ELISA methods for detection of the antibody to NP49and25AA.25AAfrom Nsp2region of PRRSV and49AA from NP protein of NDV were artificially synthesized asantigen for detecting corresponding antibodies in swine sera by ELISA, the antigen concentration is500ng/hole,and serum dilution degree is1:40. NP49-ELISA method was used to detect679samplesfrom rHN4-Δ25+NP49vaccined pig, the results of NP49-ELISA and IDEXX PRRSV detection kitcoincided in87.9%. On the other hand,25AA-ELISA method was used to detect200unknown clinical samples, the results of25AA-ELISA and IDEXX PRRSV detection kit coincided in94.84%. Then25AA-ELISA was used to detect HuN4-F112immunized serum samples from1to126dpi, specificantibody against25AA can be detected from21dpi to126dpi, and it is same with the results detected byIDEXX PRRSV detection kit.Taken together, in this study we established a RT-PCR method to differentiate rHN4-Δ25+NP49strain from other PRRSV strains. The vaccine-immunized pigs could be differentiated from naturallyinfected ones by two ELISAs detecting antibodies against NP49or25AA peptide, respectively.Therefore, this study set up a foundation for further investigation of the differentiation diagnosis for aDual-marker vaccine rHN4-Δ25+NP49.