Development For Differential Diagnosis Of TGEV/PRCV And Preparation Of Monoclonal Antibodies Against TGEV N Protein

Porcine transmissible gastroenteritis virus (TGEV) is the causative agent of transmissible gastroenteritis of swine, which causes piglets vomit, diarrhea and death (100% high mortality). It is a world disease and causes great harm to swine industry. Porcine respiratory coronavirus (PRCV) is a natural deletion mutant of TGEV, whose homoly of nucleotide sequence reaches 96% compared to TGEV. Compared to TGEV, there are 621～681 nucleotides deletion at the N-terminal of S gene of PRCV, making PRCV lose two antigen sites. Because of similar antigenicity and morphology, TGEV couldn't be differentiated from PRCV by traditionary serodiagnosis and electron microscope. Today,more and more swine herds infect PRCV all over the world. So that, it is very necessary to constrcuct a quick and sensitive detection method.Thus, we developed a nested RT-PCR method targeted to the deletion in Spike gene of PRCV to differentiate and diagnose TGEV from PRCV. Two pairs of primers, P1 and P2, P3 and P4, were designed based on S gene nucleotide sequences of TGEV and PRCV, after sequences compared between PRCV published and TGEV attenuated-H strain. Nested RT-PCR was developed to amplify specific sequences, after extracting RNA from TGEV and PRCV cell cultures. The amplified segment with P1 and P2 of TGEV gene was 1336bp, P3 and P4 936bp and that of PRCV gene was 717bp, 315bp. This method was optimized by Orthogonal design. Specific straps were amplificated with PRCV and TGEV as templates, but not with other viruses (PEDV,PRoV,PRRSV,PRV,HCV,SIV). And the lowest quantity of both TGEV and PRCV were 4 TCID50/mL. Results showed that the nested PCR was rapid, specific and sensitive in detecting the clinical samples.We developed a loop-mediated isothermal amplification(LAMP) method to differential diagnosis TGEV and PRCV. Comparing the S protein sequences of PRCV published and TGEV attenuated H strain, we designed a pair of primers , named Q-FIP, Q-BIP, Q-F3, Q-B3 and QX-FIP, QX-BIP, QX-F3, QX-B3, targeted different and the same sequence on S gene respectively. LAMP was developed to amplify specific sequences, after RNA extracted from TGEV and PRCV cell cultures. There were a typical ladder strap using TGEV RNA as template, and the smallest amplificated straps were 184bp. And there was a typical ladder strap using PRCV RNA as template with QX-primers, and the smallest strap was 194bp. The amplified products were analyzed by agarose gel electrophoresis or visualized with SmartGreenⅠand MgCl2 as stain. This method was optimized by Orthogonal design. The results demonstrated that the RT-LAMP assay had no cross- reaction with other swine viruses (PEDV, PRRSV, PRoV). And the lowest quantity of both TGEV and PRCV were 0.1TCID50/mL. Therefore, the RT-LAMP assay provides a specific and sensitive means for detecting TGEV and PRCV in a simple, fast, and costeffective manner. And the visualized analysis could help us tell positive products from the negative.Both nested RT-PCR and LAMP are effective to differential diagnosis TGEV and PRCV specifically and sensitively. Furthermore, the RT-LAMP assay is more sensitive and can be performed in less well- equipped laboratories as well as fields.In this research, recombinant neucleocapsid protein of transmissible gastroenteritis virus(TGEV) which was expressed with prokaryotic expression system was used to immune BALB/c mice. Cell fusion was performed previously. Indirect ELISA assay was carried out to screen hybridoma cell lines using TGEV N potein as the antigen. By limiting dilution and 4 serial of clones, 3 hybridoma cell lines(named 1A9,1G7,1H8, respectively) secreting antbodies against TGEV N protein were obtained. The antibodies of all the three hybridoma cell lines belong to IgG1 subgroup. After extended culture and several freeze-thaw cycles, the monoclonal antibodies wre still stably secreted. The average number of chromosomes in 3 hybridomas was 84～100. Investigated through indirect ELISA, the titers of 3 cell-cultured antibodies were measured to be 1:640,1:320,1:800, and the titers of antbodiees produced in ascities were 1:6400,1:6400,1:12800, respectively. Immunofluorescence assays were performed with ST cell monolayers infected with TGEV probed by the antibodies or supernatant of SP2/0 cell culture.After staining with FITC-labeled secondary antbodies, strong yellow-green fluorescence was observed in plasma and on the membrane of cells. However, no fluorescence was detected with the addition of supernatant of SP2/0 cell culture. With recombinant N protein as immunogen, antibodies were found to specifically bind to pathogens with the confirmation of indirect immunofluorescence assays. Using purified TGEV and TGEV N protein as antigen, westernblot was performed to determine the correlation between the monoclonal antbodies and TGEV/TGEV N protein. After staining with fluorescein-labeled secondary antbodies, it was revealed that the supernatants of 3 hybridomas but not supernatant of SP2/0 cell culture recognized TGEV and TGEV N protein. The specificity of antibodies to virus was further ensured.The monoclonal antibodies against TGEV N protein could provide foundation for the development of blocking-ELISA of differential diagnosis TGEV and PRCV.