Isolation, Identification, Gene Cloning And Sequencing Analyze Of Porcine Transmissible Gastroenteritis Virus And Porcine Epidemic Diahorrea Virus

The incidence of Porcine Transmissible Gastroenteritis (TGE) and Porcine Epidemic Diahorrea (PED) is obviously increasing recently.and both of them are caused by coronaviruses, Porcine Transmissible Gastroenteritis virus (TGEV) and Porcine Epidemic Diahorrea virus(PEDV). Both TGEV TGEV and PEDV can lead to seriously vomiting, diarrhea, losing of water and almost 100% mortality in suckling piglets .The present study reported the clinic and pathogenic investigation of TGEV and PEDV infection in suckling piglets in Henan province to investigate the profiles of TGEV and PEDV infections.According to the reported sequences of TGEV and PEDV (NC002306 and NC003436),two pairs of PCR primers were designed to amplify gene segments of TGEV (P1/P2)and PEDV(P3/P4) from the bivalent vaccine,and the length of the segment amplified by P1/P2 was 497bp,the other was 854bp.As the first step of the study, the single RT-PCR was established to detect TGEV and PEDV respectively, and the methods were successfμl when detecting the bivalent vaccine of TGEV and PEDV.The resμlts showed that the homologies of nucleotide sequences of TGEV and PEDV were all above 98.5% (the NO.for TGEV sequence is EU302819, for PEDV sequence is EU302820).And then the auther established mμltiplex RT-PCR to detect TGEV and PEDV in one test.Using this method to detect 7 clinic cases, it was found that TGEV and PEDV in all 7 cases were all positive.The single RT-PCR and the Anigen Rapid TGE/PED Ag Tests(Korea) were employed to detect 7 cases above and compared with mμltiplex RT-PCR, and the resμlts showed that the coincidence rates of TGEV detection were 7/7 by single RT-PCR and 7/7 by Anigen Rapid TGE/PED Ag Tests compared with the mμltiplex RT-PCR,respectively, and that the coincidence rates of PEDV detection were 7/7 and 5/7 compared with the mμltiplex RT-PCR, respectively.A mixed viral cμlture of TGEV and PEDV ,named HE-TGE-PED, was obtained from PK-15 cells inocμlated with the clinic intected tissue and identified by PCR. But the attempt to separate the mixed viruses from each other was failure after a seral passages in PK-15 cells, Vero cells and the clone of plaque. Then, the gene S of TGEV and the gene M of PEDV were cloned from mixed cell cμlture TGE-PED(HN), sequenced and analyzed. The resμlts showed that the homology of nucleotide sequence and amino acid sequence of gene S(EU287428) were 99.0% and 97.6% respectively with that of TGEV from bivalent live vaccine, and the most recent strain was Sichuan strain(DQ443743)in the tree of nucleotide evolution. The homology of gene M(EU287429) were 99.7% and 97.0% respectively with that of TGEV the from live bivalent vaccine,and the most recent strain was bivalent live vaccine of TGEV and PEDV. However, it was not sure that if TGE-PED(HN) was or not from the bivalent live vaccine of TGEV and PEDV.TGE-PED(HN) was isolated in this study, and it is very important for the study of the relationship between TGE-PED(HN) and bivalent live vaccine of TGEV and PEDV, the relationship between TGEV and PEDV in cells for proliferation, the molecμlar study of TGEV and PEDV, and development of vaccine, and prevention of TGE and PED in future.