Isolation And Identification Of Transmissible Gastroenteritis Viruses And Their Genomic Sequencing And Analysis

Transmissible gastroenteritis virus(TGEV) is an enveloped signal-strand positive RNA virus belonging to Coronaviridae(CoV) family and Nidovirales order Alpha-CoV. It is the pathogen that replicates in the cytoplasm of villous epithelial cells of the swine small int estine, which causes transmissible gastroenteritis(TGE). Now in the swine farms, vaccination is the preferred measure to prevent and control TGE. However, TGEV has the characteristics of high mutation, recombination in practice and produces antigenic variation and shift. The vaccination with tranditional vaccine has difficulties in providing efficient immune protection. So isolation and identification of current clinical epidemic TGEV strains and illumination of their biological characteristics is the foundation of new efficient vaccine study.Patholigical materials of small intestines from suspected TGEV-infected pig in Qiqihaer City were processed sterile then inoculated in swine testis(ST) serially, and stable typical cytopathic effect(CPE) was observed. According to results from the RT-PCR and indirect immunofluorescence assay, it was proved that two TGEV field strains were successfully isolated, and named HE-1 and HE-4 respectively. With electron microscope, classical Co V morphological character could be observed.According to TGEV-HX genomic sequence in the GenBank, 13 pairs specific primers were designed to mulitify TGEV structure and non-stucture genes. At the same time RACE Kit was used to clone the non-coding regions of the genome 5’UTR and 3’UTR. Finally, the total genomic sequences of TGEV HE-1 and HE-4 strain were obtained and analyzed with bioinformatic software. The results showed that the TGVE isolates HE-1and HE-4 had high homology with others reference TGEV strains, whose genomic informatio ns had been submitted to Gen Bank before. They had highest similarity 99.8% with China strains AYU, HX, WH-1 and tranditional strain of Purdue P115, and at the same time they had lowest similiruty of 97.4% with variant strain of PRCV ISU-1. Phylogenetic analysis based on the complete viral nucleotide sequence showed HE-1 and HE-4 had closer relationship with TGEV Purdue strain, but not Miller strain. However, they were varied for the phylogenetic analysis based on the different single genes, and these might be the result of different conservation and diversity of these genes in the evolutionary process. TGEV isolates HE-1 and HE-4, their whole genome are composed of 28, 580 nucleotides, have the typical characteristics of TGEV gene sequences. 5’ and 3’ UTR is relatively conservative. Unstructured genes of ORF1 a, ORF1 b, ORF3 a, ORF3 b, and ORF7 have no insertion and deletion, and the nearly same results could be found in the structure protein membrane protein(M), nucleoprotein(N) and small membrane protein(E) gene,except 4, 2, and 1 amino acids mutated in the respective genes. However, it was found that spike protein(S) of TGEV HE-1 and HE-4 isolates, their 562-564 aa NNT glycosylation site was deleted compared with the classical TGEV Purdue strain; and their 375-377 aa NDT glycosylation site deleted with the TGEV TS strain. In addition, it was found that HE-4 strain has mutation at 385 aa(S-N) at the major antigen site D in S protein. In summary, the successful isolation, identification and genomic analysis o f TGEV HE-1 and HE-4 strains, this will provide material and theoretical basis for more profound study the biological characterics of TGEV and development of new and effective vaccine against TGE.