key: cord-0727899-j9338csa authors: Li, Jian-qiang; Cheng, Jie; Lan, Xi; Li, Xue-rui; Li, Wei; Yin, Xiang-ping; Li, Bao-yu; Yang, Bin; Li, Zhi-yong; Zhang, Yun; Liu, Ji-xing title: Complete genomic sequence of transmissible gastroenteritis virus TS and 3′ end sequence characterization following cell culture date: 2010-06-06 journal: Virol Sin DOI: 10.1007/s12250-010-3108-2 sha: 5c947a9467200e2f74640d07b83debd64cd6645d doc_id: 727899 cord_uid: j9338csa The complete genome sequence of transmissible Gastroenteritis virus (TGEV) strain TS, previously isolated from Gansu province, was cloned and compared with published sequence data from other TGEV strains. Phylogenetic tree analysis based on the amino acid and nucleotide sequences of the S gene showed that the TGEV strains were divided into 3 clusters. TGEV TS showed a close evolutionary relationship to the American Miller cluster but had a 5′ non-translated region (NTR) sequence closely related to the American Purdue cluster. Continued culture in different cell types indicated that TGEV TS virulence could be attenuated after fifty passages in Porcine kidney (PK-15) cells, and that the Porcine kidney cell line IB-RS-2 (IBRS) was not suitable for culture of the TGEV strain TS. highly conserved core sequence (CS), 5'-CUAAAC-3', or a related sequence, are present at sites immediately upstream of the TGEV genes [1] . The S glycoprotein makes up the large surface projections of the virion and plays an important role in the attachment of viral particles to host cell receptors, with subsequent penetration into the cell by membrane fusion. The S glycoprotein also stimulates the production of neutralizing antibodies in the host [18] . The M and E proteins are essential for viral envelope formation and release; the M protein can also stimulate the production of interferon (IFN) [12] . The N protein participates in transcription of the viral genome, the formation of the viral core, and packaging of viral RNA [20] . The Coronavirus replicase is a multifunctional polyprotein with helicase and protease activities necessary for the transcription of viral RNA [4, 23] . Four major antigenic sites, A, B, C and D, were characterized on the N terminus of the S protein. Using monoclonal antibodies, the adjacent sites A and B were mapped to a region of approximately 200 amino acids (aa) beginning from residue 506 at its N terminal boundary. The two antigenic sites also overlapped with the S protein domain encompassing aa 522-744 that mediates aminopeptidase N (APN) receptor binding [5, 6, 8] . After continuous passage in cell culture, TGEV isolates gradually lose their virulence and may shift from enteric to respiratory tropism [14] . Attenuation of virulence and tropism shift can also occur in nature, an example being the naturally occurring s and orf3 gene deletion mutant, the Porcine respiratory coronavirus (PRCV), which has both reduced pathogenicity and a predominantly respiratory tropism [14] . Although it is generally accepted that deletions in the spike and orf3 genes contribute to tropism change and attenuation in PRCV, other genes may also be involved. For instance, amino acid mutations in the M protein affect the ability of attenuated Purdue TGEV P115 to induce IFN-alpha, implying a potential role for the M protein in altered host response and virulence [12] . In this study, the complete genome of TGEV TS strain isolated in Gansu province has been cloned. In addition, the virus was continuously propagated in different cell lines and the 3' end genome sequences were also cloned. These data are useful for further study of the molecular structure of the TGEV strains prevalent in China, and the characterization of the long-term TGEV genome stability in different host cells. TGEV strain TS was isolated from a suburb in Gansu province, China. Swine testicle (ST), PK-15 and IBRS cells were grown as monolayers in DMEM (GIBCO, USA) containing 10% fetal calf serum (GIBCO, USA) and 5% CO 2 in air. Samples were obtained by passaging the first TGEV field isolate 10 times in swine intestine and named TS-ST. Total RNA was isolated from infected cell samples using a RNA extraction kit (Qiagen, Germany) following the manufacturer's instructions. The RT-PCR amplifications were carried out using seventeen primer sets (Table 1) and an RT-PCR amplification Kit (Toyobo, Japan) according to the manufacturer's instructions (Fig. 1 ). Phylogenetic analysis was performed for the TGEV TS strain compared to other coronavirus strains retrieved The complete genome of the TGEV TS strain The replicase genes were designated genes 1a and 1b, and share 43 overlapping nucleotides. The replicase 1a gene was predicted to encode a protein of 4017 aa Table 2 ). The protein of 261 amino acid in length. The nucleotide and predicted amino acid sequences (Table 3) . In group 2 (Fig. 2) . The tree based on the deduced amino acid sequences of the TGEV replicase genes revealed that the strains compared were divided into 2 groups: the Miller group and the Purdue group (Fig. 3) . The tree based on the nucleotide and amino acid sequences of the TGEV s genes showed that all the TGEV strains analyzed were divided into 3 clusters, and that the TGEV TS strain shared the closest relationship with Miller M6 and HN2002 (Fig. 4) . Another Position TS TS-ST TS-PK 158 T T C 202 G T T 290 G C C 295 C C T 952 T T C 1253 T T T 1691 C C A 1753 T T G s gene 3629 T C C 50 G A A 85 G G A 176 C T T 660 T T G The uORF sequences of HCoV-299E and IBV were found to be eleven codons in length, while those of MHV and PEDV were eight and twelve codons, respectively. Meanwhile, the uORF of TGEV encodes a peptide of only three-amino acids [11] . In this study, we demonstrated that the TS strain has the same three-amino acid uORF peptide as other TGEV strains, indicating that uORF is highly conserved in TGEV. Moreover, most of the 5' NTR sequences containing uORF were conserved, except for the first few nucleotides. In this study, we used two sense PCR primers beginning with the sequences "ACTTTTAAA sequences showed that they were highly conserved except for nucleotide deletions in some strains (Fig. 6) . We speculate that incomplete sequencing is responsible for the presence of these deletions, and propose that the first several nucleotides of the 5' residues are sufficient for TGEV RNA replication [2] . The principal component of the Coronavirus genome is the replicase gene, which contains two large open reading frames, orf1a and orf1b, from which two products are translated. The latter is synthesized via a ribosomal frameshifting mechanism, facilitated by a pseudoknot structure [7] . orf1b sequences in two Chinese TGEV strains, TS and SC-Y, were predicted to encode a protein of 2 678 aa. In fact, because the ribosomal frameshifting mechanism was not accounted for, two amino acids were deleted in ORF1b of TS and SC-Y. In common with other TGEV strains, the predicted orf1a sequences of TS and SC-Y extended from nucleotide 315 to 12 054. This resulted in a 4 017-codon orf that overlapped orf1b from nucleotide 12 011 to nucleotide 20 368, with the capacity to encode a polypeptide of 2 680 amino acids (Fig. 7) . The orf3 and s genes have been hypothesized to be important in the virulence and pathogenesis of TGEV infections [19] . Analysis of TGEV ORF3 sequences revealed that TS had equal numbers of ORF3 nucleotides and amino acids as the virulent Miller M6 strain. The G residue at nucleotide position 655 of the spike protein was essential to maintain enteric tropism of the TGEV strain PUR46-MAD, and mutation at this nucleotide caused a shift from enteric to respiratory tropism [3] . The T to G mutation at nucleotide 1 753 of the s gene is regarded to be a signal of attenuation [25] . this deletion was also thought to play a role in attenuation [18] . Comparison of the attenuated Miller of TGEV had been considered to affect virulence [13] , we found no deletion among the TGEV strains and [25] . In this study, we confirmed that PRCV-ISU-1 had a closer relationship with Miller strains than the Purdue strains. The TS strain shared 100% aa homology with PRCV-ISU-1 in the e gene, indicating that TS may have the closest relationship with the PRCV-ISU-1. Interestingly, the phylogenetic tree based on the s gene sequence showed that PRCV-ISU-1 had the closest relationship with TSX, which belongs to the British cluster. 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