Dataset Information

Cloning and Sequence Analysis of N Gene of Transmissible Gastroenteritis Virus HYM-09 Isolated from Dog in China.

ABSTRACT: Transmissible gastroenteritis virus (TGEV) is the etiological agent of TGE, and dogs are potential carriers of TGEV. In this study, genomic RNA were extracted from TGEV designated HYM-09 isolated from dog naturally infected with TGEV. The nucleocapsid (N) gene of HYM-09 was amplified by RT-PCR and cloned into pMD18-T vector. The N gene cDNA was sequenced and encompassed an open reading frame of 1,149 nucleotides, encoding a 382-amino acids protein. Sequence analyses of the N genes were performed, including homologous comparison, phylogenetic tree analysis and residue substitution analysis. The results showed that there existed some unique mutations in the HYM-09 isolate N gene, but HYM-09 N gene shared over 96 % homologous identities compared with 12 TGEV reference strains derived from other regions or countries respectively. The phylogenetic tree analysis revealed that the HYM-09 branched into the most strains group. This study shows that the nucleotide sequence analysis can form a base or further study on the mutation trend of non-porcine TGEV.

SUBMITTER: Man C

PROVIDER: S-EPMC3550793 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature

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Cloning and Sequence Analysis of N Gene of Transmissible Gastroenteritis Virus HYM-09 Isolated from Dog in China.

Man Chaolai C Yu Xiaolong X

Indian journal of virology : an official organ of Indian Virological Society 20120814 3

Transmissible gastroenteritis virus (TGEV) is the etiological agent of TGE, and dogs are potential carriers of TGEV. In this study, genomic RNA were extracted from TGEV designated HYM-09 isolated from dog naturally infected with TGEV. The nucleocapsid (N) gene of HYM-09 was amplified by RT-PCR and cloned into pMD18-T vector. The N gene cDNA was sequenced and encompassed an open reading frame of 1,149 nucleotides, encoding a 382-amino acids protein. Sequence analyses of the N genes were performed ...[more]

PMID: 24293826

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Project description:We have recently isolated a transmissible gastroenteritis virus (TGEV) infectious construct designated TGEV 1000 (B. Yount, K. M. Curtis, and R. S. Baric, J. Virol. 74:10600-10611, 2000). Using this construct, a recombinant TGEV was constructed that replaced open reading frame (ORF) 3A with a heterologous gene encoding green fluorescent protein (GFP). Following transfection of baby hamster kidney (BHK) cells, a recombinant TGEV (TGEV-GFP2) was isolated that replicated efficiently and expressed GFP. Replicon constructs were constructed that lacked either the ORF 3B and E genes or the ORF 3B, E, and M genes [TGEV-Rep(AvrII) and TGEV-Rep(EcoNI), respectively]. As the E and M proteins are essential for TGEV virion budding, these replicon RNAs should replicate but not result in the production of infectious virus. Following cotransfection of BHK cells with the replicon RNAs carrying gfp, GFP expression was evident by fluorescent microscopy and leader-containing transcripts carrying gfp were detected by reverse transcription-PCR (RT-PCR). Subsequent passage of cell culture supernatants onto permissive swine testicular (ST) cells did not result in the virus, GFP expression, or the presence of leader-containing subgenomic transcripts, demonstrating the single-hit nature of the TGEV replicon RNAs. To prepare a packaging system to assemble TGEV replicon particles (TGEV VRP), the TGEV E gene was cloned into a Venezuelan equine encephalitis (VEE) replicon expression vector and VEE replicon particles encoding the TGEV E protein were isolated [VEE-TGEV(E)]. BHK cells were either cotransfected with TGEV-Rep(AvrII) (E gene deletion) and VEE-TGEV(E) RNA transcripts or transfected with TGEV-Rep(AvrII) RNA transcripts and subsequently infected with VEE VRPs carrying the TGEV E gene. In both cases, GFP expression and leader-containing GFP transcripts were detected in transfected cells. Cell culture supernatants, collected approximately 36 h posttransfection, were passed onto fresh ST cells where GFP expression was evident approximately 18 h postinfection. Leader-containing GFP transcripts containing the ORF 3B and E gene deletions were detected by RT-PCR. Recombinant TGEV was not released from these cultures. Under identical conditions, TGEV-GFP2 spread throughout ST cell cultures, expressed GFP, and formed viral plaques. The development of infectious TGEV replicon particles should assist studies of TGEV replication and assembly as well as facilitate the production of novel swine candidate vaccines.

Dataset Information

Cloning and Sequence Analysis of N Gene of Transmissible Gastroenteritis Virus HYM-09 Isolated from Dog in China.

Publications

Cloning and Sequence Analysis of N Gene of Transmissible Gastroenteritis Virus HYM-09 Isolated from Dog in China.

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