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Partial sequence of the spike glycoprotein gene of transmissible gastroenteritis viruses isolated in Korea.

ABSTRACT: The spike (S) glycoprotein of transmissible gastroenteritis virus (TGEV) is the predominant inducer of neutralizing antibodies and has been implicated in virulence and host cell tropism. In this study, the nucleotide and deduced amino acid sequences of the amino terminal half of the S glycoprotein gene of one Korean field TGEV strain (133) isolated in 1997 and three Korean field TGEV strains (KT2, KT3 and KT4) isolated in 2000 and HKT2 strain, KT2 passaged 104 times in ST cells, were determined. The amino terminal half of the S glycoprotein gene including antigenic sites A, B, C and D, were amplified by reverse transcriptase-polymerase chain reaction (RT-PCR). Amplified PCR products were cloned, sequenced, and compared with published sequences for non-Korean TGEV strains. Korea TGEV field strains had 98.5-99.5% nucleotide sequence and 97.2-99.0% amino acid sequence similarity with each other. They had 96.5-99.0% nucleotide sequence similarity and 94.9-97.6% amino acid sequence similarity compared to non-Korean TGEV strains. Korean TGEV strains had several specific nucleotide and amino acid sequences which were not found in foreign TGEV or PRCV strains. HKT2 strain differed by 0.89% in nucleotide and 2.03% amino acid sequences compared to original KT2 strain although the regions forming four antigenic sites were not changed. By phylogenetic tree analysis, Korean field TGEV strains were branched into different groups from non-Korean TGEV or PRCV strains. Korean TGEV field strains KT2 and 133 were branched in separate groups that were differentiated from the other Korean TGEV strains. The Korean TGEV strains seemed to be evolved from a separate lineage of TGEV strain.

SUBMITTER: Kim SJ

PROVIDER: S-EPMC7117453 | biostudies-literature | 2003 Jul

REPOSITORIES: biostudies-literature

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Partial sequence of the spike glycoprotein gene of transmissible gastroenteritis viruses isolated in Korea.

Kim Soo Joung SJ Han Jeong Hee JH Kwon Hyuk Moo HM

Veterinary microbiology 20030701 3

The spike (S) glycoprotein of transmissible gastroenteritis virus (TGEV) is the predominant inducer of neutralizing antibodies and has been implicated in virulence and host cell tropism. In this study, the nucleotide and deduced amino acid sequences of the amino terminal half of the S glycoprotein gene of one Korean field TGEV strain (133) isolated in 1997 and three Korean field TGEV strains (KT2, KT3 and KT4) isolated in 2000 and HKT2 strain, KT2 passaged 104 times in ST cells, were determined. ...[more]

PMID: 12814887

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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.

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Partial sequence of the spike glycoprotein gene of transmissible gastroenteritis viruses isolated in Korea.

Publications

Partial sequence of the spike glycoprotein gene of transmissible gastroenteritis viruses isolated in Korea.

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