Transmissible Gastroenteritis Virus Isolates Research Articles

English

Open reading frame (ORF7) of transmissible gastroenteritis virus (TGEV) plays important functions in regulating virus virulence. In this study, the ORF7s were cloned from non-porcine TGEV isolates (HYM-09, HYM-09-1 and HYM-09-2), respectively. Sequence analyses showed that there existed some new unique mutations in the three non-porcine TGEV ORF7s, and which had relative farther genetic relationship and evolution distance to ORF7s of other TGEV strains. The study can serve as a foundation for further insight into the virulence change or mutation trend of TGEVs. Key words: Non-porcine transmissible gastroenteritis virus (TGEV), open reading frame (ORF7), phylogenetic analysis, homology modeling.

New mutations existing in the nucleocapsid (N) gene of non-porcine TGEV strains isolated in China

Transmissible gastroenteritis virus (TGEV) is the etiological agent of TGE, and non-porcine hosts are potential carriers of TGEVs. In this study, genomic RNA were extracted from TGEVs designated HYM-09, HYM-09-1 and HYM-09-2 isolated respectively from non-porcine hosts (dogs, cats and foxes) naturally infected with TGEVs. The nucleocapsid (N)genes of the three non-porcine TGEV strains were amplified by RT-PCR and cloned into pMD18-T vector respectively. Each N cDNA was sequenced and encompassed an open reading frame of 1149 nucleotides, encoding a 382-amino acids protein. Sequence analyses of the N genes were performed, including homologous comparison, phylogenetic tree analysis and homology modeling. The results showed that there existed some new point mutations in the three non-porcine TGEV isolates N genes. The phylogenetic tree analysis revealed that these non-porcine TGEVs N genes had closer genetic relationships and evolution distance to the N genes of Chinese TGEV strains. The three-dimensional structure of the N protein (32-154AA (amino acid)) of HYM-09-2 generated by homology modeling was different from that of other TGEV strains (including HYM-09 and HYM-09-1).These data serve as a foundation for further insight into the virulence or mutation rates of the non-porcine TGEVs. Key words: N gene, cloning, TGEV, non-porcine, phylogenetic analysis, homology modeling.

A real-time TaqMan ® RT-PCR assay with an internal amplification control for rapid detection of transmissible gastroenteritis virus in swine fecal samples

A TaqMan probe-based real-time RT-PCR assay was developed for simultaneous detection of RNA of transmissible gastroenteritis virus (TGEV) in pig fecal samples and RNA of enhanced green fluorescent protein (EGFP) added exogenously as an internal amplification control. The TGEV primers and probe were designed to be specific to a portion of the S gene sequence conserved in all TGEV isolates, but absent in the closely related porcine respiratory coronaviruses. The optimized TaqMan assay detected a minimum of 2.8 copies of in vitro transcribed RNA of the target S gene and RNA extracted from 1 TCID 50/ml of TGEV. Using 113 clinical samples received at our diagnostic laboratory over a 4-year period, the performance of the assay was tested and compared with that of a previously described nested RT-PCR assay. All the fecal samples which tested positive for TGEV by the nested RT-PCR assay also tested positive by the TaqMan assay. However, approximately 9% of the samples that tested negative by the nested RT-PCR assay tested positive by the TaqMan assay. These results indicate that the developed TaqMan assay is a highly sensitive diagnostic test for rapid detection of TGEV in pig fecal samples.

Development of a novel real-time RT-PCR assay with LUX primer for the detection of swine transmissible gastroenteritis virus

Real-time RT-PCR assay, based on light upon extension (LUX) fluorogenic primer and LightCycle technology, was developed for rapid detection of transmissible gastroenteritis virus (TGEV). Viral RNA from different TGEV isolates and clinical specimens was detected. To evaluate the sensitivity of the assay, a gel-based RT-PCR method targeted at the same 101 bp sequence was also developed. Serial 10-fold dilutions of TGEV RNA were detected by the two methods. Although the real time method used only 2 μl RNA for each reaction, a 10-fold increase of sensitivity over that of the gel-based method, which used 10 μl RNA was demonstrated. The study indicates that the LUX assay reported below is rapid, reliable and sensitive and it has the potential for use as an alternative molecular method for TGEV diagnosis.

Characterisation of a recent virulent transmissible gastroenteritis virus from Britain with a deleted ORF 3a.

SummaryAnalyses of transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus (PRCV) isolates have suggested that tropism and pathogenicity are influenced by the spike protein and ORF 3. In general, enteric viruses (TGEV) have been shown to contain intact spike and ORF 3 genes, whilst respiratory isolates (PRCV) have major deletions within both regions. Virulence has been correlated to a functional ORF 3. Here, sequence analysis of a recent isolate of virulent TGEV, revealed a variant with an intact spike gene, but a large deletion in ORF 3a. This suggests that ORF 3a is not essential for enteric virulence.

Field isolates of transmissible gastroenteritis virus differ at the molecular level from the Miller and Purdue virulent and attenuated strains and from porcine respiratory coronaviruses.

The diversity in selected regions of the transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus (PRCV) genomes was analyzed among known TGEV and PRCV strains and field isolates. The N-terminal half of the spike (S) glycoprotein gene and open reading frames (ORF) 3, 3-1 and 4 were amplified by reverse transcriptase reaction and polymerase chain reaction (RT/PCR), and analyzed using restriction fragment length polymorphism (RFLP) patterns of the amplified DNA. Reference TGEV strains (Miller and Purdue) and a PRCV strain (ISU-1), and TGEV and PRCV field isolates were analyzed. Based on the size of the ORF 3, 3-1 and 4 RT/PCR products, TGEV and PRCV strains could be quickly and easily differentiated into three groups designated TGEV Miller, Purdue types and PRCV. By RFLP analysis of the N-terminal region of the S glycoprotein gene and ORFs 3, 3-1 and 4, TGEV and PRCV strains were differentiated into five groups using the restriction enzyme Sau3AI. Sequence analysis of a PCR product in the ORFs 3, 3-1 and 4 from virulent and attenuated Miller strains demonstrated additional differences in that region which have been correlated with a change in virulence of TGEV isolates.

Discrimination between transmissible gastroenteritis virus isolates.

SummaryTwenty TGEV isolates were compared by sequencing a 393–414 nucleotide stretch near the 5′ end of the S gene, after amplification by RT-PCR. This part of the S gene is known to show considerable variation between porcine, canine and feline coronaviruses and is completely deleted from porcine respiratory coronaviruses. The discrimination achieved by nucleotide sequence analysis was compared with that obtained by monoclonal antibody typing. The viruses could be split into several clusters, and recent isolates of TGEV from England, The Netherlands and Belgium showed the greatest differences compared to earlier reference types. However, not all viruses with unique isolation histories were distinct, suggesting either genetic stability over many years, laboratory cross-contaminations or repeated introductions of similar viruses into the field. Firm conclusions on evolutionary trends cannot be drawn without obtaining a larger number of isolates, preferably from outbreaks with known epidemiological links. The sequences of some field isolates from the 1980s contained both nucleotide deletions and insertions. The latter included a short sequence of fourteen nucleotides with identity to a region of the TGEV polymerase gene.

Detection of transmissible gastroenteritis virus by RT-PCR and differentiation from porcine respiratory coronavirus

An RT-PCR method was developed that amplified genetic material from the 5′ end of the S protein gene of both transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus (PRCV), but discriminated between the two by the size of the product generated. A number of restriction endonuclease enzymes were assessed for recognition of the amplicons so produced. The assay was shown to detect viral RNA from all of the 26 different TGEV and PRCV isolates examined, covering a period from 1946 to 1996. Detection of TGEV in clinical specimens was possible using a spin column method to extract RNA and sensitivity was compared to virus isolation and antigen detection ELISA. The method could provide a means of confirming positive results from immunological screening tests such as FAT and ELISA, reducing the need for virus isolation and convalescent serology.

Molecular differentiation of transmissible gastroenteritis virus and porcine respiratory coronavirus strains. Correlation with antigenicity and pathogenicity.

Transmissible gastroenteritis virus (TGEV) causes an economically important enteric disease of swine. Differences in the pathogenicity, antigenicity and tissue tropism have been observed among porcine coronaviruses. Although porcine respiratory coronavirus (PRCV) is antigenically similar but not identical to TGEV isolates, these respiratory coronaviruses differ markedly in pathogenicity and tissue tropism compared to TGEV isolates. Using a reverse transcriptase/polymerase chain reaction-restriction fragment length polymorphism (RT/PCR-RFLP) assay, TGEV and PRCV isolates were assigned to several distinct groups. By RFLP analysis of the 5' region of the S gene, TGEV strains were differentiated into 4 groups using the restriction enzyme Sau3AI. A fifth Sau3AI group contained the PRCV isolates. These 5 groups correlated with antigenic groups previously defined using monoclonal antibodies in our laboratory. Several restriction enzymes could be used to differentiate the TGEV strains into Miller and Purdue types. Analysis of a PCR amplified product in the 3 and 3-1 genes indicated the RT/PCR-RFLP assay results for TGEV Miller strains could be correlated with lower virulence created by passage in cell culture.

Antigenic and biological diversity among transmissible gastroenteritis virus isolates of swine

Twenty-four field isolates of transmissible gastroenteritis virus (TGEV) were isolated and examined for antigenic and biological characteristics. Most TGEV isolates produced a typical cytopathic effect (CPE) in swine testis (ST) cell culture, which included a ballooning or lifting away of the infected cells from the cell monolayer with heavy granulation evident. Minor variations in CPE were observed with one isolate, IA-145. Protein profiles of the TGEV isolates as determined by SDS-PAGE were essentially identical, with the exception of the isolate IA-101. The TGEV isolate IA-101 presented a higher molecular mass M protein and lacked an N protein doublet that was present in all other TGEV isolates. The TGEV isolates were shown to be closely related antigenically by using hyperimmune sera in a virus neutralization (VN) test. Some antigenic diversity was detected by utilizing monoclonal antibodies (mAbs) in a VN test. Titers of the mAbs were highest with the homologous Miller TGEV, and one virus isolate, IA-156, was very poorly neutralized with the mAbs used in this study. Indirect immunofluorescence assay (IFA) results were similar to those obtained by the VN test. These studies show that some biologic and antigenic diversity exists among TGEV isolates.

Differentiation between transmissible gastroenteritis virus and porcine respiratory coronavirus using a cDNA probe.

A plasmid, pG3BS, containing a cDNA clone from the 5' coding region of the peplomer glycoprotein gene appears to be specific for enteric transmissible gastroenteritis virus (TGEV) strains and for live-attenuated TGEV vaccines. This cDNA probe is used to differentiate porcine respiratory coronavirus (PRCV) isolates from TGEV field and vaccine strains by a slot blot hybridization assay. Probe pG3BS also hybridizes to canine coronavirus (CCV) RNA but does not hybridize to antigenically related feline infectious peritonitis virus (FIPV) RNA. The RNAs of 13 enteric TGEV isolates from the United States, Japan, and England, 4 US-licensed live-attenuated TGEV vaccines, and antigenically closely related CCV were detected by pG3BS. The RNAs of FIPV and 3 US isolates of PRCV did not react with pG3BS but were detected by a TGEV-derived plasmid, pRP3. Pigs infected with either PRCV or TGEV test serologically positive for TGEV antibody by the serum neutralization test. Characterization of the virus circulating in a swine herd by the pG3BS probe will differentiate between an enteric TGEV and a respiratory PRCV infection.

Sequence of the S gene from a virulent British field isolate of transmissible gastroenteritis virus

Subgenomic mRNA from a virulent field isolate of porcine transmissible gastroenteritis virus (TGEV), strain FS772/70, was used to produce cDNA. The cDNA from three overlapping clones was sequenced by the chain termination method and two open reading frames (ORFs) were identified. The largest ORF, 4350bp, encoded a polypeptide of 1449 amino acids of relative molecular mass (M r) 159811, which contained 33 potential N-linked glycosylation sites, a cysteine-rich region, and a potential transmembrane region. The C-terminal half of this ORF showed homology to the S proteins of four other coronaviruses. The other ORF consisted of the 3'-end of a gene with homology to the carboxyl terminus of the F2 subunit of infectious bronchitis virus (IBV) RNA polymerase.

The integral membrane protein from a virulent isolate of transmissible gastroenteritis virus: molecular characterization, sequence and expression in Escherichia coli

SummarySubgenomic mRNA from a virulent isolate of porcine transmissible gastroenteritis virus (TGEV) was used to produce cDNA clones. Part of a new clone and a previously reported clone were sequenced and used to construct the viral gene for integral membrane protein. A single open reading frame (ORF) encoding a polypeptide of 262 amino acids, relative molecular mass (Mr) 29459, was identified. The positive identification of the polypeptide as the integral membrane protein was demonstrated by the production in E. coli of a chimaeric protein comprising most of the ORF encoding the Mr 29459 polypeptide and β‐galactosidase. The chimaeric protein reacted with a specific monoclonal antibody to viral integral membrane protein and antibodies raised against the chimaeric protein immune precipitated the viral protein. Comparison with the sequence of an avirulent isolate indicates amino acid residues that may be important in pathogenicity.

Sequence of the nucleoprotein gene from a virulent British field isolate of transmissible gastroenteritis virus and its expression in Saccharomyces cerevisiae.

SummarySubgenomic mRNA from a virulent isolate of porcine transmissible gastroenteritis virus (TGEV) was used to produce cDNA which was sequenced. Two non‐overlapping open reading frames (ORFs) were identified. The largest, encoding a polypeptide of 382 amino acids (relative molecular mass (Mr) 43 483), was shown to be the viral nucleoprotein gene. The second ORF, found 3’to the larger ORF, encodes a polypeptide of 78 amino acids (Mr 9068) which has yet to be assigned to a viral product. The nucleoprotein gene was expressed in yeast cells under the control of two types of yeast promoters: the constitutive PGK promoter, and the inducible GAL1 promoter. Yeast cells containing recombinant plasmids, with the nucleoprotein gene in the correct orientation, produced a polypeptide of M, 47000, identical to the viral product, that reacted with a specific monoclonal antibody.