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PubMed1994

Antiviral activity of interferon against transmissible gastroenteritis virus in cell culture and ligated intestinal segments in neonatal pigs.

Segments of jejunum in 5 to 6 days old piglets were surgically ligated, inoculated with transmissible gastroenteritis virus (TGEV) and 18 hours later the segments were fixed for histology or suspensions were prepared for plaque assay in swine testis (ST) cell cultures to determine the yield of virus. When the virulent Purdue strain of TGEV was used, villous atrophy was seen and TGEV antigen was demonstrated immunohistochemically in the villous enterocytes. The Miller M6 strain of virus produced less extensive lesions in the segments, but since it was titratable by plaque assay it was used in the subsequent yield reduction assays to determine the antiviral activity of interferon. When intestinal segments were inoculated simultaneously with either 3200 units of natural porcine interferon-alpha or up to 1000,000 units of recombinant human interferon-alpha 2 a, and TGEV, there no reductions in virus yield, although the same cytokines exerted an antiviral effect in ST cells treated in a similar way. However, virus yields were significantly reduced in intestinal segments in piglets treated parenterally with the synthetic interferon inducer polyinosinic: polycytidylic acid 6 hours before challenge of the segments with TGEV. There was also a trend for the antiviral effects of interferon induction before challenge to be augmented by the inclusion of interferon with the virus inoculum. It was concluded that interferon would be ineffective as a therapeutic for TGEV, although it might be useful prophylactically.

Animals↗

PubMed1986

Antigenic structure of transmissible gastroenteritis virus. I. Properties of monoclonal antibodies directed against virion proteins.

Thirty-two hybridoma cell lines producing monoclonal antibodies (MAbs) against the three major structural proteins of transmissible gastroenteritis virus (TGEV) have been isolated. Radioimmunoprecipitation of intracellular viral polypeptides showed that 17 hybridomas recognized both the peplomer protein [E2, 220 X 10(3) mol. wt. (220K)] and a lower mol. wt. species (E'2, 175K), which was characterized as a precursor of E2. Six MAbs selectively immunoprecipitated the E'2 protein. Four hybridomas were directed against the low mol. wt. envelope protein (E1, 29K), and three against the nucleoprotein (N, 47K). All major neutralization-mediating determinants were found to be carried by the peplomers. Several anti-E2 MAbs displayed an intrinsic neutralizing activity close to that of the most potent anti-TGEV polyclonal reagents tested (including ascitic fluid of feline infectious peritonitis virus-infected cats). None of the anti-E'2 MAbs induced significant neutralization, although this protein might be incorporated to some extent into the virions. Immunofluorescence patterns obtained with MAbs directed against either the envelope glycoproteins or the nucleocapsid revealed distinctly different distributions of these antigens within the cells. Comparison of nine TGEV strains using our panel of MAbs confirmed their close antigenic relationship, but revealed the occurrence of distinct antigenic differences.

Animals↗

PubMed1977

Lipids of transmissible gastroenteritis virus and their relation to those of two different host cells.

The lipids of two cell types (primary pig kidney and secondary adult pig thyroid) and those of transmissible gastroenteritis virus (TGEV) grown in these cells were studied using 14C-palmitic acid. Differences were demonstrated between the incorporation of isotopically labelled lipid precursors in the two cell types and it was found that the phospholipid and glycolipid profiles of purified TGEV closely resembled those of the host cell in which it was grown.

Animals↗

PubMed2001

Complete genome sequence of transmissible gastroenteritis coronavirus PUR46-MAD clone and evolution of the purdue virus cluster.

The complete sequence (28580 nt) of the PUR46-MAD clone of the Purdue cluster of transmissible gastroenteritis coronavirus (TGEV) has been determined and compared with members of this cluster and other coronaviruses. The computing distances among their S gene sequences resulted in the grouping of these coronaviruses into four clusters, one of them exclusively formed by the Purdue viruses. Three new potential sequence motifs with homology to the alpha-subunit of the polymerase-associated nucleocapsid phosphoprotein of rinderpest virus, the Bowman-Birk type of proteinase inhibitors, and the metallothionein superfamily of cysteine rich chelating proteins have been identified. Comparison of the TGEV polymerase sequence with that of other RNA viruses revealed high sequence homology with the A-E domains of the palm subdomain of nucleic acid polymerases.

Amino Acid Sequence↗

PubMed1988

The amino-terminal signal peptide on the porcine transmissible gastroenteritis coronavirus matrix protein is not an absolute requirement for membrane translocation and glycosylation.

cDNA clones mapping within the first 2601 bases of the 3' end of the porcine transmissible gastroenteritis corona-virus (TGEV) genome were sequenced by the method of Maxam and Gilbert and an open reading frame yielding a protein having properties of the matrix (M or E1) protein was identified. It is positioned at the 5' side of the nucleocapsid (N) gene from which it is separated by an intergenic stretch of 12 bases. The deduced M protein comprises 262 amino acids, has a molecular weight of 29,544, is moderately hydrophobic, and has a net charge of +7 at neutral pH. Thirty-four percent of its amino acid sequence is homologous with the M protein of the bovine coronavirus (BCV), 32% with that of the mouse hepatitis coronavirus (MHV), and 19% with that of the avian infectious bronchitis coronavirus (IBV). Judging from alignment with the BCV, MHV, and IBV M proteins, the amino terminus of the TGEV M protein extends 54 amino acids from the virion envelope which compares with only 28 for BCV, 26 for MHV, and 21 for IBV. Eleven of the sixteen amino-terminal amino acids are hydrophobic and the positions of charged amino acids around this sequence suggest that the first 16 amino acids comprise a potentially cleavable signal peptide for membrane insertion. A similar sequence is not found in the M proteins of BCV, MHV, or IBV. When mRNA from infected cells, or RNA prepared by in vitro transcription of the reconstructed M gene, was translated in vitro in the presence of microsomes, the M protein became translocated and glycosylated. When a protein without the amino-terminal signal peptide was made by translating a truncated version of the M gene transcript, some translocation and glycosylation also occurred suggesting that the amino-terminal signal peptide on the TGEV M protein is not an absolute requirement for membrane translocation. Interestingly, the amino-terminal peptide did not appear to be cleaved during in vitro translation in the presence of microsomes suggesting that a step in virion assembly may be required for proper exposure of the cleavage site to the signal peptidase.

Amino Acid Sequence↗

PubMed1981

Laboratory evaluation of selected disinfectants as virucidal agents against porcine parvovirus, pseudorabies virus, and transmissible gastroenteritis virus.

Of a variety of disinfectants evaluated, only sodium hypochlorite and sodium hydroxide inactivated porcine parvovirus (PPV) after a 5-minute incubation period. After the same incubation time, pseudorabies and transmissible gastroenteritis viruses were inactivated by all of the disinfectants tested. When the incubation time was increased to 20 minutes, 2% glutaraldehyde and a double-strength concentration of a commercial formaldehyde preparation also inactivated PPV. Formaldehyde vapor and ultraviolet radiations inactivated PPV also, but relatively long exposure times were required.

Animals↗

PubMed1982

Serum antibody responses of neonatal and young adult pigs to transmissible gastroenteritis coronavirus.

Serum titers of virus-neutralizing (VN) antibody were 10 to 16 times higher in neonatal pigs than in young adult pigs, after single oral doses of virulent transmissible gastroenteritis virus (TGEV). To determine the reason for this higher response, sera from neonatal and young adult pigs, 18 to 21 days after exposure to TGEV, were collected and assayed for VN antibody by plaque reduction. In addition, sera of VN-positive and VN-negative neonatal pigs were analyzed for immunoglobulin classes by radial immunodiffusion technique. The competence of neonatal pigs to produce VN antibody with increased IgG levels was demonstrated. The higher antibody response seen in neonatal pigs, when compared to sera of young adult pigs, may be attributed to the increased replication of TGEV in the intestinal tracts of neonatal pigs or to the lack of other immunogens that may interfere or compete with the production of specific antibody.

Aging↗

PubMed1989

Isolation of peplomer glycoprotein E2 of transmissible gastroenteritis virus and application in enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay (ELISA) based on peplomer glycoprotein E2 was developed for the detection of antibodies to transmissible gastroenteritis virus (TGEV). Purified preparations of E2 were isolated by solubilization of the viral membrane with nonion detergent Nonidet P-40, followed by sucrose density gradient sedimentation. ELISA optical density values with E2 antigen significantly exceeded the indices when other TGEV protein or intact virion antigen was used. It was shown that a virus protein concentration in the E2 preparation of 500 ng per well is sufficient to sensitize the solid phase of microplates. A comparison of the ELISA and the virus neutralization test for the detection of TGEV antibodies was conducted. A significant correlation between the ELISA and the virus neutralization test was shown (r = 0.97). This serological test may be successfully used for various immunologic investigations.

Animals↗

PubMed1998

Structure and intracellular assembly of the transmissible gastroenteritis coronavirus.

Coronaviruses have been described as pleomorphic, round particles with a helical nucleocapsid as the unique internal structure under the virion envelope. Our studies on the organization of the transmissible gastroenteritis coronavirus (TGEV) have shown that the structure of these viruses is more complex. Different electron microscopy techniques, including cryomicroscopy of vitrified viruses, revealed the existence of an internal core, most probably icosahedral, in TGEV virions. Disruption of these cores induced the release of elongated ribonucleoprotein complexes. Ultrastructural analysis of freeze-substituted TGEV-infected swine testis (ST) cells showed characteristic intracellular budding profiles as well as two types of virions. While large virions with an electron-dense internal periphery are seen at perinuclear regions, smaller viral particles exhibiting compact internal cores of poligonal contours are more abundant in areas closer to the plasma membrane of the cell. These data strongly suggest that maturation events following the budding process are responsible for the formation of the internal core shell, the new structural element that we have recently described in extracellular infectious TGEV virions.

Animals↗

PubMed1995

Cloning and sequencing of a 8.4-kb region from the 3'-end of a Taiwanese virulent isolate of the coronavirus transmissible gastroenteritis virus.

The nucleotide sequence (8396 nucleotides) was determined, from the 3'-end of the putative polymerase gene to the poly(A) tail, for a Taiwanese virulent isolate, TFI, of transmissible gastroenteritis virus (TGEV). The TFI nucleotide sequence had very high identity to the British virulent field isolate FS772/70 (98.3%), the attenuated Purdue 115 (96.7%) and from the S gene to ORF-4 gene region, to the low passaged virulent Miller (98.3%) strains of TGEV. Comparison of the TFI S protein sequence with those determined from other TGEV strains and those of the TGEV variant, porcine respiratory coronavirus, isolated from Europe and North America showed that they had changed very little over a period of 4 decades. The two extra amino acids found to be present in the spike proteins of the virulent FS772/70 and Miller strains when compared to the avirulent Purdue strain were found to be present in the TFI strain. The genomic organisation of the TFI strain was the same as that of the other TGEV viruses.

Animals↗

PubMed1990

Four major antigenic sites of the coronavirus transmissible gastroenteritis virus are located on the amino-terminal half of spike glycoprotein S.

Four major antigenic sites have been delineated on the spike protein (S) of the porcine enteric coronavirus transmissible gastroenteritis virus (TGEV) in previous topological studies using monoclonal antibodies (MAbs). Correlation of these sites with the physical structure of the protein was achieved by use of different approaches. Recombinant pEX plasmids directing the synthesis of various fused S polypeptides were constructed. A hybrid protein containing nine S-specific residues (363 to 371) was shown to express site C epitopes. The other sites were localized through study of the antigenic activity of fragments generated by controlled cleavage of the native protein with different endopeptidases. Two identified cleavage products of 26K and 13K, immunoreactive to site A-B- and site D-specific MAbs respectively, could be aligned on the S primary structure according to N-terminal sequence data. This led us to propose that the major neutralization domain A-B is contained in a region of approximately 200 residues with residue 506 as its N boundary. Similarly, site D epitopes should be located within a stretch of 130 residues, starting at 82 residues from the N terminus. Point mutations identified by direct RNA sequencing of neutralization-resistant mutants were consistent with the proposed location of these sites.

Amino Acid Sequence↗

PubMed1992

Antigenic variation among transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus strains detected with monoclonal antibodies to the S protein of TGEV.

Five nonneutralizing monoclonal antibodies (MAb) generated to the virulent Miller strain of transmissible gastroenteritis virus (TGEV) and specific for the S protein were characterized. Competition assays between purified and biotinylated MAb indicated that MAb 75B10 and 8G11 mapped near a new subsite, designated V and 2 MAb, 44C11 and 45A8, mapped to a previously designated subsite D. A fifth MAb mapped between subsites V and E. These MAb were tested with 3 previously characterized MAb to subsites A, E, and F in fixed-cell ELISA and cell culture immunofluorescent assays against 5 reference and 9 field strains of TGEV and 2 US strains (ISU-1 and ISU-3) of porcine respiratory coronavirus (PRCV). Subsites A, E, and F were conserved on all TGEV and PRCV strains examined. The 2 MAb to subsite V, 8G11 and 75B10, reacted only with the Miller TGEV strains (M5C, M6, and M60), except that 75B10 also recognized field strain U328. The MAb 11H8 did not react with 4 field strains or the Purdue strains of TGEV. The 2 MAb to subsite D reacted with all TGEV strains examined, but not with 2 US PRCV strains, 2 European PRCV strains, 1 feline infectious peritonitis virus strain, and 1 canine coronavirus strain. Because of this specificity for TGEV, but not PRCV, these latter 2 subsite D MAb may be useful for the development of competition ELISA to differentiate serologically between TGEV and PRCV infections in swine, similar to the currently used European subsite D MAb.

Animals↗

PubMed1997

Two amino acid changes at the N-terminus of transmissible gastroenteritis coronavirus spike protein result in the loss of enteric tropism.

To study the molecular basis of TGEV tropism, a collection of recombinants between the PUR46-MAD strain of transmissible gastroenteritis coronavirus (TGEV) infecting the enteric and respiratory tracts and the PTV strain, which only infects the respiratory tract, was generated. The recombinant isolation frequency was about 10(-9) recombinants per nucleotide and was 3.7-fold higher at the 5'-end of the S gene than in other areas of the genome. Thirty recombinants were plaque purified and characterized phenotypically and genetically. All recombinant viruses had a single crossover and had inherited the 5'- and 3'-halves of their genome from the enteric and respiratory parents, respectively. Recombinant viruses were classified into three groups, named 1 to 3, according to the location of the crossover. Group 1 recombinants had the crossover in the S gene, while in Groups 2 and 3 the crossovers were located in ORF1b and ORF1a, respectively. The tropism of the recombinants was studied. Recombinants of Group 1 had enteric and respiratory tropism, while Group 2 recombinants infected the respiratory, but not the enteric, tract. Viruses of both groups differed by two nucleotide changes at positions 214 and 655. Both changes may be in principle responsible for the loss of enteric tropism but only the change in nucleotide 655 was specifically found in the respiratory isolates and most likely this single nucleotide change, which leads to a substitution in amino acid 219 of the S protein, was responsible for the loss of enteric tropism in the closely related PUR46 isolates. The available data indicate that in order to infect enteric tract cells with TGEV, two different domains of the S protein, mapping between amino acids 522 and 744 and around amino acid 219, respectively, are involved. The first domain binds to porcine aminopeptidase N, the cellular receptor for TGEV. In the other domain maps a second factor of undefined nature but which may be the binding site for a coreceptor essential for the enteric tropism of TGEV.

Animals↗

PubMed1990

The multiplication of transmissible gastroenteritis viruses in several cell lines originated from porcine kidney and effects of trypsin on the growth of the viruses.

Plaque formation, replication and related cytopathic function of 9 strains of transmissible gastroenteritis (TGE) virus were examined in primary cells and cell lines such as CPK, IB-RS-2, ESK, and PK-15 originated from porcine kidney and the effects of trypsin on the replication of TGE virus were examined in CPK cells. All strains produced a cytopathic effect and grew well in CPK cells as well as in primary porcine kidney cells. The effect of trypsin on the plaque formation was different from strains. The number of plaques produced by strains TO-163, Ukiha and Niigata increased from 2.6 to 3.52 times when trypsin was present in the medium during incubation at 37 degrees C for 1 hr after adsorption of the virus at 4 degrees C for 2 hr. The plaque sizes of TO-163, h-5, Ukiha and Niigata became larger from 1.4 to 1.7 times, when trypsin was present in the agar MEM overlay.

Cell Line↗

PubMed1992

The 9-kDa hydrophobic protein encoded at the 3' end of the porcine transmissible gastroenteritis coronavirus genome is membrane-associated.

The open reading frame potentially encoding a 78 amino acid, 9101 Da hydrophobic protein (HP) and, mapping at the 3' end of the porcine transmissible gastroenteritis coronavirus (TGEV) genome, was shown to be expressed during virus replication. The cloned HP gene was placed in a plasmid under control of the T7 RNA polymerase promoter and in vitro translation of transcripts generated in vitro yielded a 9.1-kDa protein that was immunoprecipitable with porcine hyperimmune anti-TGEV serum. Antiserum raised in rabbits against a 31 amino acid synthetic polypeptide that represented the central hydrophilic region of HP specifically immunoprecipitated HP from TGEV-infected cells. HP was further shown to become associated with microsomal membranes during synthesis in vitro and was found to be closely associated with the endoplasmic reticulum and cell surface membranes in infected cells. The intracellular location of HP suggests that it may play a role in the membrane association of replication complexes or in virion assembly.

Amino Acid Sequence↗

PubMed1994

Characterization of the IgA and subclass IgG responses to neutralizing epitopes after infection of pregnant sows with the transmissible gastroenteritis virus or the antigenically related porcine respiratory coronavirus.

In this study, we have investigated the characteristics of secreted IgA and other classes of Ig induced after vaccination of sows with transmissible gastroenteritis virus (TGEV) or the antigenically related porcine respiratory coronavirus (PRCV). Both viruses induced the secretion of neutralizing antibodies of different classes in the sows' milk, but these protected suckling piglets against TGEV to different degrees. Quantitative differences in the induction of IgA by both viruses were found among the different viral antigenic sites and subsites of glycoprotein S. In TGEV-vaccinated sows, antigenic subsite A was the best inducer of IgA, followed by antigenic site D. After vaccination with PRCV, lower levels of IgA were detected on colostrum and milk, antigenic site D and subsite Ab being the immunodominant sites. This quantitative difference in epitope recognition could explain the differences in newborn piglet protection found using Ig classes purified from the milk of sows immunized with both viruses. Apparently only IgA recognizing at least antigenic sites A and D confers good protection in vivo, whereas any Ig class recognizing only one antigenic site may neutralize the virus in cell culture. These results indicate that the formulation of a subunit vaccine against TGEV has to consider the inclusion of more than one antigenic site involved in virus neutralization.

Animals↗

PubMed1996

A continuous epitope from transmissible gastroenteritis virus S protein fused to E. coli heat-labile toxin B subunit expressed by attenuated Salmonella induces serum and secretory immunity.

Antigenic site D from the spike protein of transmissible gastroenteritis virus (TGEV), which is a continuous epitope critical in neutralization, has been expressed as a fusion protein with E. coli heat-labile toxin B subunit (LT-B) in attenuated S. typhimurium. Synthetic peptides containing the sequence of site D induced TGEV neutralizing antibodies when inoculated subcutaneously in both rabbits and swine. A synthetic oligonucleotide encoding residues 373-398 of TGEV S protein, including antigenic site D, was cloned in frame with the 3' end of LT-B gene, into a plasmid used to transform S. typhimurium delta asd chi 3730. A collection of 6 recombinant plasmids designated pYALTB-D I-VI encoding LTB-site D fusions with a variable number of site D sequences were selected. Four of the 6 LTB-site D fusion products expressed in S. typhimurium chi 3730 formed oligomers (pentamers) that dissociated at > 70 degrees. S. typhimurium chi 3730 (pYALTB-D) V and VI expressed the oligomer forming products with higher antigenicity. Partially purified LTB-site D fusion product expressed from S. typhimurium chi 3730 (pYALTB-D) V induced anti-TGEV neutralizing antibodies in rabbits. Recombinant vaccine strain S. typhimurium delta cya delta crp delta asd chi 3987 transformed with plasmid pYALTB-D V expressed constitutively products that formed oligomers presumably containing 20 copies of site D, and showed a high stability in vitro. This recombinant strain was orally inoculated in rabbits and induced TGEV specific antibodies in both serum and intestinal secretion.

Amino Acid Sequence↗

PubMed1991

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.

Animals↗