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PubMed1989

The polypeptide of Mr 14,000 of porcine transmissible gastroenteritis virus: gene assignment and intracellular location.

Synthetic oligopeptides, corresponding to an amino acid sequence encoded by a potential Mr 9000 product's open reading frame (ORF-4) at the 3' terminus of the transmissible gastroenteritis virus genome, were used to generate rabbit antiserum. These antibodies produced immune complexes with an Mr 14,000 (14K) polypeptide in infected cells. The 14K product was shown by immune fluorescence to become associated with the cell nucleus, correlating with the onset of nuclear vacuolation, and suggesting a role in pathogenesis for the ORF-4 gene.

Amino Acid Sequence↗

PubMed1986

Neutralizing secretory IgA and IgG do not inhibit attachment of transmissible gastroenteritis virus.

Secretory IgA (sIgA) and IgG from porcine milk and serum, respectively, [3H]uridine-labelled virus, swine testis and pig kidney cell lines were used to examine the neutralized virus-cell interaction. Transmissible gastroenteritis virus (TGEV), 99.99% neutralized by immunoglobulin, was able to attach to the cells. Moreover, sIgA enhanced virus attachment. However, the neutralized virus was unable to enter cells, as demonstrated by the action of proteinase K which removed it from the cell surface. It was also found that pre-attached virus was still neutralizable and that IgG and sIgA had similar TGEV-neutralizing capacities.

Animals↗

PubMed1975

The demonstration of transmissible gastroenteritis viral antigens by immunoelectrophoresis and counterimmunoelectrophoresis.

Immunoelectrophoresis of alkaline intestinal extracts, or the supernatants after precipitation of these extracts with 60% ammonium sulfate, prepared from piglets experimentally infected with the DL or Purdue strains of transmissible gastroenteritis virus, revealed up to three antigens. Two antigens migrated towards the anode, and the third migrated towards the cathode. Antigens with anodal or cathodal mobility were also demonstrated in the same materials by counterimmunoelectrophoresis, and the procedure for counterimmunoelectrophoresis was modified to detect both antigens in a single test.

Animals↗

PubMed1992

Pathogenicity and antigen detection of the Nouzilly strain of transmissible gastroenteritis coronavirus, in 1-week-old piglets.

We compared the pathogenicity and the sites of multiplication of the attenuated Nouzilly strain, with the highly passaged Purdue-115 and the virulent Gep II strains of transmissible gastroenteritis (TGE) coronavirus, in 1-week-old weaned piglets. The immunohistochemical peroxidase technique, with an antiviral nucleoprotein monoclonal antibody, was used for the localization of the multiplication sites, in the intestine and other organs. The Gep II and the Purdue-115 strains, administered orally to piglets, caused clinical signs and lesions of TGE. These strains multiplied within the intestinal tract in the enterocytes of the jejunum and ileum, Peyer's patches and mesenteric lymph nodes. In view of the small numbers of infected cells in the tonsils, spleen, kidney, liver and lung, these tissues are not considered to be preferential multiplication sites. The attenuated Nouzilly strain multiplies only in the ileum and the mesenteric lymph nodes. The variation in the tropism for particular parts of the intestine (with the preferential localization of the virus in the ileum rather than the jejunum), could be related to the high degree of attenuation of the Nouzilly strain.

Animals↗

PubMed1991

Differentiation of transmissible gastroenteritis virus from porcine respiratory coronavirus and other antigenically related coronaviruses by using cDNA probes specific for the 5' region of the S glycoprotein gene.

Two cDNA clones prepared from the virulent Miller strain of transmissible gastroenteritis virus (TGEV) were identified, and their nucleotide sequences were determined. The clones were nonoverlapping and located in the 5' region of the S glycoprotein gene. Their nucleotide and predicted amino acid sequences were compared with published sequences of the attenuated Purdue strain of TGEV and feline infectious peritonitis virus (FIPV). TGEV clone pE21 contained 381 bp of the S glycoprotein gene and had greater than 98% nucleotide and amino acid sequence homology with Purdue TGEV and over 87% nucleotide and amino acid sequence homology with FIPV. TGEV clone pD24 contained 267 bp of the S glycoprotein gene. It had greater than 98% nucleotide and amino acid sequence homology with Purdue TGEV but only 54% nucleotide sequence homology and 24% amino acid sequence homology with FIPV. A probe prepared from pD24 could differentiate TGEV from porcine respiratory coronavirus and other antigenically related coronaviruses, FIPV, feline enteric coronavirus, and canine coronavirus in a dot blot hybridization assay.

Amino Acid Sequence↗

PubMed1990

Kinetics of the in vitro antibody response to transmissible gastroenteritis (TGE) virus from pig mesenteric lymph node cells, using the ELISASPOT and ELISA tests.

A method is described for in vitro studies of viral humoral immune responses in the pig. After oral immunization with transmissible gastroenteritis (TGE) coronavirus, antibody production from primed mesenteric lymph node cells was revealed by an in vitro boost with viral antigen. For the latter the leukocytes were co-cultured with UV-inactivated virus using a variety of different methods of antigenic stimulation. Enumeration of specific antibody-secreting cells (ASC) and titration of secreted anti-virus antibodies were performed with ELISASPOT (using 3-amino 9-ethyl carbazole as the peroxidase chromogen) and ELISA tests respectively, according to the Ig isotype. The results showed a close relationship between ASC numbers and secreted antibody titres. The best in vitro antibody synthesis was observed when the sensitized cells were maintained in contact with virus during the whole culture period. Antibody responses were defined by a kinetic profile characterized by a narrow peak, with a maximum occurring after 4 and 6 days of culture and with the IgA response appearing earlier than the IgG. This methodology, which analyses specific antibody responses at the cellular level, may permit studies on the mechanisms of Ig isotype regulation. Extended to leukocytes from other organs of the immune system, it may also constitute an in vitro model to study antibody responses expressed in different lymphoid tissues of the pig.

Animals↗

PubMed1989

Epitope mapping and the detection of transmissible gastroenteritis viral proteins in cell culture using biotinylated monoclonal antibodies in a fixed-cell ELISA.

A fixed-cell ELISA was developed using swine testicle (ST) cells infected with the virulent Miller strain of transmissible gastroenteritis virus (TGEV) and purified biotinylated monoclonal antibodies (b-MAbs). Five of the b-MAbs were specific for the peplomer (E2), five reacted to the nucleocapsid (N), and one reacted to the E 1 protein of the Miller strain of TGEV. Protein A-Sepharose purification of MAbs yielded protein concentrations ranging from 0.40 to 3 mg per ml of ascites. Separate pools of N-MAbs and E 2-MAbs, and the E 1-MAb were used to monitor synthesis of TGE viral antigen in ST cells from 0 to 16 h post-infection at various multiplicities of infection (MOI). Epitopes of N proteins appeared sooner and at a lower MOI than those for the E 1 and E 2 proteins. The fixed-cell ELISA was also used to examine relative binding affinities of TGEV MAbs. Concentrations of b-MAbs producing a half-maximal signal ranged from 0.11 to 3.8 microgram/ml for E 2-MAbs, from 0.05 to 0.82 microgram/ml for N-MAbs, and 6 micrograms/ml for the E 1-MAb. The assay was used to determine the 50% neutralization concentrations for four neutralizing E 2-MAbs (0.1 microgram/ml to 6.9 micrograms/ml) and one E 1-MAb (1.2 micrograms/ml). Competition assays between b-MAbs and unlabeled competitors indicated that at least two major antigenic sites exist on the E 2-protein and 2 to 3 antigenic sites are present on the N-protein of Miller TGEV.

Animals↗

PubMed1998

Expression of immunogenic glycoprotein S polypeptides from transmissible gastroenteritis coronavirus in transgenic plants.

The use of transgenic plants as vaccine production systems was described recently. We report on the immunological response elicited by two recombinant versions of the glycoprotein S from the swine-transmissible gastroenteritis coronavirus (TGEV) expressed in transgenic plants. Arabidoposis plants were genetically transformed with cDNAs constructs encoding either the N-terminal domain (amino acid residues 1-750) or the full-length glycoprotein S of TGEV, responsible for the neutralizing antibody induction against the virus, under the control of the cauliflower mosaic virus 35S (CaMV 35S) promoter. Genomic DNA and mRNA analyses of leaf extracts from transformed plants demonstrated the incorporation of the foreign cDNA into the arabidopsis genome, as well as their transcription. Expression of recombinant polypeptides were observed in most transgenic plants by ELISA using specific antibodies. Mice immunized with leaf extracts from transgenic plants developed antibodies that reacted specifically with TGEV in ELISA, immunoprecipitated the virus-induced protein, and neutralized the virus infectivity. From these results, we conclude that transgenic plants expressing glycoprotein S polypeptides may possibly be used as a source of recombinant antigen for vaccine production.

Animals↗

PubMed1992

Effect of fixation on the detection of transmissible gastroenteritis coronavirus antigens by the fixed-cell immunoperoxidase technique.

The effect of various fixatives and detergents on the in vitro detection of the viral determinants which are expressed in swine testis cells infected with the transmissible gastroenteritis coronavirus (TGEV) was studied using a microwell immunoperoxidase technique. When compared with glutaraldehyde and formaldehyde, 0.1% paraformaldehyde was found to be the fixative of choice for the detection of these determinants on the membranes of infected cells. Among dehydrating fixatives, 80% acetone or a mixture of acetone and ethanol or of acetone, methanol and ethanol were found to be the best fixatives for the detection of these viral determinants which are expressed in infected cells. In the case of acetone, the temperature of fixation and its concentration in the fixative preparation were found to be important. The treatment of 0.05% glutaraldehyde-fixed, infected cells with 0.1% saponin or 0.1% paraformaldehyde-fixed, infected cells with 1%NP-40 led to satisfactory detection of viral determinants. Using Triton X-100 to render cells permeable, the quantities of N and M antigen detected in TGEV-infected cells prefixed with either 0.05% glutaraldehyde or 0.1% paraformaldehyde were equal to those of 80% acetone-fixed, TGEV-infected cells while the quantity of S antigen detected was diminished. The effect of other detergents such as zwittergent, empigen BB, Chaps and N-lauroylsarcosine on the detection of viral determinants was also studied.

Acetone↗

PubMed1995

Membrane protein molecules of transmissible gastroenteritis coronavirus also expose the carboxy-terminal region on the external surface of the virion.

The binding domains of four monoclonal antibodies (MAbs) specific for the M protein of the PUR46-MAD strain of transmissible gastroenteritis coronavirus (TGEV) have been located in the 46 carboxy-terminal amino acids of the protein by studying the binding of MAbs to recombinant M protein fragments. Immunoelectron microscopy using these MAbs demonstrated that in a significant proportion of the M protein molecules, the carboxy terminus is exposed on the external surface both in purified viruses and in nascent TGEV virions that recently exited infected swine testis cells. The same MAbs specifically neutralized the infectivity of the PUR46-MAD strain, indicating that the C-terminal domain of M protein is exposed on infectious viruses. This topology of TGEV M protein probably coexists with the structure currently described for the M protein of coronaviruses, which consists of an exposed amino terminus and an intravirion carboxy-terminal domain. The presence of a detectable number of M protein molecules with their carboxy termini exposed on the surface of the virion has relevance for viral function, since it has been shown that the carboxy terminus of M protein is immunodominant and that antibodies specific for this domain both neutralize TGEV and mediate the complement-dependent lysis of TGEV-infected cells.

Animals↗

PubMed1975

Transmissible gastroenteritis (TGE) of swine: effect of age of swine testes cells culture monolayers on plaque assays of TGE virus.

A continuous line of swine testes cell culture monolayers was infected at various ages with both cell culture-adapted transmissible gastroenteritis (TGE) virus and tissue infected with TGE virus. Both produced increasing numbers of plaques as the cell monolayers aged from two to five days. Therefore, allowing the swine testes cell monolayer to age five to six days before inoculation should increase the likelihood of detecting TGE virus by plaque assay.

Animals↗

PubMed1978

[Transmissible gastroenteritis of swine as a model for infectious diarrhea].

Transmissible gastro-enteritis is a virus-caused diarrhoeal disease of swine which may result in up to a total loss among newborn animals. Changes are, primarily, of pathologicoanatomic nature and take place in the small intestine. The epithelium of the intestinal villi is completely destroyed, and diarrhoea is the result. Deformation in blood composition is a secondary sequel. The diarrhoea proper causes dehydration and acidosis which, in conjunction with abnormal heart function due to hyperkalaemia, cause death of the piglets affected. Reference is made to the intrinsic immunological conditions in swine, and possibilities are discussed for immunoprophylactic control of transmissible gastro-enteritis by induction of effective lactogenic immunity. The approaches taken to vaccine application should largely correspond to the natural route of infection.

Animals↗

PubMed1994

Major receptor-binding and neutralization determinants are located within the same domain of the transmissible gastroenteritis virus (coronavirus) spike protein.

The spike glycoprotein (S) of coronavirus, the major target for virus-neutralizing antibodies, is assumed to mediate the attachment of virions to the host cell. A 26-kilodalton fragment proteolytically cleaved from transmissible gastroenteritis virus (TGEV) S protein was previously shown to bear two adjacent antigenic sites, A and B, both defined by high-titer neutralizing antibodies. Recombinant baculoviruses expressing C-terminal truncations of the 26-kilodalton region were used to localize functionally important determinants in the S protein primary structure. Two overlapping 223- and 150-amino-acid-long products with serine 506 as a common N terminus expressed all of the site A and B epitopes and induced virus-binding antibodies. Coexpression of one of these truncated protein S derivatives with aminopeptidase N (APN), a cell surface molecule acting as a receptor for TGEV, led to the formation of a complex which could be immunoprecipitated by anti-S antibodies. These data provide evidence that major neutralization-mediating and receptor-binding determinants reside together within a domain of the S protein which behaves like an independent module. In spite of their ability to prevent S-APN interaction, the neutralizing antibodies appeared to recognize a preformed complex, thus indicating that antibody- and receptor-binding determinants should be essentially distinct. Together these findings bring new insight into the molecular mechanism of TGEV neutralization.

Amino Acid Sequence↗

PubMed1995

A transmissible gastroenteritis coronavirus nucleoprotein epitope elicits T helper cells that collaborate in the in vitro antibody synthesis to the three major structural viral proteins.

Four strong T cell epitopes have been identified studying the blastogenic response of lymphocytes from haplotype-defined transmissible gastroenteritis virus (TGEV) immune miniswine to sixty-one 15-mer synthetic peptides. Three of these epitopes are located on the nucleoprotein (N46, amino acids 46 to 60; N272, amino acids 272 to 286; and N321, amino acids 321 to 335), and one on the membrane protein (M196, amino acids 196 to 210). N321 peptide induced the highest T cell response and was recognized by immune miniswine lymphocytes with haplotypes dd, aa, and cc. T lymphocytes from peptide N321-immune miniswine reconstituted the in vitro synthesis of TGEV-specific antibodies by complementing CD4- TGEV-immune cells. This response was directed at least against the three major structural proteins. The synthesized antibodies specific for S protein preferentially recognized discontinuous epitopes and neutralized TGEV infectivity. These results show that peptide N321 defines a functional T helper epitope eliciting T cells capable of collaborating with B cells specific for different proteins of TGEV.

Amino Acid Sequence↗

PubMed1994

Coronavirus transmissible gastroenteritis virus-mediated induction of IFN alpha-mRNA in porcine leukocytes requires prior synthesis of soluble proteins.

We studied the expression of interferon alpha (IFN alpha)-mRNA in porcine non-adherent peripheral blood mononuclear cells (PBMC) after induction by the coronavirus transmissible gastroenteritis virus (TGEV). We found that protein synthesis inhibition by cycloheximide (CHX) blocked IFN alpha-mRNA expression, except when PBMC were preincubated with a conditioned medium as a potential source of cytokines. These data indicate that IFN alpha-mRNA induction by TGEV requires de novo synthesis of proteins. Moreover, they suggest that IFN alpha-mRNA induction in porcine leukocytes by TGEV involves mechanisms identical to those described for the herpes simplex virus in humans. In addition, experiments performed with a TGEV mutant, dm 49-4, previously characterized for its low ability to induce IFN alpha, showed that addition of a conditioned medium could not normalize its IFN alpha-inducing ability. Therefore, the defect of the dm49-4 mutant may be at the level of the final triggering signal to PBMC.

Actins↗

PubMed1994

Evidence for a putative second receptor for porcine transmissible gastroenteritis virus on the villous enterocytes of newborn pigs.

Aminopeptidase-N (APN) has been identified [B. Delmas, J. Gelfi, R. L'Haridon, L. K. Vogel, H. Sjostrom, O. Noren, and H. Laude, Nature (London) 357:417-420, 1992] as a major receptor for porcine transmissible gastroenteritis virus (TGEV). Binding of TGEV to villous enterocytes from the jejuna of newborn pigs is saturable and at a higher level than that of binding of virus to newborn cryptal enterocytes or to enterocytes from older piglets (H. M. Weingartl and J. B. Derbyshire, Vet. Microbiol. 35:23-32, 1993). The distribution of APN in enterocytes in the jejuna of neonatal and 3 week-old-piglets, as determined by the measurement of enzymatic activity and by labeling of the cells with an anti-APN monoclonal antibody, did not correspond with the reported distribution of saturable binding sites on enterocytes. Monoclonal antibodies, which were prepared against plasma membranes derived from enterocytes harvested from the upper villi of newborn pigs, blocked the replication of TGEV, but not the porcine respiratory coronavirus, in ST cells and immunoprecipitated a 200-kDa protein in ST cell lysates. This protein was demonstrated by immunohistochemistry and by fluorescence-activated cell scanning to be present on the villous enterocytes of newborn pigs but to be lacking on the cryptal enterocytes of newborn pigs and on the villous and cryptal enterocytes of 3-week-old piglets. Since this distribution of the protein corresponds to the previously demonstrated distribution of saturable binding sites, we conclude that the 200-kDa protein may be an additional receptor for TGEV which is restricted to the villous enterocytes of newborn pigs and which contributes to the age sensitivity of these animals to the virus.

Animals↗

PubMed1987

Antigenic variation of porcine transmissible gastroenteritis virus detected by monoclonal antibodies.

Mouse myeloma cells (SP2/O) were fused with spleen cells from BALB/c mice immunized with detergent-solubilized antigen of purified virus, and 21 monoclonal (MC) antibodies reactive in enzyme-linked immunosorbent assay with the TO-163 strain of porcine transmissible gastroenteritis (TGE) virus were obtained. Of these MC antibodies, 14, 6 and 1 were IgG1, IgG2a and IgM, respectively. All of the MC antibodies contained light chains of the kappa type. Of these MC antibodies, 8 were found to have neutralization (NT) activity against the TO-163 strain. Comparison of 7 strains of TGE virus by NT tests using our panel of MC antibodies confirmed their close antigenic relationships, but also revealed the occurrence of distinct antigenic differences. These results suggest that there may be at least 6 different epitopes involved in NT reaction on the virion of the TO-163 strain. This notion was confirmed by the competitive binding assay.

Animals↗

PubMed1997

An Escherichia coli CS31A fibrillum chimera capable of inducing memory antibodies in outbred mice following booster immunization with the entero-pathogenic coronavirus transmissible gastroenteritis virus.

CS31A fibrillae are thin, flexible, heteropolymeric proteinaceous appendages exposed as a capsule-like material around the cell surface of certain Escherichia coli strains. Two antigenic peptides of the S spike glycoprotein (TGEV-S) amino acids (aa) 363-371 and 521-531 of the transmissible gastroenteritis virus (TGEV) were tandemly introduced in the loop-structured, variable region aa 202-218 of the major ClpG subunit protein composing the bulk of CS31A. The resulting hybrid fibrillae with a 25 aa heterologous peptide were produced at the cell surface. Using a monoclonal antibody (Mab) specific for the TGEV epitopes, purified hybrid fibrillae were analysed in Western blotting under native conditions, which showed that the two viral epitopes were recognized immunologically as an integral part of the hybrid fibrillae, and therefore that they were antigenically active. The immunogenicity of the fusion construct was evaluated with live recombinant bacteria, purified hybrid ClpG monomers, and purified chimeric CS31A polymers. Whatever the form of hybrid used as antigen, intraperitoneally immunized outbred mice elicited serum anti-TGEV peptides antibodies (Abs) with significant titres and capable of recognizing native TGEV particles, indicating that the epitopes are exposed in an immunogenic conformation in all cases. However, virus neutralization titres were only obtained after immunization with either purified polymers or monomers. Furthermore, 4 months after an ultimate immunization with 20 micrograms of hybrid fibrillae mice developed a strong anamnestic Ab response against the two TGEV peptides following booster inoculation with virions. We conclude that CS31A fibrillae carrying a combination of TGEV epitopes as insert can induce an immunological memory in outbred animals infected with TGEV, and therefore that hybrid CS31A fibrillae may prove efficient as components of a subunit vaccine.

Adhesins, Escherichia coli↗