Transmissible Gastroenteritis Virus Strains Research Articles

Induction of the 2-5A System by Interferon and Transmissible Gastroenteritis Virus

Interferon (IFN) treatment increased the level of 2',5'-oligoadenylate (2-5A) synthetase and inhibited replication of transmissible gastroenteritis virus (TGEV) in cultured swine testicular cells. Despite a minimal increase in TGEV titer in IFN-treated swine testicular cells, there was rapid cellular destruction. IFN-treated swine testicular cells had detectable levels of 2-5A (3.6 nM 6 h post-infection) after infection with 30 pfu TGEV/cell. Infection of neonatal pigs with a virulent strain of TGEV caused a significant increase in serum IFN and enterocyte 2-5A synthetase activity, as compared to control pigs. The level of enterocyte 2-5A synthetase in TGEV-infected pigs was increased 25-fold before viral-induced damage of the intestine consistently was present. 2-5A was not detected in enterocytes of either TGEV-infected or control pigs (less than 0.5 nM). Preparations of enterocytes contained two subpopulations of cells, one of which does not support replication of the virus. The considerable dilution of TGEV-infected cells (villous enterocytes) with uninfected cells (crypt cells) may be responsible for our inability to detect 2-5A in enterocytes from TGEV-infected pigs. These results indicate that the 2-5A system in porcine enterocytes and cultured testicular cells is modulated by TGEV infection and/or interaction with IFN.

Sequence of the coding regions from the 3.0 kb and 3.9 kb mRNA

SummarySubgenomic mRNA from a virulent isolate of porcine transmissible gastroenteritis virus (TGEV) was used to produce cDNA clones covering the genome region from the 3′ end of the pelomer gene to the start of the integral membrane protein gene. The nucleotide sequence of this area was determined using clone pTG11 and a previously reported cDNA clone pTG22. Three open reading frames (ORFs) were identified encoding putative polypeptides of relative molecular masses (Mr) 6,600, 27,600, and 9,200. The sequence encoding the Mr 9,200 polypeptide was found to be present on the “unique” 5′ region of the 3.0 kb mRNA species whereas the other two ORFs mapped on the 3.9 kb mRNA species. Differences between the ORFs from this strain of TGEV and those from a previously reported avirulent strain of TGEV were compared.

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

SummaryA 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 E1 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 E2-MAbs, and the E1-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 E1 and E2 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 µg/ml for E2-MAbs, from 0.05 to 0.82 µg/ml for N-MAbs, and 6 µg/ml for the E1-MAb. The assay was used to determine the 50% neutralization concentrations for four neutralizing E2-MAbs (0.1 µg/ml to 6.9 µg/ml) and one E1-MAb (1.2 µg/ml). Competition assays between b-MAbs and unlabeled competitors indicated that at least two major antigenic sites exist on the E2-protein and 2 to 3 antigenic sites are present on the N-protein of Miller TGEV.

A competitive inhibition ELISA for the differentiation of serum antibodies from pigs infected with transmissible gastroenteritis virus (TGEV) or with the TGEV-related porcine respiratory coronavirus

A competitive inhibition ELISA was developed to detect non-neutralizing antibodies to the peplomer protein of transmissible gastroenteritis virus (TGEV) in porcine sera using a monoclonal antibody as an indicator. It was demonstrated that field strains of the TGEV-related porcine respiratory coronavirus (PRCV) did not induce this antibody, whereas the Miller strain and field strains of TGEV did. The sensitivity of the competitive inhibition ELISA appeared to be similar to that of the virus neutralization (VN) test. The test enables differentiation of pigs which were previously infected with TGEV or PRCV and which cannot be distinguished by the classical anti-TGEV neutralization test. The present test is useful for selective serodiagnosis.

Transmissible gastroenteritis (TGE) of swine: In vitro virus attachment and effects of polyanions and polycations

Four transmissible gastroenteritis virus (TGEV) strains (Purdue-115, D-52, 188-SG and Gep-II) and two cell lines (swine testis-ST and pig kidney-RPD) were used to study virus attachment and cell susceptibility. Virus attachment was partially thermodependent and the rate varied, depending on the strain. Identical TGEV inocula produced a higher plaque number by plaque assay in the swine testis cell line (ST) than in the pig kidney cell line (RPD) but [ 3H]uridine-labèlled virus was found associated equally well with both cell lines. A field TGEV strain (Gep-II), which was unable to multiply in cell cultures, appeared able to inhibit the attachment of radiolabelled cell-passaged virus. Therefore, the susceptibility to TGEV infection was apparently not determined at the virus-to-cell attachment stage. The attachment sites on the cell surface were specific, however, differences in TGEV attachment determinant between strains were not observed. Attachment of all the virus strains tested was enhanced by DEAE-dextran and inhibited by dextran sulfate, poly- L-lysine (PLL), poly- L-α-ornithine (PLO) and protamine sulfate.

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 occurence 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.

Isolation of hemagglutinating encephalomyelitis virus from respiratory tract of pigs in Japan.

Four strains of cytopathic agents with syncytium formation were isolated from the respiratory tract of pigs affected with respiratory diseases in Niigata and Osaka Prefectures in 1984. These viruses agglutinated with erythrocytes from mouse, hamster, rat and chicken. Multiplication of the isolated viruses in cell culture was not inhibited by IUdR. The viruses were inactivated with ether and chloroform, and were also unstable at pH 3.0. The isolated viruses passed readily through a membrane filter of 220 nm pore size, but not through one of 100 nm pore size. Coronavirus-like particles of 120-160 nm in diameter were observed by the electron microscope. In the serum neutralization test, the 4 isolated viruses were immunologically related with the 67N strain of hemagglutinating encephalomyelitis virus (HEV) and were slightly related with the No. 66 strain of bovine coronavirus (BCV). None of them were found to be immunologically correlated with the T0160 strain of transmissible gastroenteritis virus (TGEV). From these resuflts, the 4 isolated viruses were identified to be as the HEV. Conventional and colostrum-deprived piglets were inoculated with isolated HEV by the intranasal or ocular route. They showed mild coughing, sneezing with nasal discherge with some other mild clinical sign. The viruses were successfully recovered from the respiratory tract and lungs during 1 to 9 days after virus exposure. On farms N and T, the field investigations revealed that there was an increase in hemagglutination-inhibiting (HI) antibody in breeding pigs 3 to 4 months old.

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.

High interferon titer in newborn pig intestine during experimentally induced viral enteritis.

We looked for the presence of interferon in the digestive tract of newborn piglets infected with a virulent strain of transmissible gastroenteritis virus (Coronaviridae). High levels of type 1 interferon activity were found early in the disease in jejunal and ileal parts of the intestine as well as in the serum. Enterocytes appeared to be involved in the interferon synthesis. These findings raise the question of the role of interferon in the pathogenesis of viral enteritis.

Genome of porcine transmissible gastroenteritis virus.

The Purdue strain of transmissible gastroenteritis virus, a porcine coronavirus, was grown to titers of greater than 10(8) PFU/ml in a swine testicle cell line, and the RNA was isotopically labeled with [3H]uridine. The RNA was extracted from purified virus and was found to have the following properties. (i) It consisted primarily of a homogeneous large-molecular-weight species which electrophoretically migrated with an apparent molecular weight of 6.8 X 10(6) under denaturing conditions. (ii) It migrated electrophoretically at the same rate on nondenaturing gels before and after heat denaturation, suggesting that it does not consist of subunits. (iii) It was susceptible to pancreatic RNase A digestion in high (0.3 M) NaCl. (iv) It was polyadenylated to the extent that greater than 60% of the native RNA bound to oligodeoxythymidilic acid-cellulose under conditions of high (0.5 M) NaCl. RNA extracted from virions was infectious. This coronavirus can therefore be characterized as a positive-strand RNA virus.

Multiplication of low and high cell culture passaged strains of transmissible gastroenteritis virus in organs of newborn piglets

Distribution and persistence of four different strains of transmissible gastroenteritis (TGE) virus in newborn piglets were compared. The piglets inoculated with high-passaged TO-163 strain did not show any clinical signs of TGE on any days postinoculation (DPI), but the piglets inoculated with one of the other three strains, SH-14, SH-164 or TO-16, had soft feces or diarrhea. In the latter cases, the virus was isolated mainly from respiratory organs, lymph nodes, and digestive tract on any DPI, but was rarely detected in the digestive tract of piglets inoculated with the TO-163 strain. The frequency of virus recovery from the tissues was the highest till 4 DPI in all of the piglets inoculated with one of the four virus strains, and it was markedly reduced thereafter in the piglets inoculated with high-passaged strains. The TO-163 strain was subjected to serial passage in newborn piglets for seven passages. There was no evidence of regained pathogenicity with advance in passage, and detection of virus was restricted to lymph nodes and lung of these piglets. In gnotobiotic piglets inoculated with the TO-163 strain, frequent virus recovery and high titers of virus from the tissues were obtained on up to the 4th DPI. The viruses in high titer were found in the digestive tract of some of the piglets; however, none of them showed any clinical signs of TGE.

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.

Observations on the Experimental Infection and. Cellular Pathology of Transmissible Gastroenteritis in Piglets

SUMMARY Hysterotomy-derived, colostrum-deprived piglets were inoculated orally with a strain of transmissible gastroenteritis virus that had been isolated originally in the United Kingdom. Piglets which were injected when 5, 9 or 11 days old developed the clinical disease, but piglets which were 19 days old at infection failed to show symptoms. The changes observed at post-mortem examination were consistent with acute infection, as were the histological changes. A variety of ultrastructural changes in intestinal epithelial cells were recorded including alterations to microvilli, mitochondria, endoplasmic reticulum and other cytoplasmic components. Virus particles were identified in ultrathin sections from one piglet.