Miller Strain Research Articles

Characterization and reactivity of monoclonal antibodies to the Miller strain of transmissible gastroenteritis virus of swine

SUMMARY Hybridomas secreting monoclonal antibodies (mab) to transmissible gastroenteritis virus (tgev) were produced by fusion of SP2/0 myeloma cells and splenic lymphocytes of BALB/c mice immunized with the virulent cell-passaged Miller strain of tgev. The mab secreted by these hybridomas were partially characterized; 4 of them (MA4, MA5, MH11, MB2) had high-neutralization titer for tgev. The remaining 7 (MC6, MD9, ME5, MG5, MF2, ME9, MG7) did not neutralize tgev at 1:25 dilution. All 4 neutralizing and 2 of the nonneutralizing mab reacted with the E2 protein of tgev in a radioimmunoprecipitation assay. The remaining 5 mab reacted with the E1 protein of tgev. Reactivity of the mab was tested in an indirect immunofluorescent assay with 3 cell culture-adapted strains of tgev (Miller, Purdue, and Illinois) and 13 wild-type isolates of tgev. Neutralizing mab reacted with all 13 wild-type isolates and the 3 cell culture-adapted strains of tgev. In contrast, nonneutralizing mab that reacted with the Miller strain of TGEV varied in their reactivity with the wild-type tgev isolates. Reactivity of neutralizing mab was also tested, using plaque-reduction neutralization assays with Miller, Purdue, and Illinois strains and 5 wild-type isolates. All 4 neutralizing mab neutralized the 8 virus isolates, but the neutralization titer was higher with the homologous virus than with the heterologous virus isolates. However, neutralization titers of the 4 neutralizing mab were 4 to 16 times higher for the homologous Miller strain of tgev than for the heterologous Illinois and Purdue strains, and were 4 to 1,000 times higher than for the wild-type isolates. Extensive antigenic heterogeneity was observed among tgev isolates on epitopes recognized by the nonneutralizing mab directed against either E1 or E2 protein.

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.

Intestinal permeability to macromolecules in piglets infected with transmissible gastroenteritis virus.

The permeability of the intestine of specific pathogen free piglets was investigated by measuring the concentration of 125-I in the blood after oral administration of 125-I polyvinylpyrrolidone (125-I PVP, MW = 40,000 Da) and the concentration of 131-I in the faeces after intravenous administration of 131-I porcine albumin (131-I PA, MW = 68,000 Da). The tests were performed one day before and up to two days after the piglets were infected with the Miller strain of transmissible gastroenteritis (TGE) virus. Biopsies of the jejunum were taken at the end of the experiment and blood samples were taken six-hourly. The piglets became anorexic and had diarrhoea 12 hours after infection; the packed cell volume decreased and the concentrations of urea and total serum proteins increased slightly after infection. However, the marked villous atrophy was not accompanied by an increased permeability of the intestine to PVP or PA.

Identification of a 17000 molecular weight antigenic polypeptide in transmissible gastroenteritis virus-infected cells.

Pulse labelling of cells with [35S]methionine at different times after infection followed by SDS-PAGE was used to resolve and to identify polypeptides designated as specific to transmissible gastroenteritis virus (TGEV)-infected swine testicular (ST) cell cultures. The major TGEV structural proteins, with apparent molecular weights of 200,000 (200K), 47K and 30K were detected in radiolabelled cell extracts by 6 h postinfection. Additionally, a 17K major polypeptide was present in infected cells but not in mock-infected control cultures. Labelling with [3H]glucosamine revealed only the 200K and 30K proteins to be glycosylated. TGEV-primed porcine lymphocytes, secondarily stimulated in vitro with sucrose gradient-purified virus, produced antibody only to the two glycoproteins (gp) indicating that the 17K polypeptide is not a surface feature of the virion. Two pigs were infected oronasally with the virulent Miller strain of TGEV and their sera were analysed by immunoprecipitation. At 25 days postinfection convalescent sera responded strongly to gp30 and gp200 and there was a weak initial response to the 17K polypeptide. Serum immunoglobulins at 60 days postinfection reacted strongly to the 17K protein while the antibody response to gp30 was significantly reduced and that to gp200 was slightly reduced.

A viral subunit immunogen for porcine transmissible gastroenteritis

An immunogenic component was isolated from both the Illinois (propagated in young swine) and the Miller (cell culture-adapted) strains of porcine transmissible gastroenteritis (TGE) virus. The viral subunit was released from the virion by sonication and was separated from intact virus and other viral components by isopycnic centrifugation. It had a buoyant density in sucrose of 1.02 g/ml. Further purification consisted of gel filtration through Sephadex G200, in which process the immunogen, with a molecular weight of approximately 25 000, was the last component to be eluted. A group of ten young, weaned swine, inoculated intramuscularly with two or three 1-mg doses of the viral subunit were protected against challenge with virulent TGE virus, probably by the induction of secretory IgA. The immunogen also induced a humoral immune response of variable magnitude (titers ranging from 8 to 5 625) in the animals. These antibodies are not believed to be directly related to protection against infection. They can, however, be easily identified by serologic procedures and may serve as a convenient indicator of responsiveness to the TGE viral immunogen.