Critical epitopes in transmissible gastroenteritis virus neutralization
Journal of Virology
Purified transmissible gastroenteritis (TGE) virus was found to be composed of three major structural proteins having relative molecular weights of 200,000, 48,000, and 28,000. The peplomer glycoprotein was purified by affinity chromatography with the monoclonal antibody (MAb) 1D.G3. A collection of 48 MAbs against TGE virus was developed from which 26, 10, and 3 were specific for proteins E2, N, and El, respectively. A total of 14 neutralizing MAbs of known reactivity were E2 protein specific.
... 2 protein specific. In addition, MAb lB.C11, of upknown specificity, was also neutralizing. These MAbs reduced the virus titer 102_ to 109-fold. Six different epitopes critical in TGE virus neutralization were found, all of which were conformational base4d on their immunogenicity and antigenicity. Only the epitope defined -by MAb 1G.A7 was resistant to sodium dodecyl sulfate treatment, although it was destroyed by incubation ih the presence of both the detergent and P-mercaptoethanol. The frequency of MAb-resistant (mar) mutants selected with four MAbs (1G.A7, 1B.C11, 1G.A6, and 1E.F9) ranged from 10-6 to 10-7, whereas the frequency of the putative mar mutant defined by MAb lB.B11 was lower than 10-9. Furthermore, the epitopes defined by these MAbs and by MAbs 1H.C2 and 1A.F10, were present in 11 viral isolated with different geographical locations, years of isolation, and passage numbers (with the exception of two epitopes absent or modified in the TOY 56 viral isolate), suggesting that the critical epitopes in TGE virus neutralization were highly conserved. * Corresponding author. t Present address: BIOKIT S.A., 08025 Barcelona, Spain. in TGE virus neutralization, (ii) their immunogenicity and antigenicity, and (iii) the variability of these epitopes, by isolating mar mutants and studying the presence of the critical epitopes in viral isolates with different origins and dates of isolation. MATERIALS AND METHODS Cells. The epithelial swine testicle (ST) cell line developed by McClurkin (38) was obtained from Dr. Kemeny, National Animal Disease Center, Ames, Iowa. Cells were grown as monolayers in growth medium consisting of Dulbecco modified Eagle (DME) medium (GIBCO Europe) and 10% newborn calf serum (Flow Laboratories, Inc.). Animals. BALB/c mice, originally obtained from R. A. Fox, Frederick Cancer Research Center, Frederick, Md., were used for immunization and as a source of thymocytes and peritoneal macrophages, when the mice were approximately 10, 5, and 16 weeks old, respectively. Viruses. The characteristics of all the TGE virus isolates used in this study are summarized in Table 1 . Virus titration and plaque isolation. TGE virus was titrated on ST cells as described by Brian et al. (8), with minor modifications. Briefly, cells were seeded in 24-well tissue culture plates (Costar). Serial dilutions (10-fold) of virus were made in DME medium containing 2% newborn calf serum and 40 pug of DEAE-dextran per ml (Pharmacia). Portions containing 50 ,ul of each dilution were applied to cells. After 1 h of virus adsorption, the inoculum was replaced with medium containing 2% newborn calf serum, 40 ,ug of DEAE-dextran per ml, and 0.1% agarose, and the cells were incubated at 37°C for 2 to 3 days in a humidified CO2 incubator. Cells were fixed with 10% formaldehyde and stained with 0.1% crystal violet, and the plaques were counted.