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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.

The 87,000-dalton glycoprotein (gp87) of bovine herpesvirus type 1 (BHV-1) was found to selectively attach to susceptible cells. The attachment of gp87 to the cells was markedly decreased by the prior adsorption of intact virions. Anti-gp87 (site Ia) monoclonal antibody, which inhibited BHV-1 adsorption to the cells and neutralized the virus without complement [Okazaki et al., Virology 250: 260-264], was effective in inhibiting the adsorption of gp87. Only the same antibody was able to inhibit the hemagglutination activity of BHV-1. Other monoclonal antibodies to the glycoproteins of BHV-1, including antibodies directed to sites Ib and Ic on gp87, were ineffective in inhibiting either virus adsorption or hemagglutination. The results of this study indicate that site Ia of gp87 molecule is the critical site of virus attachment for initiation of infection as well as the hemagglutination of BHV-1.

Monoclonal antibodies directed to different antigenic sites on bovine herpesvirus type 1(BHV-1) glycoproteins were used to study the mechanisms of neutralization of the virus. Three nonoverlapping neutralizing antigenic sites, designated Ia, Ib, and Ic, were defined on gp87. Antibodies to site la which mediated viral neutralization without complement were effective on inhibition of virus adsorption. Antibodies to a single neutralization site on gp71, designated IIa, were able to neutralize the virus without complement even when they were incubated with the virus which had already adsorbed onto the cells. Antibodies directed against gp117 and antibodies against sites Ib and Ic on gp87 required complement for virus neutralization.

Monoclonal antibodies against 3 different glycoproteins of bovine herpesvirus type 1 (BHV-1) involved in virus neutralization were used in indirect immunofluorescence (IIF) tests to characterize the appearance and transport to the plasma membrane of virus antigens in the infected cells. Antibodies against gp 117 and gp 71 glycoproteins first showed pronounced ring-like nuclear fluorescence at 4 hours post-infection (PI), followed by staining of the perinuclear region, presumably the Golgi apparatus. In contrast, antibody against gp 87 produced staining in cell-to-cell junctional areas at 3 hours PI before any staining close to the nucleus. The expression of the 3 glycoproteins at the surface of the infected cells was confirmed by the use of monoclonal antibodies having neutralizing activity, but not by non-neutralizing antibodies against gp 117 and gp 71. Non-neutralizing antibody against gp 87 detected the surface fluorescence only in those cells showing marked degeneration. Inhibition of glycosylation of the viral glycoproteins with tunicamycin (TM) was followed by interference with transport of gp 117 and gp 87 to the plasma membrane. On the other hand, gp 71 was incorporated into the plasma membrane despite the lack of N-linked glycosylation.