Tetsuo Kumagai

Mechanisms of neutralization by monoclonal antibodies to different antigenic sites on the bovine herpesvirus type 1 glycoproteins

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 Ia 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.

A new in vitro method (END) for detection and measurement of hog cholera virus and its antibody by means of effect of HC virus on newcastle disease virus in swine tissue culture

A newin vitro method of neutralization test for hog cholera virus by means of the END method was established. The test is based on the finding that immune serum against hog cholera virus specifically inhibits the exalting effect of hog cholera virus on Newcastle disease virus in swine testicular cell culture. The serum-virus mixtures are incubated at 37° C for 60 minutes or at 4° C overnight and tested for infectivity by the END method described in the previous papers of this series. The test is not only simple enough for the routine use, but also highly reproducible and specific. The test may achieve wide application in studies of hog cholera virus.

Bovine herpesvirus type 1 gp87 mediates both attachment of virions to susceptible cells and hemagglutination.

SummaryThe 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.

Intracellular localization and transport of three different bovine herpesvirus type 1 glycoproteins involved in neutralization

SummaryMonoclonal 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.

Technical Improvement of the END Method

The END method is a new in vitro method for detecting and measuring hog cholera (HC) virus and its neutralizing antibody by means of an enhancing effect of HC virus on Newcastle disease (ND) virus in swine testicular (ST) cell cultures (4--6, 8). ST cell monolayers infected with HC virus, which exerts no cytopathic effect (CPE), develop CPE when challenged with ND virus after growth of HC virus for some days, whereas ND virus produces no CPE in ST cell cultures not infected with HC virus. Although the reproducibility and specificity of the method were found to be satisfactory enough, an improvement of the method is obviously desirable part iculary regarding the sensitivity for slow growing HC virus strains and in view of the difficulty in obtaining sera suitable for the culture medium (6). We wish to describe here a technical improvement of the method in these respects. The materials and methods employed were essentially the same as used in the previous studies (5, 6, 8). In a previous s tudy (5) the difficulty in finding the suitable bovine sera was reduced by increasing the serum concentration of the culture medium from 10~o to 20%, which made more sera suitable for the test. This was confirmed in the present study: many of the bovine sera showing poor maintenance of ST cells in a concentration of 10 ~o maintained the cells in a good condition when used at 20~o, and no or only slight CPE was produced after challenge with ND virus in HC uninoculated cell cultures. Thns, of 16 sera tested 12 were found to be suitable for the test. Of the remaining 4 sera, one was unsuitable because of poor maintenance of the cells, and 3 because of inhibitory act ivi ty to the END effect.