A NEUTRALIZING MONOCLONAL-ANTIBODY TO BOVINE ROTAVIRUS VP8 NEUTRALIZES ROTAVIRUS INFECTION WITHOUT INHIBITING VIRUS ATTACHMENT TO MA-104 CELLS

VP8, the N-terminal cleavage product of rotavirus VP4, contains the v irus neutralizing epitopes in the hemagglutination domain. To map the neutralizing epitope, we developed monoclonal antibodies specific for VP4 of bovine rotavirus C486 (BRV). A neutralizing escape mutant was g enerated by one of these monoclonal antibodies (2E8) and a point mutat ion (Glu --> Asp) was found at aa 116 of VP8. To investigate the effe ct of this mutation on the cellular binding and hemagglutination activ ities, the VP8 genes of the escape mutant and wild type (WT) virus we re expressed in E. coli and their functional activities were compared. Both the escape mutant and WT virus VP8 showed hemagglutination and MA-104 cell binding activities. However, hemagglutination activity of the WT virus VP8 was inhibited by 2E8, but that of the escape mutant VP8 was not. These data indicate that the neutralizing epitope is loc ated in the HA domain but is not critical for rotavirus attachment to MA-104 cells. To understand virus neutralization, radiolabelled BRV wa s incubated with 2E8 and the distribution of radioactivity in a CsCl d ensity gradient was analysed as was the morphology of the virions in p eak fractions. Interaction of 2E8 with rotavirus led-to virus morpholo gical changes with a concomitant shift in buoyant density. These data suggest that aa 116 influences the binding of 2E8 which in turn may al ter virus integrity.