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 virus 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 generated by one of these monoclonal antibodies (2E8) and a point mutation (Glu-->Asp) was found at aa 116 of VP8*. To investigate the effect of this mutation on the cellular binding and hemagglutination activities, the VP8* genes of the escape mutant and wild type (WT) virus were 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 located in the HA domain but is not critical for rotavirus attachment to MA-104 cells. To understand virus neutralization, radiolabelled BRV was incubated with 2E8 and the distribution of radioactivity in a CsCI density gradient was analysed as was the morphology of the virions in peak fractions. Interaction of 2E8 with rotavirus led to virus morphological changes with a concomitant shift in buoyant density. These data suggest that aa 116 influences the binding of 2E8 which in turn may alter virus integrity.
Lee
J
J
Veterinary Infectious Disease Organization, University of Saskatchewan, Saskatoon.
Babiuk
L A
LA
Yoo
D
D
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D000918:Antibody Specificity; D002213:Capsid / Q000737:chemistry / Q000276:immunology*; D036022:Capsid Proteins; D002417:Cattle; D002460:Cell Line; D019976:Cloning, Organism; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D004926:Escherichia coli; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology; D012401:Rotavirus / Q000276:immunology / Q000502:physiology*; D012680:Sensitivity and Specificity; D010948:Viral Plaque Assay
D000911:Antibodies, Monoclonal; D036022:Capsid Proteins; D000939:Epitopes; D011994:Recombinant Proteins