ENTRY KINETICS AND MOUSE VIRULENCE OF ROSS RIVER VIRUS MUTANTS ALTERED IN NEUTRALIZATION EPITOPES

Previously we identified the locations of three neutralization epitope s (a, b1, and b2) of Ross River virus (RRV) by sequencing a number of variants resistant to monoclonal antibody neutralization which were fo und to have single amino acid substitutions in the E2 protein (S. Vrat i, C. A. Fernon, L. Dalgarno, and R. C. Weir, Virology 162:346-353, 19 88). We have now studied the biological properties of these variants i n BHK cells and their virulence in mice. While variants altered in epi topes a and/or b1 showed no differences, variants altered in epitope b 2, including a triple variant altered in epitopes a, b1, and b2, showe d rapid penetration but retarded kinetics of growth and RNA and protei n synthesis in BHK cells compared with RRV T48, the parent virus. Vari ants altered in epitopes a and/or b1 showed no change in mouse virulen ce. However, two of the six epitope b2 variants examined had attenuate d mouse virulence. They had a four- to fivefold-higher 50% lethal dose (LD(50)), although no change in the average survival time of infected mice was observed. These variants grew to titers in mouse tissues sim ilar to those of RRV T48. The LD(50) of the triple variant was unchang ed, but infected mice had an increased average survival time. This var iant produced lower levels of viremia in infected mice. On the basis o f these findings we propose that both the receptor binding site and ne utralization epitopes of RRV are nearby or in the same domain of the E 2 protein.