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Previously we identified the locations of three neutralization epitopes (a, b1 and b2) of Ross River virus (RRV) by sequencing a number of variants resistant to monoclonal antibody neutralization which were found to have single amino acid substitutions in the E2 protein (S. Vrati, C.A. Fernon, L. Dalgarno, and R.C. Weir, Virology 162:346-353, 1988). We have now studied the biological properties of these variants in BHK cells and their virulence in mice. While variants altered in epitopes a and/or b1 showed no difference, variants altered in epitope b2, including a triple variant altered in epitopes a, b1, and b2, showed rapid penetration but retarded kinetics of growth and RNA and protein synthesis in BHK cells compared with RRV T48, the parent virus. Variants altered in epitopes a and/or b1 showed no change in mouse virulence. However, two of the six epitope b2 variants examined had attenuated mouse virulence. They had a four- to fivefold-higher 50% lethal dose (LD50), although no change in the average survival time of infected mice was observed. These variants grew to titers in mouse tissues similar to those of RRV T48. The ID50 of the triple variant was unchanged, but infected mice had an increased average survival time. This variant produced lower levels of viremia in infected mice. On the basis of these findings we propose that both the receptor binding site and neutralization epitopes of RRV are nearby or in the same domain of the E2 protein.

Entry kinetics and mouse virulence of Ross River virus mutants altered in neutralization epitopes