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Serial passage of Ross River virus in chick embryo fibroblasts selected for virus variants altered in their reactions to neutralizing monoclonal antibodies. The E1 and E2 genes of each antigenic variant were sequenced; single nucleotide changes were found in E2 leading to amino acid substitutions at either residue 4 (Glu → Lys) or 218 (Asn → Lys); no changes were found in the E1 gene. Variants with the substitution at E2 residue 218 replicated less efficiently in 1-day-old mice than did the parental strain. The variant changed at E2 residue 4 showed little alteration in replication efficiency in mice. Similar genotypic or phenotypic changes were not found in virus passaged serially in human or mosquito cell lines.