Genetic and phenotypic studies on Ross River virus variants of enhanced virulence selected during mouse passage

We have passaged Ross River virus (RRV) in mice to generate variants with increased mouse virulence and attempted to relate changes in virulence to genome sequence changes. RRV NBO (zero passage in mice) is a plaque-purified clone of the mouse-avirulent strain RRV NB5092, and is of low virulence for day-old mice. During RRV NBO replication in infant mice, its virulence for day-old mice increased markedly with time. By 7 days postinfection the LD50 value of harvested virus (passage level one) was congruent to 10(4)-fold less than that of the parental virus. No further decrease in LD50 followed 10 serial passages in infant mice. However, 10th passage level virus showed increased clinical effects in week-old mice by comparison with virus from passage levels one and two. The growth kinetics of RRV variants in mice suggested that the rate and extent of RRV replication in the brain tissue determined the enhanced mouse virulence of serially passaged virus. Seven out of eight independently passaged, 10th passage level variants had changes in the E2 gene leading to one or two amino acid substitutions. The changes were at residues 212, 232, 234, 251, 341, 27 and 172, and 72 and 134 in these variants; all changes except two were nonconservative. Residues 212, 234, and 251 form part of a neutralization determinant in RRV. Changes in epitope b2 (which includes amino acids 246, 248, and 251) alter the kinetics of RRV entry into cells (P. Kerr, R. C. Weir, and L. Dalgarno, unpublished data). First and second passage level virus of enhanced virulence was unchanged in E2 or E1 gene sequences from RRV NBO. However, 1st, 2nd, and 10th passage level virus induced higher levels of virus-specific RNA synthesis than did RRV NBO in cultured BHK cells. We propose a model for the mechanism of virulence enhancement on passaging RRV NBO in mice.