In vitro and in vivo growth comparisons of rJ and rJ.S_W513 variant viruses. A, 17Cl-1 cells were infected with rJ and rJ.S_W513 variant viruses at an MOI of 1.0. Cells and supernatants were harvested at the indicated times, and titers were measured by plaque assay on Hela-MHVR cells as described in Materials and Methods. Data are means ± SEM for three independent experiments. B, Representative sequencing chromatograms from RT-PCR analyses of brain RNA samples in which only rJ (top), a mixture of rJ and rJ.S_W513G (middle), or only rJ.S_W513G (bottom) virus templates were detected. C, 17Cl-1 cells were infected with equal PFU of rJ and each variant (MOI of 1) and propagated for four passages. Total RNA was harvested from infected wells at passages 2 (left panel) and 4 (right panel) and relative representation of rJ or variant was determined as described in Materials and Methods. Data for passages 2 and 4 are pooled results obtained by analyzing 6 wells for each virus mixture. D, B6 mice (left panel) and BALB/c (right panel) mice were infected with virus mixtures consisting of equal PFU of rJ and each variant. Seven days later total RNA was harvested from the brains of infected mice and relative representation of virus template was determined via RT-PCR and direct sequencing of PCR products as described in Materials and Methods. Numbers in parentheses indicate the number of mice examined for each group. E, Survival analysis of maternal Ab-protected suckling mice infected with rJ or rJ.S_W513 variant viruses. Mice were scored as deceased after developing clinical signs of hindlimb paralysis/paresis (day of tissue harvest).