Because we observed that a subpopulation of S598 Q600Y -primed cells cross-reacted with the native determinant, we next determined whether this subpopulation also exhibited high functional avidity. For this purpose, we isolated cells from the CNS of rJ-and rJ.S Q600Y -infected adult B6 mice and stimulated them with 10-fold dilutions of native S598 peptide. S598 Q600Y -primed cross-reacting cells exhibited 7-fold higher functional avidity, relative to those primed by the native determinant ( Figure 3C , D) and are therefore distinct from CTL primed by the native S598 determinant. Consistent with the presence of a distinct population of cells, there were modest differences in Vb chain utilization when total populations from rJ and rJ.S Q600Y -infected mice and when total and cross-reacting cell populations from rJ.S Q600Y -infected mice ( Figure S2 ) were compared. In all instances, Vb5.1/5.2 expression was relatively over-represented, but some Vb elements were preferentially utilized by specific responding populations (Vb11, Vb13 and Vb14). Also consistent with this observation, alanine scanning mutagenesis of the two determinants revealed that the CTL response to each cognate peptide following infection was also subtly different reflecting the altered repertoire. The CTL response to each cognate peptide was very sensitive to mutation at every position except 2 and 9 although mutations in the S598 Q600Y determinant were tolerated slightly better than changes in the S598 epitope ( Figure S2C -E).