Sites other than nucleotide 234 determine cardiovirulence in natural isolates of coxsackievirus B3

The genetic site(s) that naturally determine the cardiovirulence phenotype of coxsackievirus B3 (CVB3) have yet to be mapped. Using two closely related CVB3 strains that differed in terms of cardiovirulence phenotype in mice, we previously reported the difference in phenotype mapped to a single site, nucleotide 234 (nt234) in the 5' non-translated region (NTR) of the CVB3 genome. When nt234 was C, the virus was attenuated and when U, the virus was cardiovirulent. To determine whether this finding was applicable to other strains of CVB3, we examined 13 different naturally occurring CVB3 strains isolated in different years in the United States. We determined that only two isolates induced severe inflammatory heart muscle disease in C3H/HeJ male mice. Using PCR products as sequencing templates, we determined the 5' NTR sequence from each viral genome. Alignment of these sequences and other published CVB3 5' NTR sequences suggests as many as four separate lineages, with commonly used laboratory strains clustering closely in one branch. An examination of the sequences showed that regardless of cardiovirulence phenotype, nt234 was invariably uridine. Thus, the previously reported cytidine at nt234 is most likely the result of a rare mutation and is not a naturally occurring variation and other sites must account for the variance in virulence seen in natural isolates of CVB3.