Echoviruses (ECVs), members of the genus enterovirus(polioviruses [PV], group A and B coxsackieviruses [CAV and CBV], ECV and enteroviruses), cause syndromes ranging from febrile exanthems to myocarditis and meningitis. The PV1-3 and CBV3 5'NTR, the major viral regulatory region, has been shown to contain virulence determinants important in pathogenesis. Despite their clinical significance, few studies have explored the role of the ECV 5'NTR in pathogenesis. Murine models exist for the study of the PV and CBV. No such models exist for the ECV due to the inability of these viruses to replicate in mice. Because the ECV 5'NTR is similar to CBV, a possible approach to the study of the ECV 5'NTR, using a murine model, is to create chimeric viruses in which the 5'NTR of CBV is replaced with that of the ECV. We constructed such ECV/CBV chimeras and report their viability, growth kinetics and ability to retain parental virulence phenotype. ECV serotypes 9 and 12 were selected based on clinical virulence phenotype; ECV9 infection more frequently results in overt clinical disease, while ECV12 infection results in subclinical seroconversion. Nucleotides 62-753 of the ECV9 and 12 5'NTRs were RT-PCR amplified, directly sequenced, cloned and used to replace the homologous region of a cloned, transcriptionally controlled, full-length, noncardiovirulent CBV3. HeLa cell transfection of run-off RNA transcripts from the chimeric constructs resulted in viable virus. Single passage chimeric virus was sequenced prior to further characterization. No variation from the parental ECV 5'NTR sequence was found. One-step viral growth studies revealed that while both chimeras achieved similar final titers, the ECV12/CBV3 chimera was slower in doing so. CH3/He mice inoculated with the chimeric viruses were examined for evidence of myocarditis as a marker of virulence phenotype. The ECV9/CBV3 chimera produced myocardial lesions while ECV12/CBV3 failed to do so. These studies indicate that 5'NTR ECV/CBV3 chimeras are viable, appear to retain the parental ECV virulence phenotype and provide evidence for ECV 5'NTR virulence determinants.