An outbreak of CVB2 provided a unique opportunity to explore the degree of genetic drift exhibited by this virus during an epidemic and its genetic relationship to isolates from previous years. From 8.27-9.27 1994 consultations on 4 infants less than 2 months of age (9-54 days) with severe CVB2 infection were made: 2 had severe myocarditis, resulting in death in one and cardiac transplantation in the other; a third presented with severe hepatitis and DIC; the last had meningitis and hepatitis. Isolates from these and 6 other 1994 cases (including the first and last isolates for 1994) along with isolates from 1947 (prototype strain Ohio), 1986, 1987, 1991, were obtained. All were retyped to confirm they were CVB2. Using primers targeted to nucleotides 64-88 and 742-756 of the 5′ nontranslated region(5′NTR) a 696 bp fragment was amplified using a high fidelity reverse transcription (RT)-PCR procedure and cycle sequenced.

Nine isolates from 6.27-9.27 exhibited 99.9% identity to each other, indicating minimal genetic drift had occurred. The final isolate of 1994(Nov-94) revealed only 86% identity to those clustering from June-Sept(JS-94). The degree of divergence was greater than would have been predicted based on the 9 earlier isolates. To explore the extent of divergence permitted in the 5′NTR, the Ohio strain and 3 other isolates were compared. Strains from 1947-1991 shared a 83.5-88.5% identity, Ohio (1947) was the most distantly related. Comparison of pre-1994 isolates with those from 1994 revealed that the JS-94 cluster had 93.8% identity with the 1987 isolate while the Nov-94 isolate had 95.5% identity to the 1991 isolate. These findings suggest that the degree of divergence exhibited by the Nov-94 isolate was not the result of genetic drift from the JS-94 cluster, but rather due to the introduction of a CVB2 strain closely related to that of 1987. They also support the concept of recirculation of the enteroviruses in the epidemiology of infections with these agents. RT-PCR is a powerful and rapid procedure that permitted us to identify the introduction of a second CVB2 strain into the community and to draw genetic ties between 1994 strains and those from previous years.