CHARACTERIZATION OF GENOMIC RNA OF COXSACKIEVIRUS B3 IN MURINE MYOCARDITIS - RELIABILITY OF DIRECT SEQUENCING OF REVERSE TRANSCRIPTION-NESTED POLYMERASE CHAIN-REACTION PRODUCTS

SWR mice develop viral myocarditis histologically similar to the human disease following inoculation with a cardiovirulent Coxsackievirus B3 (CVB3), reactivated from a sequenced cDNA clone of Nancy strain. A se quence of 215 nucleotides, or 628 nucleotides in representative cases, of the 5'non-translated region (5'NTR) of CVB3 genome was amplified f rom myocardial samples of the infected mice by reverse transcription-n ested polymerase chain reaction (RT-NPCR). In order to verify the vira l nucleotide sequence and detect the mutation frequency of the viral R NA, the nucleotide sequence of NPCR products were determined by direct sequencing in both orientations. The amplified products from mouse he art on day 1-13 post-inoculation were sequenced and, in each case, the consensus sequence was identical to the published sequence of CVB3 (N ancy strain). To evaluate further the reproducibility of these techniq ues, three tissue samples from the same infected mouse heart were proc essed independently. Sequences of their RT-NPCR products were identica l to each other as well as to the published sequence. When two attenua ted CVB3 mutants were amplified and sequenced, single mutations were d etected. To evaluate the overall fidelity of these two combined techni ques, genomic RNA of a different CVB3 Nancy strain stock, Coxsackievir us A9 or poliovirus sabin 1 was amplified and the NPCR products sequen ced. Each product showed 100% homology with its published sequence. Th ese results demonstrate that the coupled technique of the enterovirus RT-NPCR with direct sequencing of NPCR products generates accurate con sensus sequence data and this technique proved to be useful in verific ation of enteroviral amplicons and in detection of nucleotide mutation s. In addition, a low mutation frequency was found in the 5'NTR of CVB 3 detected in myocardial samples of immunocompetent mice up to 13 days . (C) 1997 Elsevier Science B.V.