Characterisation 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 sequence of 215 nucleotides, or 628 nucleotides in representative cases, of the 5'non-translated region (5'NTR) of CVB3 genome was amplified from myocardial samples of the infected mice by reverse transcription-nested polymerase chain reaction (RT-NPCR). In order to verify the viral nucleotide sequence and detect the mutation frequency of the viral RNA, the nucleotide sequence of NPCR products were determined by direct sequencing in both orientations. The amplified products from mouse heart on day 1-13 post-inoculation were sequenced and, in each case, the consensus sequence was identical to the published sequence of CVB3 (Nancy strain). To evaluate further the reproducibility of these techniques, three tissue samples from the same infected mouse heart were processed independently. Sequences of their RT-NPCR products were identical to each other as well as to the published sequence. When two attenuated CVB3 mutants were amplified and sequenced, single mutations were detected. To evaluate the overall fidelity of these two combined techniques, genomic RNA of a different CVB3 Nancy strain stock, Coxsackievirus A9 or poliovirus sabin 1 was amplified and the NPCR products sequenced. Each product showed 100% homology with its published sequence. These results demonstrate that the coupled technique of the enterovirus RT-NPCR with direct sequencing of NPCR products generates accurate consensus sequence data and this technique proved to be useful in verification of enteroviral amplicons and in detection of nucleotide mutations. In addition, a low mutation frequency was found in the 5'NTR of CVB3 detected in myocardial samples of immunocompetent mice up to 13 days.