Variability in molecular typing of Coxsackie A viruses by RFLP analysis and sequencing

The aim of the present study was to develop an assay capable of classifying the Coxsackie A virus (CAV) prototype strains on the basis of restriction fragment length polymorphism (RFLP) analysis of 5'-UTR-derived reverse transcription polymerase chain reaction (RT-PCR) amplicons, and to determine how these data could be used for typing wild-type CAV isolates. Moreover, sequencing of the amplified genomic fragments of the clinical isolates, and comparison with all the published sequences of the respective genomic region of enterovirus reference and wild-type strains were attempted for typing of the isolates. Twenty-four prototype CAV strains from the 23 currently recognized serotypes were studied; most of them were successfully differentiated with the aid of four restriction endonucleases: HaeIII, HpaII, DdeI, and StyI. It was not possible to differentiate between CAV5, 7, and 16, or between CAV15 and 18 in this way, but the members of each of these two groups were satisfactorily differentiated with the aid of single-strand conformational polymorphism (SSCP) analysis of their RT-PCR amplicons. Fifteen clinical isolates, 13 of them of known CAV serotype, were also studied with the same four endonucleases and the results were compared with the data obtained from the RFLP analysis of the reference strains. The experimental results showed that only two clinical samples of previously known identity had an identical restriction pattern with the respective prototype strains. The sequences of the amplicons of the clinical isolates had the greatest percentage of alignment with enterovirus strains of a different serotype, indicating variability in the 5'-UTR and the inability to use the whole sequence of the amplicons for typing CAVs. The significance of the findings in relation to the possible usefulness of the RFLP-based method is discussed.