Development of a simple restriction fragment length polymorphism assay for subtyping of coxsackie B viruses

Coxsackie B viruses (genus, Enterovirus; family, Picornaviridae) can cause aseptic meningitis, encephalitis, pleurodynia, myocarditis and are implicated in the pathogenesis of dilated cardiomyopathy. The differentiation of the group B coxsackieviruses into their subtypes has potential clinical and epidemiological implications. In the present study, a simple restriction fragment length polymorphism (RFLP) assay was developed for typing of group B coxsackieviruses into subtypes 1-6. It is a two step process, first, virus isolation and identification by virus neutralization assay, using pools of polyclonal antisera, second, the reverse transcription polymerase chain reaction (RT-PCR) using a single primer pair selected from the conserved 5'-untranslated region (5'-UTR) of enterovirus genome followed by RFLP. A 440 bp product was amplified from the reference strains of each subtype of group B coxsackievirus and 29 clinical isolates (positive for group B coxsackieviruses by neutralization assay). The amplified products were subjected to restriction endonuclease digestion by enzyme BsaJI. The assay was able to distinguish all six serotypes of coxsackie B viruses. The results were comparable to serotyping and showed that due to the relatively conserved nature of 5'-UTR in enterovirus genome, this region can be used for subgeneric molecular identification of enteroviruses.