Detection of enterovirus RNA in experimentally infected mice by molecular hybridisation: Specificity of subgenomic probes in quantitative slot blot and in situ hybridisation

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Abstract
Subgenomic cDNA clones representing defined regions of the genome of Coxsackie B3 virus were used as hybridisation probes to detect RNA of various enteroviruses in cell culture and mouse model systems. Radiolabelled probes were used in slotblots to quantitate the RNA in samples; biotinylated probes were used to localise virus RNA at the cellular level by in situ hybridisation with monolayers of infected cells or thin sections of tissue samples. Probes derived from the 5′ or 3′ terminal regions of Coxsackie virus RNA, which are highly conserved in the genus Entero‐virus, detected RNA of various serotypes in infected cell cultures, but failed to hybridise with hepatitis A virus (HAV) RNA. Although HAV is clearly a Picornavirus, our data support the view that its taxonomic position within the enteroviruses should be reconsidered. The biotinylated probes were also used to detect in situ virus RNA in paraffin‐embedded tissue samples from experimental mouse models of Coxsackie B3 virus‐induced myocarditis or Coxsackie B1 virus‐induced myositis. Since the integrity of the tissues was preserved during the process, and viral RNA was localised in the affected muscle fibres, this has enabled us unequivocally to relate the infecting virus to the underlying tissue injury.