Detection of Coxsackievirus B3 RNA in mouse myocarditis by nested polymerase chain reaction

Abstract A majority of cases of viral myocarditis are associated with group B Coxsackieviruses (CVB) and the persistence of these viruses in the myocardium is associated with the progression of acute myocarditis to chronic dilated cardiomyopathy. A highly sensitive nested polymerase chain reaction (NPCR) is required to study the mechanisms of viral persistence in the myocardium. To develop an enterovirus group-specific NPCR system, to compare it to the reverse-transcription PCR (RT-PCR) plus Southern hybridisation and to investigate the dynamics of viral RNA in a murine model of myocarditis induced by CVB3. Primers corresponding to the conserved sequences in the 5′-nontranslated region of enteroviruses were designed to ensure a broad specificity. The specificity of PCR products was confirmed by Southern hybridisation. The sensitivity of RT-PCR or NPCR was assessed using reconstructed infected muscle samples. The myocardial samples of the SWR murine model of CVB3-myocarditis were collected from day 1 to 30 after infection. The presence of viral RNA was detected by the RT-PCR or NPCR and infectious virus was isolated by cell culture. Both RT-PCR and NPCR could detect all 11 representative enteroviruses. The NPCR could detect as few as 0.01 plaque forming unit of virus, 100 times more sensitive than the RT-PCR. Virus was isolated from the myocardium in acute phase, but was no longer recoverable after 9 days. Viral RNA was detected by the NPCR technique throughout the studied period. An enterovirus group-specific NPCR system was developed and was much more sensitive than the RT-PCR technique. It can replace the Southern hybridisation of RT-PCR products. The presence of viral RNA in the myocardium after acute phase indicates a possibility of CVB3 shifting to persistent infection in the SWR mice.