DETECTION OF COXSACKIEVIRUS B3 RNA IN MOUSE MYOCARDITIS BY NESTED POLYMERASE CHAIN-REACTION

Background: A majority of cases of viral myocarditis are associated wi th group B Coxsackieviruses (CVB) and the persistence of these viruses in the myocardium is associated with the progression of acute myocard itis to chronic dilated cardiomyopathy. A highly sensitive nested poly merase chain reaction (NPCR) is required to study the mechanisms of vi ral persistence in the myocardium. Objectives: To develop an enterovir us group-specific NPCR system, to compare it to the reverse-transcript ion PCR (RT-PCR) plus Southern hybridisation and to investigate the dy namics of viral RNA in a murine model of myocarditis induced by CVB3. Study design: Primers corresponding to the conserved sequences in the 5'-nontranslated region of enteroviruses were designed to ensure a bro ad specificity. The specificity of PCR products was confirmed by South ern hybridisation. The sensitivity of RT-PCR or NPCR was assessed usin g 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. Results: Bo th RT-PCR and NPCR could detect all 11 representative enteroviruses. T he 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 myoc ardium in acute phase, but was no longer recoverable after 9 days. Vir al RNA was detected by the NPCR technique throughout the studied perio d. Conclusions: An enterovirus group-specific NPCR system was develope d and was much more sensitive than the RT-PCR technique. It can replac e the Southern hybridisation of RT-PCR products. The presence of viral RNA in the myocardium after acute phase indicates a possibility of CV B3 shifting to persistent infection in the SWR mice.