Development and evaluation of quantitative-competitive PCR for quantitation of coxsackievirus B3 RNA in experimentally infected murine tissues

A method is described for quantitation of enterovirus RNA in experimentally infected murine tissues. Viral RNA was extracted from tissue samples and a mplified by reverse transcriptase PCR in the presence of an internal standa rd RNA. The ratio of PCR product derived from viral RNA and internal;standa rd RNA was then determined using specific probes in a post-PCR electrochemi luminescent hybridization assay. This provided an estimate of the viral RNA copy number in the original sample, and detection of PCR product derived f rom internal standard RNA validated sample processing and amplification pro cedures. RNA copy number correlated with viral infectivity of cell culture- derived virus, and one tissue culture infective dose was found to contain a pproximately 10(3) genome equivalents. The ratio of RNA copy number to infe ctivity in myocardial tissue taken from mice during the acute phase of coxs ackievirus B3 myocarditis was more variable ranging from 10(4)-10(7), and w as dependent on the stage of infection, reflecting differential rates of cl earance for viral RNA and viral infectivity. The assay is rapid, and could facilitate investigations which currently rely upon enterovirus quantitatio n by titration in cell culture. This would be useful for experimental studi es of viral pathogenesis, prophylaxis and antiviral therapy. (C) 1999 Elsev ier Science B.V. All rights reserved.