Real-time PCR for rapid determination of antiviral drug susceptibility of Adenovirus

GAINOTTI ROZANA; RICARTE CARMEN; VIDELA CRISTINA; EBEKIAN BEATRIZ; CARBALLAL GUADALUPE; DAMONTE ELSA; ECHAVARRIA MARCELA

Antimicrobial Agents

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Año: 2010 vol. 164 p. 30 - 34

Human adenoviruses (AdV) are associated with respiratory, ocular and gastrointestinal infections as well as potentially fatal disseminated disease in highly immunocompromised patients. Currently, there is no formally approved antiviral therapy against AdV infections. Furthermore, antiviral assays for AdV are not standardized and most of them are time consuming. The objective of this study was to evaluate a real-time PCR assay for rapid determination of antiviral drug susceptibility of AdV. The nucleoside analogue stavudine (d4T) was used as test compound in A549 cells infected with AdV serotype 5. The antiviral assay measured the reduction of the AdV DNA levels in culture supernatants by real-time PCR using a specific TaqMan probe for a conserved region at the 5’ end of the hexon gene. The compound d4T was shown to be a selective inhibitor of AdV5 with values of effective concentration 50% (EC50) determined by real-time PCR in the range 0.08-0.12 mM at MOIs between 0.001 and 1. Furthermore, the EC50 obtained with this test correlated well with those obtained in parallel with plaque reduction and virus yield inhibition assays (r2 = 0.9938 and r2 = 0.9468, respectively). In conclusion, the real-time PCR based antiviral assay is rapid, reproducible and could replace classical and more labor-intensive infectivity assays.