Journal of Virological Methods 125 (2005) 95–98 Short communication Persistence of DNA in cell cultures may jeopardize the analysis of human herpesvirus 6 dynamics by means of real-time PCR Pascale Bonnafous a , Agn` es Gautheret-Dejean a,b , David Boutolleau a,c , Delphine Ca¨ ıola a , Henri Agut a,∗ a Laboratoire de Virologie, UPRES EA2387, CERVI, Groupe Hospitalier Piti´ e-Salpˆ etri` ere, 75013 Paris, France b Laboratoire de Microbiologie, Facult´ e des Sciences Pharmaceutiques et Biologiques, 75006 Paris, France c Laboratoire de Bact´ eriologie-Virol´ ogie, Facult´ e de M´ edecine Paris sud, CHU de Bicˆ etre, 94275 Le Kremlin Bicˆ etre, France Received 7 September 2004; received in revised form 30 November 2004; accepted 1 December 2004 Available online 28 January 2005 Abstract The use of real-time PCR has been described previously for analysing both the replication kinetics and antiviral susceptibility of human herpesvirus 6 in MT4 cells. It is now reported that viral DNA persists in infected cell culture long after the end of lytic virus replication. Consequently, high levels of DNA may correspond to an absence of infectivity and late readout occurring after the exponential phase of virus growth may lead to misinterpretation of the results of susceptibility assays. These limitations must be borne in mind when using real-time PCR. © 2004 Elsevier B.V. All rights reserved. Keywords: Virus cell load; Virus growth kinetics; Susceptibility assay; Antiviral drug Human herpesvirus 6 (HHV-6) is a betaherpesvirus closely related to human cytomegalovirus (HCMV). The primary in- fection causes febrile illnesses including exanthem subitum (Yamanishi et al., 1988). The virus then persists in the host lifelong with both a latent state with no infectious particles produced except during episodes of reactivation, and chronic replication with continuous or frequent production of infec- tious virus. Reactivation may cause various diseases, such as encephalitis, multiple sclerosis or be associated with HCMV disease (Ablashi et al., 2000; Nash et al., 2004; Razonable et al., 2003). HHV-6 replication is inhibited by antiviral com- pounds such as ganciclovir (GCV), cidofovir (CDV) and fos- carnet (PFA) in vitro (Agut et al., 1989; Akesson-Johansson et al., 1990; Long et al., 2003; Manichanh et al., 2000; Reymen et al., 1995). The treatment of severe HHV-induced diseases by these compounds is now carried out in vivo (Denes et ∗ Corresponding author. Tel.: +33 1 42 17 74 00; fax: +33 1 42 17 74 17. E-mail address: henri.agut@psl.ap-hop-paris.fr (H. Agut). al., 2004), and there is the possible emergence of resistance (Manichanh et al., 2001; Safronetz et al., 2003). This concern has led to the development of susceptibility assays for detect- ing resistance and replication dynamics tests for examining the fitness of resistant viruses. Real-time PCR has been applied previously for the de- velopment of such assays (Mac´ e et al., 2003). The princi- ple of these assays was to measure the intracellular HHV-6 DNA content and use it as a marker for HHV-6 replication. This approach was tested for the study of HHV-6 replica- tion on MT4 cells: it proved to be successful both for the detection of resistance and analysis of replicative capacity; it was also convenient because of a micro-format culture sup- port, an early readout and a good tolerance to the variations of multiplicity of infection (MOI). However, two facts re- mained unexplained: (i) the lack of significant inhibition of virus replication in the presence of an antiviral at a concen- tration close to its 50% inhibitory concentration (IC 50 ) when the time of culture exceeded 2 weeks and (ii) the biphasic 0166-0934/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2004.12.001