Methods in Anti-HCMV Research

The study of strategies for the treatment of human cytomegalovirus (HCMV) infection starts with the discovery of compounds that are potent and selective inhibitors of HCMV replication. Selectivity means that the window between the concentrations that block viral replication on the one hand and those that affect host cell functions on the other is sufficiently wide. Because HCMV induces an obvious cytopathic effect (CPE) in human fibroblast cultures, potential antiviral activity can easily be scored microscopically. For this type of assay, a relatively low input of virus should be used, so that the CPE does not appear within the first 2-3 d after infection. If too high a multiplicity of infection (MOI) is used, the input virus will result in a substantial amount of CPE shortly (i.e., within 24 h) after infection. As the CPE induced by this input virus will not be blocked by compounds that inhibibit new progeny virus formation, the use of a high MOI will mask a potential inhibitory effect of a given compound on HCMV replication. Scoring of CPE can be done either by counting the HCMV-induced plaques (although this is a rather labor-intensive method for large-scale screening purposes) or simply by scoring the CPE on a scale of 1-5 (or 10). Each assay should always be validated by using a reference compound with proven anti-HCMV activity (e.g., ganciclovir, cidofovir, foscarnet).