Dengue virus is sensitive to inhibition prior to productive replication

Uncovering vulnerable steps in the life cycle of viruses supports the rational design of antiviral treatments. However, information on viral replication dynamics obtained from traditional bulk assays with host cell populations is inherently limited as the data represent averages over a multitude of unsynchronized replication cycles. Here, we use time-lapse imaging of virus replication in thousands of single cells, combined with computational inference, to identify rate-limiting steps for dengue virus (DENV), a widespread human pathogen. Comparing wild-type DENV with a vaccine candidate mutant, we show that the viral spread in the mutant is greatly attenuated by delayed onset of productive replication, whereas wild-type and mutant virus have identical replication rates. Single-cell analysis done after applying the broad-spectrum antiviral drug, ribavirin, at clinically relevant concentrations revealed the same mechanism of attenuating viral spread. We conclude that the initial steps of infection, rather than the rate of established replication, are quantitatively limiting DENV spread.