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In vitro RNA-dependent RNA polymerase assays revealed that the JEV replication complex (RC) synthesized viral RNA utilizing a semiconservative and asymmetric mechanism. Peak viral replicase activity and levels of viral RNA observed 15–18 h postinfection (h p.i.) preceded maximum viral titers in the culture medium seen 21 h p.i. Among divalent cations, Mg2+ was essential and exhibited cooperative binding for its two replicase-binding sites. Mn2+, despite sixfold higher affinity for the replicase, elicited only 70% of the maximum Mg2+-dependent activity, and deficit of either cation led to synthesis of incomplete RNA products. We also determined as a first instance for a flavivirus RC, kinetic parameters using cytoplasmic “virus-induced heavy membranes” after depleting endogenous nucleotides. Exhaustive trypsin treatment, which degraded the bulk of NS3 and NS5, had no effect on replicase activity, suggesting that the active flaviviral RC resides behind a membrane barrier and recruits minuscule proportions of the replicase proteins. [Copyright &y& Elsevier]