Beta-d-N4-hydroxycytidine is an anti-VEEV (venezuelan equine encephalitis virus) agent with EC ₅₀ , EC ₉₀ , and EC ₉₉ are 0.426, 1.036, and 2.5 μM, respectively.Beta-d-N4-hydroxycytidine inhibits CHIKV replicon activity and the 50% effective concentration (EC ₅₀ ) s 0.8 μM in the Huh-7–CHIKV replicon cell line. Similar results is presented with the replicon in BHK-21 cells (EC ₅₀ =1.8 μM). NHC has no cytotoxicity for NHC in the Huh-7 cell culture system until up to 100 μM using MTT assays. The 50% cytotoxic concentration (CCsub>50) values for NHC are determined to be 30.6 μM, 7.7 μM, and 2.5 μM in peripheral blood mononuclear (PBM), Vero, and CEM cells, respectively.NHC behaves as a pyrimidine analog, NHC-mediated inhibition of the CHIKV replicon can be abrogated by the addition of exogenous nucleosides, such as pyrimidines C and U, but dA, dC, dG, dU, or T has no impact on the replicon. Pyrimidines A and G contributes to replicon inhibition both in the presence and in the absence of NHC.