Dear Editor,

Chikungunya virus (CHIKV) is a single-strand positive-sense RNA virus belonging to the family Togaviridae. Since 2004, CHIKV outbreaks have become more frequent and widespread, affecting millions of people and establishing Chikungunya disease as a significant global public health issue.1 In Asia, the Asian lineage (CHIKV-Asian) and the East-Central-South-African lineage (CHIKV-ECSA) have become the most prevalent. In July 2025, a large-scale outbreak occurred in Guangdong Province, China, resulting in more than 9000 confirmed cases.2 To date, no drug has been approved for clinical therapy, highlighting the urgent need to develop effective treatments against CHIKV.

Nucleos(t)ide analogs are among the most promising broad-spectrum inhibitors of viral RNA-dependent RNA polymerase (RdRp) for combating RNA viruses. In this study, we set out to apply a recombinant CHIKV-nluc infectious clone to rapidly screen a nucleos(t)ide analog library for identifying active inhibitors in cells (supplementary Fig. 1). Among these nucleotide analogs, we found that 4’-fluorouridine (4’-FlU), β-d-N4-hydroxycytidine (NHC), and ribavirin exhibited >90% inhibitory effects at a concentration of 10 µM against the reporter virus (Fig. 1a). Favipiravir (T-705) did not show obvious activity even at concentrations as high as 20 µM determined by detecting viral copies, but it showed activity with EC50 of 3.3 µM to decrease the plaques caused by infectious progeny viruses (supplementary table 1). T-705 has been reported to significantly suppress CHIKV in mice at doses of 200–400 mg/kg (mpk).3 4’-FlU and NHC are broad-spectrum antivirals that inhibit RNA viruses, including alphaviruses,4 which they showed comparable activity to ribavirin against a rescued CHIKV of EC50 in nanomole ranges in BHK-21, HEK-293T, and Huh-7 cells (supplementary table 1). In contrast, remdesivir, GS-441524, and sofosbuvir exhibited weak antiviral activity in our cell-based assays.

Fig. 1
figure 1

A screening of nucleotide analogs targeting CHIKV identified the oral nucleotide VV261, which showed significant efficacy in both in vitro and in vivo models. a Rapid antiviral activity screening of nucleotide analogs for CHIKV inhibitors. CHIKV-Nluc infected BHK-21 cells at an MOI of 0.01. The viral replication was evaluated by luciferase assay. b The inhibitory effects of VV261 on CHIKV-Asian, CHIKV-ECSA, and SINV were evaluated in Huh-7 cells. The viral yield in the cell supernatant was quantified by qRT-PCR. c Nucleoside competition assay. BHK-21 cells infected with CHIKV were exposed to 10 µM VV261 and increasing concentrations of exogenously added natural nucleosides. Values were normalized to DMSO-treated (vehicle) controls. d The anti-CHIKV activity of VV261 in vivo. A129 mouse (n = 5 per group) was inoculated 103 PFU CHIKV-Asian in the left rear footpad and orally treated with indicated compounds or vehicle. Viral infectious titer in ipsilateral ankles was determined in each treatment group. The error bars present in the in vitro results denote mean ± sd of three independent replicates. The in vivo viral loads were statistically analyzed with one-way ANOVA tests performed in GraphPad Prism software 9.0. **P < 0.01; ****P < 0.0001. Other supportive data are available in Figshare repository at https://doi.org/10.6084/m9.figshare.30918995

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Additionally, we identified a novel nucleotide analog, VV261, a double prodrug of 4’-FlU, showing a remarkable antiviral efficacy in a rapid screen with 90% inhibitory effect against CHIKV (Fig. 1a). VV261 was designed to enhance the chemical stability and pharmacokinetic properties of 4’-FlU, the original nucleoside that is susceptible to aqueous solutions and obscure to be druggability, and our previous study has demonstrated that VV261 has potent activity against severe fever with thrombocytopenia syndrome virus (SFTSV).5 In 2024, VV261 has been approved for Phase I clinical trials in China for the treatment of severe fever with thrombocytopenia syndrome (SFTS). Due to the favor oral bioavailability and safety profiles of VV261 in rodent and non-human primate,5 we were motivated to further investigate its potential as a drug candidate against CHIKV. Cell-based assays indicate that VV261 potently inhibits CHIKV-Asian, CHIKV-ECSA, and the rescued recombinant CHIKV in BHK-21, HEK-293T, Huh-7, and Rhabdomyosarcoma (RD) cells, and has activity against another alphavirus Sindbis virus (SINV) (Fig. 1b, supplementary Fig. 2). A CHIKV-Rluc replicon assay demonstrates that VV261 blocks viral replication in a dose-dependent manner (supplementary Fig. 3). This result aligns with the time-of-addition assays, which demonstrated that VV261 predominantly inhibits CHIKV replication when the compound is added to cells between 1 hour and 12 hours post infection (h.p.i.) (supplementary Fig. 3), suggesting that VV261 functioned at viral post-entry stage. The nucleoside competition assay reveals that the antiviral effect of VV261 against CHIKV can be competitively antagonized by the addition of exogenous uridine and cytidine (Fig. 1c), thereby supporting the hypothesis that VV261 functions as a pyrimidine analog disrupting the synthesis of nascent CHIKV RNA. Moreover, our findings demonstrate that VV261 suppresses CHIKV replication without inducing lethal mutations; in contrast, treatment with T‑705 at 5 µM promoted the accumulation of C-U transitions and elevated the transition‑to‑transversion ratio in progeny viral genomes across two passages in HEK‑293 T cells (supplementary Fig. 4).

To evaluate the antiviral potential of VV261 in vivo, we conducted an efficacy study against CHIKV infection in IFNAR knockout (A129) mice aged 8–10 weeks. T-705 (400 mpk) was used as positive control as it has been demonstrated efficacy against CHIKV in mouse models. Mice infected with CHIKV started to show footpad swelling at 2 d.p.i, while the swelling was attenuated followed by VV261 treatment (supplementary Fig. 5). T-705 treatment showed significant protection from swelling at 2 d.p.i, despite the edema rebounding afterwards. Cytokinetic and inflammatory factor examination revealed that VV261 and T-705 treatments resulted in the reduction of IL-6, TNF-α, IL-1β, CCL2, CXCL10, ISG15 and IFNAR1 expression in ankles, wrists, and spleens compared to that of vehicle treatment, while the expression of IL-6 and IL-1β in contralateral ankles remained high with T-705 treatment (supplementary Fig. 5). Then, we wish to know whether VV261 is capable of suppressing viral replication through determining the viral loads in target tissues among treatment groups. The results showed that the viral titers in the ipsilateral ankle, contralateral ankle, and ipsilateral wrist were remarkably reduced in VV261 treatment groups in a dose-dependent manner compared to that in the vehicle group, and the dose of 10 mpk significantly reduced the viral titers under the limit of detection (LOD) (Fig. 1d, supplementary Fig. 6). T-705 treatment at 400 mpk effectively inhibited CHIKV replication in mice as what the VV261-10 mpk did. Notably, the reduced viral RNA copies in the serum after the compound administration implied that both VV261 and T-705 can relieve the viremia in blood (supplementary Fig. 6), which would benefit for ceasing CHIKV spread from an infected person to its mosquito vector. The viral RNA copy reductions in the target tissues are consistent with the readouts of viral titers (supplementary Fig. 6). Additionally, the results of clinical sign scoring on muscle weakness of limbs, reduced activity, and piloerection revealed that compound VV261 (5 and 10 mpk) and T-705 (400 mpk) could significantly reduce disease severity (supplementary table 2). These in vivo outcomes demonstrate that VV261 is potency against CHIKV in the target tissues and serum at low doses and can attenuate inflammation caused by infection.

Overall, this positive preclinical study of the oral nucleoside prodrug VV261 highlights its potential as a promising anti-CHIKV drug candidate. VV261 not only effectively inhibits the replication of CHIKV strains in cells but also suppresses viral loads and inflammation at low doses in vivo. Acuter toxicity studies in Sprague-Dawley (SD) rats indicated that 500 mpk was the lowest dose associated with significant adverse effects, such as weight loss and hepatotoxicity,5 which is much higher than 2.5–10 mpk used in this study. Considering the preclinical pharmacokinetic studies in mice and monkeys,5 it is estimated that after oral administration of 10 mpk VV261 in humans, the maximum blood concentration of 4’-FlU would be approximately 2000 ng/mL, which is about 9000-fold higher than the EC50 value for inhibiting CHIKV. Given the urgent clinical need for treatments against CHIKV, clinical studies should be conducted to investigate its efficacy for Chikungunya fever therapy.