Identification and characterization of anti-chikungunya virus compounds using a biosafe toolkit

Yang Gao; Guojie Wang; Hanhua Zhang; Sicheng Tian; Yanqun Wang; Jincun Zhao; Xing Liu; Rong Ye; Rong Zhang

doi:10.1016/j.virs.2026.08.005

August 2026

. doi: 10.1016/j.virs.2026.08.005

Citation: Yang Gao, Guojie Wang, Hanhua Zhang, Sicheng Tian, Yanqun Wang, Jincun Zhao, Xing Liu, Rong Ye, Rong Zhang. Identification and characterization of anti-chikungunya virus compounds using a biosafe toolkit .VIROLOGICA SINICA, 2026, 41(4) : 779-790. http://dx.doi.org/10.1016/j.virs.2026.08.005

利用生物安全的实验工具筛选和鉴定抗基孔肯雅病毒的化合物

高阳 ^a ,
汪国杰 ^a ,
张汉华 ^b ,
田思成 ^a ,
王延群 ^c ,
赵金存 ^c ,
刘星 ^b,, ,
叶荣 ^a,, ,
张荣 ^a,,

a 复旦大学上海医学院基础医学院病原生物学系, 上海市重大传染病和生物安全研究院及病原微生物与感染前沿科学中心, 医学分子病毒学教育部/卫健委/医科院重点实验室;
b 广东省感染免疫与炎症重点实验室, 深圳大学医学院病原生物学系;
c 广州医科大学附属第一医院, 广州呼吸健康研究院, 国家呼吸系统疾病临床医学研究中心, 呼吸疾病国家重点实验室

通讯作者： 刘星, xingliu1@szu.edu.cn; 叶荣, yerong24@fudan.edu.cn; 张荣, rong_zhang@fudan.edu.cn
收稿日期： 2026-04-30
录用日期： 2026-08-06

摘要

基孔肯雅病毒（Chikungunya virus，CHIKV）是一种重新流行的蚊媒传播甲病毒，目前尚无特异性抗病毒治疗手段。2025年7月，中国广东省佛山市发生大规模暴发疫情，CHIKV迅速扩散至周边地区，累计确诊病例超过16,000例。本研究选取此次暴发中的流行毒株作为参考序列，建立了一套互补的生物安全工具体系，用于抗病毒化合物筛选及作用机制研究。首先构建了基于CHIKV的病毒复制子颗粒（virus replicon particle，VRP）系统，并用于化合物库筛选，成功鉴定出三种候选抗病毒化合物：MDL-12330A、bazedoxifene acetate和anidulafungin。为进一步验证其抗病毒活性并解析潜在作用机制，随后建立了多种CHIKV功能评价系统，包括用于病毒入侵研究的水疱性口炎病毒（VSV）和鼠白血病病毒（MLV）假病毒系统、用于入侵后复制相关过程研究的复制子RNA系统、源自亲本复制子RNA并引入nsP4突变以阻断复制的复制缺陷型nsP4突变复制子RNA系统（用于初始翻译过程分析），以及用于病毒颗粒组装与出芽评估的病毒样颗粒（virus-like particle，VLP）系统。利用上述互补系统，评估了三种候选化合物在CHIKV生命周期不同阶段的抗病毒特征，揭示了其具有阶段特异性的抑制模式，并为其潜在抗病毒机制提供了线索。尽管上述结果均基于生物安全替代体系获得，仍需通过真实CHIKV感染实验进一步验证其转化应用潜力。
- 基孔肯雅病毒
- , 抗病毒化合物
- , 假病毒
- , 病毒复制子颗粒
- , 复制子RNA

Identification and characterization of anti-chikungunya virus compounds using a biosafe toolkit

Yang Gao ^a ,
Guojie Wang ^a ,
Hanhua Zhang ^b ,
Sicheng Tian ^a ,
Yanqun Wang ^c ,
Jincun Zhao ^c ,
Xing Liu ^b,, ,
Rong Ye ^a,, ,
Rong Zhang ^a,,

a. Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), Shanghai Institute of Infectious Disease and Biosecurity, Shanghai Frontiers Science Center of Pathogenic Microorganisms and Infection, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China;
b. Guangdong Provincial Key Laboratory of Infection Immunity and Inflammation, Department of Pathogen Biology, Shenzhen University Medical School, Shenzhen 518060, China;
c. State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou Institute of Respiratory Health, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou 510182, China

Corresponding author: Xing Liu, xingliu1@szu.edu.cn Rong Ye, yerong24@fudan.edu.cn Rong Zhang, rong_zhang@fudan.edu.cn
Received Date: 30 April 2026
Accepted Date: 06 August 2026

Abstract

Chikungunya virus (CHIKV) is a re-emerging mosquito-borne alphavirus for which no specific antiviral therapy is currently available. During the large outbreak in Foshan, Guangdong Province, China, in July 2025, CHIKV rapidly spread to neighboring regions and caused more than 16,000 confirmed cases. In this study, the predominant outbreak strain of CHIKV was selected as the reference sequence to establish a panel of complementary biosafe tools for antiviral compound screening and mechanistic investigation. A virus replicon particle (VRP) system for CHIKV was first constructed and applied to compound library screening, resulting in the identification of three candidate antiviral compounds: MDL-12330A, bazedoxifene acetate, and anidulafungin. To further validate their antiviral activities and investigate their potential mechanisms, CHIKV functional evaluation systems were subsequently established, including vesicular stomatitis virus (VSV)- and murine leukemia virus (MLV)-based pseudovirus systems for viral entry, a replicon RNA system for post-entry replication-associated processes, a replication-defective nsP4 mutant replicon RNA system for primary translation, and a virus-like particle (VLP) system for viral particle assembly and budding assessment. Using these complementary systems, we systematically evaluated the antiviral profiles of the three candidate compounds across multiple stages of the CHIKV life cycle. This analysis revealed distinct stage-specific inhibitory patterns and provided insights into their potential antiviral mechanisms, which warrant validation using authentic CHIKV infection to assess their translational potential.
- Chikungunya virus (CHIKV)
- , Antiviral compound
- , Pseudovirus
- , Viral replicon particle
- , Replicon RNA

References
1. Alves V, Mulholland CV, Zamith-Miranda D, Frases S, Berney M, Nosanchuk JD. 2026. Caspofungin Reshapes the Extracellular Vesicles Metabolome of Candidozyma (Candida) auris, Altering Amino Acid and Nucleotide Metabolism. Journal of Fungi, 12: 156.
2. Briolant S, Garin D, Scaramozzino N, Jouan A, Crance JM. 2004. In vitro inhibition of Chikungunya and Semliki Forest viruses replication by antiviral compounds: synergistic effect of interferon-alpha and ribavirin combination. Antiviral Res, 61: 111-117.
3. Cenedella RJ. 2009. Cholesterol synthesis inhibitor U18666A and the role of sterol metabolism and trafficking in numerous pathophysiological processes. Lipids, 44: 477-487.
4. Chmielewski D, Schmid MF, Simmons G, Jin J, Chiu W. 2022. Chikungunya virus assembly and budding visualized in situ using cryogenic electron tomography. Nat Microbiol, 7: 1270-1279.
5. Cho IJ, Woo NR, Shin IC, Kim SG. 2009. H89, an inhibitor of PKA and MSK, inhibits cyclic-AMP response element binding protein-mediated MAPK phosphatase-1 induction by lipopolysaccharide. Inflamm Res, 58: 863-872.
6. Cunha MS, Costa PAG, Correa IA, de Souza MRM, Calil PT, da Silva GPD, Costa SM, Fonseca VWP, da Costa LJ. 2020. Chikungunya Virus: An Emergent Arbovirus to the South American Continent and a Continuous Threat to the World. Front Microbiol, 11: 1297.
7. Delang L, Segura Guerrero N, Tas A, Querat G, Pastorino B, Froeyen M, Dallmeier K, Jochmans D, Herdewijn P, Bello F, Snijder EJ, de Lamballerie X, Martina B, Neyts J, van Hemert MJ, Leyssen P. 2014. Mutations in the chikungunya virus non-structural proteins cause resistance to favipiravir (T-705), a broad-spectrum antiviral. J Antimicrob Chemother, 69: 2770-2784.
8. Grupp G, Grupp IL, Johnson CL, Matlib MA, Rouslin W, Schwartz A, Wallick ET, Wang T, Wisler P. 1980. Effects of RMI 12330A, a new inhibitor of adenylate cyclase on myocardial function and subcellular activity. Br J Pharmacol, 70: 429-442.
9. Hellstrom K, Kallio K, Utt A, Quirin T, Jokitalo E, Merits A, Ahola T. 2017. Partially Uncleaved Alphavirus Replicase Forms Spherule Structures in the Presence and Absence of RNA Template. J Virol, 91: :e00787-17.
10. Ho YJ, Liu FC, Yeh CT, Yang CM, Lin CC, Lin TY, Hsieh PS, Hu MK, Gong Z, Lu JW. 2018. Micafungin is a novel anti-viral agent of chikungunya virus through multiple mechanisms. Antiviral Res, 159: 134-142.
11. Karlas A, Berre S, Couderc T, Varjak M, Braun P, Meyer M, Gangneux N, Karo-Astover L, Weege F, Raftery M, Schonrich G, Klemm U, Wurzlbauer A, Bracher F, Merits A, Meyer TF, Lecuit M. 2016. A human genome-wide loss-of-function screen identifies effective chikungunya antiviral drugs. Nat Commun, 7: 11320.
12. Li X, Guo Q, Gao J, Yang J, Zhang W, Liang Y, Wu D, Liu Y, Weng J, Li Q, Zhang Y. 2013. The adenylyl cyclase inhibitor MDL-12,330A potentiates insulin secretion via blockade of voltage-dependent K(+) channels in pancreatic beta cells. PLoS One, 8: e77934.
13. Liang J, Yu H, Lin Z, Tang X, Fang Z, Hon C, Hong W, Kong L, Wang Y, Chen Y, Li Y, Chen Y, Guo M, Wu S, Hu F, Qiu H, Bai H, Yan H, Jiang S, Zhang Q, Liu J, Zhou H, Tan M, Huang W, Oliveira A, Jiang J, Huang Y, Yang Z, Zhong N. 2026. Emerging local chikungunya virus transmission in a major urban area in Southern China: characteristics of clinical manifestations, viral evolution and climatic influences. Natl Sci Rev, 13: nwaf529.
14. Lin HC, Chiao DJ, Shu PY, Lin HT, Hsiung CC, Lin CC, Kuo SC. 2023. Development of a Novel Chikungunya Virus-Like Replicon Particle for Rapid Quantification and Screening of Neutralizing Antibodies and Antivirals. Microbiol Spectr, 11: e0485422.
15. Lippincott-Schwartz J, Yuan L, Tipper C, Amherdt M, Orci L, Klausner RD. 1991. Brefeldin A's effects on endosomes, lysosomes, and the TGN suggest a general mechanism for regulating organelle structure and membrane traffic. Cell, 67: 601-616.
16. Liu C, Pan S, Gao M, Zhang Y, Zhang L, Lin B, Jiang X. 2026. Recent progress in anti-Chikungunya virus drug discovery: Focus on the viral life cycle and direct-acting antivirals. Virol Sin. doi: 10.1016/j.virs.2026.07.001.
17. Ma X, Ma Y, Wang Z, Feng F, Han Y, Sun X, Li L, Gao Z, Wang M, Zhang R. 2025. The Single Amino Acid Change of R516K Enables Efficient Generation of Vesicular Stomatitis Virus-Based Crimean-Congo Hemorrhagic Fever Reporter Virus. J Med Virol, 97: e70457.
18. Martelossi-Cebinelli G, Carneiro JA, Yaekashi KM, Bertozzi MM, Bianchini BHS, Rasquel-Oliveira FS, Zanluca C, Duarte Dos Santos CN, Arredondo R, Blackburn TA, Casagrande R, Verri WA. 2025. A Review of the Biology of Chikungunya Virus Highlighting the Development of Current Novel Therapeutic and Prevention Approaches. Pathogens, 14, 1047.
19. Martin MF, Bonaventure B, McCray NE, Peersen OB, Rozen-Gagnon K, Stapleford KA. 2024. Distinct chikungunya virus polymerase palm subdomains contribute to viral protein accumulation and virion production. PLoS Pathog, 20: e1011972.
20. Maure C, Khazhidinov K, Kang H, Auzenbergs M, Moyersoen P, Abbas K, Santos GML, Medina LMH, Wartel TA, Kim JH, Clemens J, Sahastrabuddhe S. 2024. Chikungunya vaccine development, challenges, and pathway toward public health impact. Vaccine, 42: 126483.
21. McMahon R, Toepfer S, Sattler N, Schneider M, Narciso-Abraham M, Hadl S, Hochreiter R, Kosulin K, Mader R, Zoihsl O, Wressnigg N, Dubischar K, Buerger V, Eder-Lingelbach S, Jaramillo JC. 2024. Antibody persistence and safety of a live-attenuated chikungunya virus vaccine up to 2 years after single-dose administration in adults in the USA: a single-arm, multicentre, phase 3b study. Lancet Infect Dis, 24: 1383-1392.
22. Miao G, Peng H, Tang H, Liu Y, Zheng X, Liu B, Jiang L, Tang W, He Y, Liu Y, Ren H, Zhao P, Qi Z, Ding C. 2022. Antiviral efficacy of selective estrogen receptor modulators against SARS-CoV-2 infection in vitro and in vivo reveals bazedoxifene acetate as an entry inhibitor. J Med Virol, 94: 4809-4819.
23. Nuckols JT, McAuley AJ, Huang YJ, Horne KM, Higgs S, Davey RA, Vanlandingham DL. 2014. pH-Dependent entry of chikungunya virus fusion into mosquito cells. Virol J, 11: 215.
24. Pohjala L, Utt A, Varjak M, Lulla A, Merits A, Ahola T, Tammela P. 2011. Inhibitors of alphavirus entry and replication identified with a stable Chikungunya replicon cell line and virus-based assays. PLoS One, 6: e28923.
25. Powers AM, Logue CH. 2007. Changing patterns of chikungunya virus: re-emergence of a zoonotic arbovirus. J Gen Virol, 88: 2363-2377.
26. Richardson JS, Anderson DM, Mendy J, Tindale LC, Muhammad S, Loreth T, Tredo SR, Warfield KL, Ramanathan R, Caso JT, Jenkins VA, Ajiboye P, Bedell L. 2025. Chikungunya virus virus-like particle vaccine safety and immunogenicity in adolescents and adults in the USA: a phase 3, randomised, double-blind, placebo-controlled trial. Lancet, 405: 1343-1352.
27. Rudolph KE, Lessler J, Moloney RM, Kmush B, Cummings DA. 2014. Incubation periods of mosquito-borne viral infections: a systematic review. Am J Trop Med Hyg, 90: 882-891.
28. Shen S, Zhang Y, Yin Z, Zhu Q, Zhang J, Wang T, Fang Y, Wu X, Bai Y, Dai S, Liu X, Jin J, Tang S, Liu J, Wang M, Guo Y, Deng F. 2022. Antiviral activity and mechanism of the antifungal drug, anidulafungin, suggesting its potential to promote treatment of viral diseases. BMC Med, 20: 359.
29. Suhrbier A, Jaffar-Bandjee MC, Gasque P. 2012. Arthritogenic alphaviruses--an overview. Nat Rev Rheumatol, 8: 420-429.
30. Sun C, Gardner CL, Watson AM, Ryman KD, Klimstra WB. 2014. Stable, high-level expression of reporter proteins from improved alphavirus expression vectors to track replication and dissemination during encephalitic and arthritogenic disease. J Virol, 88: 2035-2046.
31. Sun C, Zhang L, Yi H, Wan S, Zhu A, Yuan B, Yan Q, Peng X, Yu C, Zhang X, Fu K, Zeng J, Shi Y, Huang L, Wang Y, Zhao J, Li B. 2025. Isolation and genomic characterization of Chikungunya virus during the 2025 outbreak in Foshan, China. Emerg Microbes Infect, 14: 2595790.
32. Szymanski M, Chmielewska S, Czyzewska U, Malinowska M, Tylicki A. 2022. Echinocandins - structure, mechanism of action and use in antifungal therapy. J Enzyme Inhib Med Chem, 37: 876-894.
33. Treffers EE, Tas A, Scholte FEM, de Ru AH, Snijder EJ, van Veelen PA, van Hemert MJ. 2023. The alphavirus nonstructural protein 2 NTPase induces a host translational shut-off through phosphorylation of eEF2 via cAMP-PKA-eEF2K signaling. PLoS Pathog, 19: e1011179.
34. Tsetsarkin KA, Vanlandingham DL, McGee CE, Higgs S. 2007. A single mutation in chikungunya virus affects vector specificity and epidemic potential. PLoS Pathog, 3: e201.
35. Tsetsarkin KA, Weaver SC. 2011. Sequential adaptive mutations enhance efficient vector switching by Chikungunya virus and its epidemic emergence. PLoS Pathog, 7: e1002412.
36. Vazeille M, Moutailler S, Coudrier D, Rousseaux C, Khun H, Huerre M, Thiria J, Dehecq JS, Fontenille D, Schuffenecker I, Despres P, Failloux AB. 2007. Two Chikungunya isolates from the outbreak of La Reunion (Indian Ocean) exhibit different patterns of infection in the mosquito, Aedes albopictus. PLoS One, 2: e1168.
37. Wang J, Zhang L. 2025. Chikungunya virus in Guangdong, China in 2025. J Travel Med, 32: taaf105.
38. Weaver SC, Lecuit M. 2015. Chikungunya virus and the global spread of a mosquito-borne disease. N Engl J Med, 372: 1231-1239.
39. Wu J, Huang W, Wang Y. 2023. Pseudotyped Viruses for the Alphavirus Chikungunya Virus. Adv Exp Med Biol, 1407: 299-312.
40. Yan X, Luo C, Yang J, Wang Z, Shao Y, Wang P, Yang S, Li Y, Dai Q, Li W, Yang X, Tao H, Ren S, Li Z, Guo X, Li S, Zhu W, Luo Y, Li J, Li S, Cao R, Zhong W. 2024. Antiviral activity of selective estrogen receptor modulators against severe fever with thrombocytopenia syndrome virus in vitro and in vivo. Viruses, 16: 1332.
41. Zaccolo M, Zerio A, Lobo MJ. 2021. Subcellular Organization of the cAMP Signaling Pathway. Pharmacol Rev, 73: 278-309.
42. Zhang R, Kim AS, Fox JM, Nair S, Basore K, Klimstra WB, Rimkunas R, Fong RH, Lin H, Poddar S, Crowe JE, Jr., Doranz BJ, Fremont DH, Diamond MS. 2018. Mxra8 is a receptor for multiple arthritogenic alphaviruses. Nature, 557: 570-574.
43. Zhu Y, Feng F, Hu G, Wang Y, Yu Y, Zhu Y, Xu W, Cai X, Sun Z, Han W, Ye R, Qu D, Ding Q, Huang X, Chen H, Xu W, Xie Y, Cai Q, Yuan Z, Zhang R. 2021. A genome-wide CRISPR screen identifies host factors that regulate SARS-CoV-2 entry. Nature Communications, 12: 961.
Proportional views