Single-cycle Rift Valley fever virus particles from stable replicon cells enable discovery of antiviral CNX-1351 for multiple RNA viruses

Zhichao Gao; Hongyuan Guo; Ziqiao Wang; Pengcheng Wang; Xinran Sun; Shimei Zhang; Fei Feng; Chao Shan; Youhua Xie; Rong Zhang

doi:10.1016/j.virs.2025.06.009

August 2025

. doi: 10.1016/j.virs.2025.06.009

Citation: Zhichao Gao, Hongyuan Guo, Ziqiao Wang, Pengcheng Wang, Xinran Sun, Shimei Zhang, Fei Feng, Chao Shan, Youhua Xie, Rong Zhang. Single-cycle Rift Valley fever virus particles from stable replicon cells enable discovery of antiviral CNX-1351 for multiple RNA viruses .VIROLOGICA SINICA, 2025, 40(4) : 636-646. http://dx.doi.org/10.1016/j.virs.2025.06.009

基于复制子稳定细胞的裂谷热病毒单轮感染颗粒平台用于发现抗RNA病毒化合物CNX-1351

高智超 ^a ,
郭宏元 ^a ,
王子樵 ^a ,
王鹏程 ^a ,
孙欣然 ^a ,
张世梅 ^a ,
冯飞 ^a ,
单超 ^b ,
谢幼华 ^a,, ,
张荣 ^a,,

a 复旦大学上海医学院基础医学院教育部、卫健委、医科院医学分子病毒学重点实验室, 上海市重大传染病和生物安全研究院, 上海市病原微生物与感染前沿科学研究基地;
b 中国科学院武汉病毒研究所, 高致病性病毒与生物安全全国重点实验室

通讯作者： 谢幼华, yhxie@fudan.edu.cn; 张荣, rong_zhang@fudan.edu.cn
收稿日期： 2025-04-06
录用日期： 2025-06-30

摘要

裂谷热病毒（RVFV）是一种高生物安全等级病原体，可导致人类严重疾病，且目前尚无获批疗法。该病毒被列为生物安全三级（BSL-3）病原体，其安全风险限制了相关研究及治疗方法的开发。本研究构建了一种稳定的复制子细胞系，可持续支持裂谷热病毒基因组片段（L和S）的复制。通过糖蛋白的反式互补，可以高效包装不同毒株的单轮感染病毒颗粒（VRPs），模拟真病毒的入侵与复制过程，同时显著降低生物安全风险。基于此平台，作者对小分子化合物库开展高通量筛选，鉴定出CNX-1351作为广谱抗RNA病毒化合物。机制研究表明，CNX-1351可能通过靶向PI3K-Akt信号通路抑制病毒复制。该单轮感染病毒颗粒平台为在生物安全限制降低的条件下研究裂谷热病毒的生物学特性、宿主互作机制、抗病毒药物及疫苗开发等提供了重要工具。
- 裂谷热病毒
- , 单轮感染病毒颗粒
- , 抗病毒化合物
- , CNX-1351

Single-cycle Rift Valley fever virus particles from stable replicon cells enable discovery of antiviral CNX-1351 for multiple RNA viruses

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, Shanghai Medical College, Fudan University, Shanghai, 200032, China;
b. Key Laboratory of Virology and Biosafety, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, 430071, China

Corresponding author: Youhua Xie, yhxie@fudan.edu.cn Rong Zhang, rong_zhang@fudan.edu.cn
Received Date: 06 April 2025
Accepted Date: 30 June 2025

Abstract

Rift Valley fever virus (RVFV) is a high-containment pathogen that causes severe diseases in humans, with no approved therapeutics available. Its classification as a biosafety level 3 (BSL-3) agent has limited research and therapeutic development due to safety concerns. In this study, we developed a stable replicon cell line maintaining the replication of L and S genomic segments of RVFV. Single-cycle viral replicon particles (VRPs) could be efficiently packaged through trans-complementation of glycoproteins from different strains, recapitulating authentic viral entry and replication while minimizing biosafety risks. Using this system, we conducted high-throughput screening of a small-molecule compound library and identified CNX-1351 as an antiviral agent for multiple RNA viruses. Mechanistic studies revealed that CNX-1351 inhibits viral replication, potentially by targeting the PI3K-Akt signaling pathway. This single-cycle VRP system provides a valuable tool for studying RVFV biology, host interactions, antiviral and vaccine development under reduced biosafety constraints.
- Rift Valley fever virus (RVFV)
- , Viral replicon particle
- , Antiviral compound
- , CNX-1351

References
1. Al-Afaleq, A.I., Hussein, M.F., 2011. The status of Rift Valley fever in animals in Saudi Arabia: a mini review. Vector Borne Zoonotic Dis, 11, 1513-1520.
2. Al-Hazmi, M., Ayoola, E.A., Abdurahman, M., Banzal, S., Ashraf, J., El-Bushra, A., Hazmi, A., Abdullah, M., Abbo, H., Elamin, A., Al-Sammani, E.-T., Gadour, M., Menon, C., Hamza, M., Rahim, I., Hafez, M., Jambavalikar, M., Arishi, H., Aqeel, A., 2003. Epidemic Rift Valley Fever in Saudi Arabia: A Clinical Study of Severe Illness in Humans. Clin Infect Dis, 36, 245-252.
3. Alrajhi, A.A., Al-Semari, A., Al-Watban, J., 2004. Rift Valley Fever Encephalitis - Volume 10, Number 3-March 2004 - Emerging Infectious Diseases journal - CDC. Emerg Infect Dis, 10, 554-555.
4. Benedict, A., Bansal, N., Senina, S., Hooper, I., Lundberg, L., Fuente, C.D.L., Narayanan, A., Gutting, B., Kehn-Hall, K., 2015. Repurposing FDA-approved drugs as therapeutics to treat Rift Valley fever virus infection. Front in Microbiol, 6, 676.
5. Blanco, J., Cameirao, C., Lopez, M.C., Munoz-Barroso, I., 2020. Phosphatidylinositol-3-kinase-Akt pathway in negative-stranded RNA virus infection: a minireview. Arch Virol, 165, 2165-2176.
6. Boer, S.M.D., Kortekaas, J., Spel, L., Rottier, P.J.M., Moormann, R.J.M., Bosch, B.J., 2012. Acid-Activated Structural Reorganization of the Rift Valley Fever Virus Gc Fusion Protein. J Virol, 86, 13642-13652.
7. Burke, C.W., Erwin-Cohen, R.A., Goodson, A.I., Wilhelmsen, C., Edmundson, J.A., White, C.E., Glass, P.J., 2022. Efficacy of Western, Eastern, and Venezuelan Equine Encephalitis (WEVEE) Virus-Replicon Particle (VRP) Vaccine against WEEV in a Non-Human Primate Animal Model. Viruses, 14, 1502.
8. Caplen, H., Peters, C.J., Bishop, D.H., 1985. Mutagen-directed attenuation of Rift Valley fever virus as a method for vaccine development. J Gen Virol, 66 ( Pt 10), 2271-2277.
9. Daubney, R., Hudson, J.R., 1931. Enzootic Hepatitis or Rift Valley Fever. An Un-described Virus Disease of Sheep, Cattle and Man from East Africa. J Pathol Bacteriol, 34, 545-579.
10. Daubney, R., Hudson, J.R., 1932. Rift Valley Fever. Lancet, 222, 611-612.
11. Dodd, K.A., Bird, B.H., Metcalfe, M.G., Nichol, S.T., Albarino, C.G., 2012. Single-dose immunization with virus replicon particles confers rapid robust protection against Rift Valley fever virus challenge. J Virol, 86, 4204-4212.
12. Easterday, B.C., 1965. Rift valley fever. Adv Vet Sci, 10, 65-127.
13. Ebogo-Belobo, J.T., Kenmoe, S., Abanda, N.N., Bowo-Ngandji, A., Mbaga, D.S., Magoudjou-Pekam, J.N., Kame-Ngasse, G.I., Tchatchouang, S., Menkem, E.Z., Okobalemba, E.A., Noura, E.A., Meta-Djomsi, D., Maidadi-Foudi, M., Kenfack-Zanguim, J., Kenfack-Momo, R., Kengne-Nde, C., Esemu, S.N., Mbacham, W.F., Sadeuh-Mba, S.A., Ndip, L., Njouom, R., 2023. Contemporary epidemiological data of Rift Valley fever virus in humans, mosquitoes and other animal species in Africa: A systematic review and meta-analysis. Vet Med Sci, 9, 2309-2328.
14. Edwards, M.R., Slater, L., Johnston, S.L., 2007. Signalling pathways mediating type I interferon gene expression. Microbes Infect, 9, 1245-1251.
15. Gabrielsen, B., Monath, T.P., Huggins, J.W., Kefauver, D.F., Pettit, G.R., Groszek, G., Hollingshead, M., Kirsi, J.J., Shannon, W.M., Schubert, E.M., Dare, J., Ugarkar, B., Ussery, M.A., Phelan, M.J., 1992. Antiviral (RNA) Activity of Selected Amaryllidaceae Isoquinoline Constituents and Synthesis of Related Substances. J Nat Prod, 55, 1569-1581.
16. Ganaie, S.S., Schwarz, M.M., Mcmillen, C.M., Price, D.A., Feng, A.X., Albe, J.R., Wang, W., Miersch, S., Orvedahl, A., Cole, A.R., Sentmanat, M.F., Mishra, N., Boyles, D.A., Koenig, Z.T., Kujawa, M.R., Demers, M.A., Hoehl, R.M., Moyle, A.B., Wagner, N.D., Stubbs, S.H., Cardarelli, L., Teyra, J., McElroy, A., Gross, M.L., Whelan, S.P.J., Doench, J., Cui, X., Brett, T.J., Sidhu, S.S., Virgin, H.W., Egawa, T., Leung, D.W., Amarasinghe, G.K., Hartman, A.L., 2021. Lrp1 is a host entry factor for Rift Valley fever virus. Cell, 184, 5163-5178.e5124.
17. Gerrard, S.R., Rollin, P.E., Nichol, S.T., 2002. Bidirectional Infection and Release of Rift Valley Fever Virus in Polarized Epithelial Cells. Virology, 301, 226-235.
18. Gharbi, Severine i., Zvelebil, Marketa j., Shuttleworth, Stephen j., Hancox, T., Saghir, N., Timms, John f., Waterfield, Michael d., 2007. Exploring the specificity of the PI3K family inhibitor LY294002. Biochem J, 404, 15-21.
19. Habjan, M., Penski, N., Wagner, V., Spiegel, M., Overby, A.K., Kochs, G., Huiskonen, J.T., Weber, F., 2009. Efficient production of Rift Valley fever virus-like particles: The antiviral protein MxA can inhibit primary transcription of bunyaviruses. Virology, 385, 400-408.
20. Halfmann, P., Ebihara, H., Marzi, A., Hatta, Y., Watanabe, S., Suresh, M., Neumann, G., Feldmann, H., Kawaoka, Y., 2009. Replication-deficient ebolavirus as a vaccine candidate. J Virol, 83, 3810-3815.
21. He, J., Qi, W.B., Wang, L., Tian, J., Jiao, P.R., Liu, G.Q., Ye, W.C., Liao, M., 2013. Amaryllidaceae alkaloids inhibit nuclear-to-cytoplasmic export of ribonucleoprotein (RNP) complex of highly pathogenic avian influenza virus H5N1. Influenza Other Respir Viruses, 7, 922-931.
22. Huggins, J.W., 1989. Prospects for Treatment of Viral Hemorrhagic Fevers with Ribavirin, a Broad-Spectrum Antiviral Drug. Rev Infect Dis, 11, S750-S761.
23. Ikegami, T., Peters, C.J., Makino, S., 2005. Rift Valley Fever Virus Nonstructural Protein NSs Promotes Viral RNA Replication and Transcription in a Minigenome System. J Virol, 79, 5606-5615.
24. Ikegami, T., Won, S., Peters, C.J., Makino, S., 2006. Rescue of infectious rift valley fever virus entirely from cDNA, analysis of virus lacking the NSs gene, and expression of a foreign gene. J Virol, 80, 2933-2940.
25. Kortekaas, J., Oreshkova, N., Cobos-Jimenez, V., Vloet, R.P.M., Potgieter, C.A., Moormann, R.J.M., 2011. Creation of a Nonspreading Rift Valley Fever Virus. J Virol, 85, 12622-12630.
26. Lin, H.-C., Chiao, D.-J., Shu, P.-Y., Lin, H.-T., Hsiung, C.-C., Lin, C.-C., Kuo, S.-C., 2023. Development of a Novel Chikungunya Virus-Like Replicon Particle for Rapid Quantification and Screening of Neutralizing Antibodies and Antivirals. Microbiol Spectr, 11, e04854-04822.
27. Linthicum, K.J., Britch, S.C., Anyamba, A., 2016. Rift Valley Fever: An Emerging Mosquito-Borne Disease∗. Annu Rev Entomol, 61, 395-415.
28. Mansfield, K.L., Banyard, A.C., Mcelhinney, L., Johnson, N., Horton, D.L., Hernandez-Triana, L.M., Fooks, A.R., 2015. Rift Valley fever virus: A review of diagnosis and vaccination, and implications for emergence in Europe. Vaccine, 33, 5520-5531.
29. Mcintosh, B.M., Russell, D., Santos, I.D., Gear, J.H., 1980. Rift Valley fever in humans in South Africa. South African medical journal = Suid-Afrikaanse tydskrif vir geneeskunde, 58, 803-806.
30. Meulen, K.V.D., Smets, G., Rudelsheim, P., 2023. Viral Replicon Systems and Their Biosafety Aspects. Applied Biosafety, 28, 102-122.
31. Moy, Ryan h., Gold, B., Molleston, Jerome m., Schad, V., Yanger, K., Salzano, M.-V., Yagi, Y., Fitzgerald, Katherine a., Stanger, Ben z., Soldan, Samantha s., Cherry, S., 2014. Antiviral Autophagy Restricts Rift Valley Fever Virus Infection and Is Conserved from Flies to Mammals. Immunity, 40, 51-65.
32. Mudhasani, R., Kota, K.P., Retterer, C., Tran, J.P., Whitehouse, C.A., Bavari, S., 2014. High Content Image-Based Screening of a Protease Inhibitor Library Reveals Compounds Broadly Active against Rift Valley Fever Virus and Other Highly Pathogenic RNA Viruses. PLoS Negl Trop Dis, 8, e3095.
33. Murakami, S., Terasaki, K., Ramirez, S.I., Morrill, J.C., Makino, S., 2014. Development of a novel, single-cycle replicable rift valley Fever vaccine. PLoS Negl Trop Dis, 8, e2746.
34. Nacht, M., Qiao, L., Sheets, M.P., Martin, T.S., Labenski, M., Mazdiyasni, H., Karp, R., Zhu, Z., Chaturvedi, P., Bhavsar, D., Niu, D., Westlin, W., Petter, R.C., Medikonda, A.P., Singh, J., 2013. Discovery of a Potent and Isoform-Selective Targeted Covalent Inhibitor of the Lipid Kinase PI3Kα. J Medl Chem, 56, 712-721.
35. Pepin, M., Bouloy, M., Bird, B.H., Kemp, A., Paweska, J., 2010. Rift Valley fever virus(Bunyaviridae: Phlebovirus): an update on pathogenesis, molecular epidemiology, vectors, diagnostics and prevention. Vet Res, 41, 61.
36. Piper, M.E., Gerrard, S.R., 2010. A novel system for identification of inhibitors of rift valley Fever virus replication. Viruses, 2, 731-747.
37. Riblett, A.M., Blomen, V.A., Jae, L.T., Altamura, L.A., Doms, R.W., Brummelkamp, T.R., Wojcechowskyj, J.A., 2016. A Haploid Genetic Screen Identifies Heparan Sulfate Proteoglycans Supporting Rift Valley Fever Virus Infection. J Virol, 90, 1414-1423.
38. Scharton, D., Bailey, K.W., Vest, Z., Westover, J.B., Kumaki, Y., Wettere, A.V., Furuta, Y., Gowen, B.B., 2014. Favipiravir (T-705) protects against peracute Rift Valley fever virus infection and reduces delayed-onset neurologic disease observed with ribavirin treatment. Antiviral Res, 104, 84-92.
39. Schultze, S.M., Hemmings, B.A., Niessen, M., Tschopp, O., 2012. PI3K/AKT, MAPK and AMPK signalling: protein kinases in glucose homeostasis. Expert Rev Mol Med, 14, e1.
40. Shirbaghaee, Z., Bolhassani, A., 2016. Different applications of virus-like particles in biology and medicine: Vaccination and delivery systems. Biopolymers, 105, 113-132.
41. Solomon, V.R., Lee, H., 2011. Quinoline as a Privileged Scaffold in Cancer Drug Discovery. Curr Med Chem, 18, 1488-1508.
42. Tan, S., Banwell, M.G., Ye, W.C., Lan, P., White, L.V., 2022. The Inhibition of RNA Viruses by Amaryllidaceae Alkaloids: Opportunities for the Development of Broad-Spectrum Anti-Coronavirus Drugs. Chem Asian J, 17, e202101215.
43. Vanhaesebroeck, B., Stephens, L., Hawkins, P., 2012. PI3K signalling: the path to discovery and understanding. Nat Rev Mol Cell Biol, 13, 195-203.
Proportional views
Electronic Supplementary Material

10.1016j.virs.2025.07.007-ESM1.docx

10.1016j.virs.2025.07.007-ESM2.xls