Synthetic genomics-based generation of the tick-borne encephalitis virus Siberian subtype prototype strain and E51K-attenuated variant for vaccine development and antiviral screening

Tolganay Kulatay; Elena Sedova; Alexander Shevtsov; Gulzat Zauatbayeva; Bakytkali Ingirbay; Viktoriya Keyer; Zhanar Shakhmanova; Maral Zhumabekova; Yergali Abduraimov; Aralbek Rsaliyev; Nurgul Sikhayeva; Irina Kozlova; Mikhail Zaripov; Alexandr V. Shustov

doi:10.1016/j.virs.2025.09.010

October 2025

Citation: Tolganay Kulatay, Elena Sedova, Alexander Shevtsov, Gulzat Zauatbayeva, Bakytkali Ingirbay, Viktoriya Keyer, Zhanar Shakhmanova, Maral Zhumabekova, Yergali Abduraimov, Aralbek Rsaliyev, Nurgul Sikhayeva, Irina Kozlova, Mikhail Zaripov, Alexandr V. Shustov. Synthetic genomics-based generation of the tick-borne encephalitis virus Siberian subtype prototype strain and E51K-attenuated variant for vaccine development and antiviral screening .VIROLOGICA SINICA, 2025, 40(5) : 812-821. http://dx.doi.org/10.1016/j.virs.2025.09.010

Synthetic genomics-based generation of the tick-borne encephalitis virus Siberian subtype prototype strain and E51K-attenuated variant for vaccine development and antiviral screening

a. National Center for Biotechnology, Astana, 010000, Kazakhstan;
b. National Holding QazBioPharm, Astana, 010000, Kazakhstan;
c. Federal State Budgetary Scientific Institution "Scientific Сentre for Family Health and Human Reproduction Problems", Irkutsk, 664003, Russian Federation;
d. Institute of Theoretical and Experimental Biophysics, Pushchino, 142290, Russian Federation

Corresponding author: Alexandr V. Shustov, shustov@biocenter.kz
Received Date: 01 April 2025
Accepted Date: 30 September 2025
Available online: 02 October 2025

Abstract

Tick-borne encephalitis virus (TBEV) is a re-emerging pathogen in Kazakhstan, where the increasing risk of its spread underscores the need for improved healthcare preparedness, including the development of local vaccines. However, the absence of reference TBEV strains in the country presented a major challenge. To address this, we generated a prototype strain (Vasilchenko) of the Siberian TBEV genotype, predominant in Kazakhstan, using synthetic genome and molecular infectious clone technology. A DNA-launched TBEV molecular clone was assembled from DNA fragments, enabling virus rescue upon plasmid transfection. During the propagation of the post-transfection virus in cell culture, a single amino acid substitution (E51K) in the envelope protein emerged, resulting in a 100-fold increase in the titer of the mutant variant. In vivo, this mutation significantly attenuated virulence: while wild-type TBEV caused 100% mortality in BALB/c mice, the E51K variant was non-lethal and exhibited reduced viremia, suggesting impaired neuroinvasiveness. To further exploit this attenuated, high-titer virus, we developed a GFP-expressing reporter TBEV variant. Using this reporter system, we demonstrated that favipiravir possesses antiviral activity against TBEV, with inhibitory concentrations within a pharmacologically relevant range. In conclusion, synthetic genomics enabled the generation of a reference TBEV strain to replenish Kazakhstan's collections. The E51K mutation enhances viral replication in vitro while attenuating pathogenicity in vivo, and the derived reporter virus is suitable for antiviral compound screening.
- Tick-borne encephalitis virus (TBEV)
- , Infectious clone
- , Virus adaptation
- , Heparan sulfate
- , Favipiravir
- , Antiviral activity

Electronic Supplementary Material

10.1016j.virs.2025.09.010-ESM1.docx
References
1. Aubry, F., Nougairede, A., de Fabritus, L., Querat, G., Gould, E.A., de Lamballerie, X., 2014. Single-stranded positive-sense RNA viruses generated in days using infectious subgenomic amplicons. J Gen Virol 95, 2462-2467.
2. Barrows, N.J., Campos, R.K., Liao, K.C., Prasanth, K.R., Soto-Acosta, R., Yeh, S.C., Schott-Lerner, G., Pompon, J., Sessions, O.M., Bradrick, S.S., Garcia-Blanco, M.A., 2018. Biochemistry and molecular biology of flaviviruses. Chem Rev 118, 4448-4482.
3. Bogovic, P., Strle, F., 2015. Tick-borne encephalitis: A review of epidemiology, clinical characteristics, and management. World J Clin Cases 3, 430-441.
4. Dobler, G., 2018. Tick-borne encephalitis and its global importance. Microbiol Aust 39, 191-194.
5. Elbe, S., 2022. Who owns a deadly virus? Viral sovereignty, global health emergencies, and the matrix of the international. Int Polit Sociol 16, 1-18.
6. Elsterova, J., Palus, M., Sirmarova, J., Kopecky, J., Niller, H.H., Ruzek, D., 2017. Tick-borne encephalitis virus neutralization by high dose intravenous immunoglobulin. Ticks Tick Borne Dis 8, 253-258.
7. Fischl, W., Elshuber, S., Schrauf, S., Mandl, C.W., 2008. Changing the protease specificity for activation of a flavivirus, tick-borne encephalitis virus. J Virol 82, 8272-8282.
8. Halabi, S., Katz, R., 2020. Viral sovereignty, technology transfer, and the changing global system for sharing pathogens for public health research. In: Halabi, S.F., Katz, R., (eds). Viral Sovereignty and Technology Transfer: The Changing Global System for Sharing Pathogens for Public Health Research. Cambridge University Press, pp.1-28.
9. Hiszczynska-Sawicka, E., Kur, J., 1997. Effect of Escherichia coli IHF mutations on plasmid p15A copy number. Plasmid 38, 174-179.
10. Ishikawa, T., Abe, M., Masuda, M., 2015. Construction of an infectious molecular clone of Japanese encephalitis virus genotype V and its derivative subgenomic replicon capable of expressing a foreign gene. Virus Res 195, 153-161.
11. Ke, X., Zhang, Y., Liu, Y., Miao, Y., Zheng, C., Luo, D., Sun, J., Hu, Q., Xu, Y., Wang, H., Zheng, Z., 2020. A Single Mutation in the VP1 Gene of Enterovirus 71 Enhances Viral Binding to Heparan Sulfate and Impairs Viral Pathogenicity in Mice. Viruses 12, 883.
12. Kwasnik, M., Rola, J., Rozek, W., 2023. Tick-Borne Encephalitis-Review of the Current Status. J Clin Med 12, 6603.
13. Lee, E., Hall, R.A., Lobigs, M., 2004. Common E Protein Determinants for Attenuation of Glycosaminoglycan-Binding Variants of Japanese Encephalitis and West Nile Viruses. J Virol 78, 8271-8280.
14. Li, P., Ke, X., Wang, T., Tan, Z., Luo, D., Miao, Y., Sun, J., Zhang, Y., Liu, Y., Hu, Q., Xu, F., Wang, H., Zheng, Z., 2018. Zika Virus Attenuation by Codon Pair Deoptimization Induces Sterilizing Immunity in Mouse Models. J Virol 92, e00701-e00718.
15. Li, X.D., Li, X.F., Ye, H.Q., Deng, C.L., Ye, Q., Shan, C., Shang, B.D., Xu, L.L., Li, S.H., Cao, S.B., Yuan, Z.M., Shi, P.Y., Qin, C.F., Zhang, B., 2014. Recovery of a chemically synthesized Japanese encephalitis virus reveals two critical adaptive mutations in NS2B and NS4A. J Gen Virol 95, 806-815.
16. Lindenbach, B.D., Thiel, H.J., Rice, C.M., 2007. Flaviviridae: The viruses and their replication. In: Knipe, D.M., Howley, P.M. (Eds.), Fields Virology, 5th ed. Lippincott-Raven Publishers, Philadelphia, pp. 1101-1152.
17. McMinn, R.J., Langsjoen, R.M., Normandin, E., Stampfer, S.D., Sabeti, P.C., Piantadosi, A., Ebel, G.D., 2023. North American Powassan virus encompasses diverse in vitro phenotypes. bioRxiv 2023.08.08, 552515.
18. Noyce, R.S., Lederman, S., Evans, D.H., 2018. Construction of an infectious horsepox virus vaccine from chemically synthesized DNA fragments. PLoS One 13, e0188453.
19. Perfilyeva, Y.V., et al., 2020. Tick-borne pathogens and their vectors in Kazakhstan - A review. Ticks Tick Borne Dis 11, 101498.
20. Pu, S.Y., Wu, R.H., Yang, C.C., Jao, T.M., Tsai, M.H., Wang, J.C., Lin, H.M., Chao, Y.S., Yueh, A., 2011. Successful propagation of flavivirus infectious cDNAs by a novel method to reduce the cryptic bacterial promoter activity of virus genomes. J Virol 85, 2927-2941.
21. Sambrook, J., Russell, D.W., 2001. Molecular Cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press, New York.
22. Schwarz, M.C., Sourisseau, M., Espino, M.M., Gray, E.S., Chambers, M.T., Tortorella, D., Evans, M.J., 2016. Rescue of the 1947 Zika virus prototype strain with a cytomegalovirus promoter-driven cDNA clone. mSphere 1, e00246-16.
23. Shan, C., Xie, X., Muruato, A.E., Rossi, S.L., Roundy, C.M., Azar, S.R., Yang, Y., Tesh, R.B., Bourne, N., Barrett, A.D., Vasilakis, N., Weaver, S.C., Shi, P.Y., 2016. An infectious cDNA clone of Zika virus to study viral virulence, mosquito transmission, and antiviral inhibitors. Cell Host Microbe 19, 891-900.
24. Shi, P.Y., Tilgner, M., Lo, M.K., Kent, K.A., Bernard, K.A., 2002. Infectious cDNA clone of the epidemic West Nile virus from New York City. J Virol 76, 5847-5856.
25. Shin, A., Tukhanova, N., Ndenkeh, J., Jr, Shapiyeva, Z., Yegemberdiyeva, R., Yeraliyeva, L., Nurmakhanov, T., Froeschl, G., Hoelscher, M., Musralina, L., Toktasyn, Y., Gulnara, Z., Sansyzbayev, Y., Aigul, S., Abdiyeva, K., Turebekov, N., Wagner, E., Peintner, L., Essbauer, S., 2022. Tick-borne encephalitis virus and West-Nile fever virus as causes of serous meningitis of unknown origin in Kazakhstan. Zoonoses Public Health 69, 514-525.
26. Syzdykova, L.R., Binke, S., Keyer, V.V., Shevtsov, A.B., Zaripov, M.M., Zhylkibayev, A.A., Ramanculov, E.M., Shustov, A.V., 2021. Fluorescent tagging the NS1 protein in yellow fever virus: Replication-capable viruses which produce the secretory GFP-NS1 fusion protein. Virus Res 294, 198291.
27. Takano, A., Yoshii, K., Omori-Urabe, Y., Yokozawa, K., Kariwa, H., Takashima, I., 2011. Construction of a replicon and an infectious cDNA clone of the Sofjin strain of the Far-Eastern subtype of tick-borne encephalitis virus. Arch Virol 156, 1931-1941.
28. Tamura, T., Igarashi, M., Enkhbold, B., Suzuki, T., Okamatsu, M., Ono, C., Mori, H., Izumi, T., Sato, A., Fauzyah, Y., Okamoto, T., Sakoda, Y., Fukuhara, T., Matsuura, Y., 2019. In vivo dynamics of reporter flaviviridae viruses. J Virol 93, e01191-19.
29. Taubenberger, J.K., Hultin, J.V., Morens, D.M., 2007. Discovery and characterization of the 1918 pandemic influenza virus in historical context. Antivir Ther 12, 581-591.
30. Tsetsarkin, K.A., Kenney, H., Chen, R., Liu, G., Manukyan, H., Whitehead, S.S., Laassri, M., Chumakov, K., Pletnev, A.G., 2016. A full-length infectious cDNA clone of Zika virus from the 2015 epidemic in Brazil as a genetic platform for studies of virus-host interactions and vaccine development. mBio 7, e01114-16.
31. Wang, T., Li, P., Zhang, Y., Liu, Y., Tan, Z., Sun, J., Ke, X., Miao, Y., Luo, D., Hu, Q., Xu, F., Wang, H., Zheng, Z., 2020. In vivo imaging of Zika virus reveals dynamics of viral invasion in immune-sheltered tissues and vertical propagation during pregnancy. Theranostics 10, 6430-6447.
32. Widman, D.G., Young, E., Yount, B.L., Plante, K.S., Gallichotte, E.N., Carbaugh, D.L., Peck, K.M., Plante, J., Swanstrom, J., Heise, M.T., Lazear, H.M., Baric, R.S., 2017. A reverse genetics platform that spans the Zika virus family tree. mBio 8, e02014-16.
33. Zhu, W., Qin, C., Chen, S., Jiang, T., Yu, M., Yu, X., Qin, E., 2007. Attenuated dengue 2 viruses with deletions in capsid protein derived from an infectious full-length cDNA clone. Virus Res 126, 226-232.
Proportional views