Single nucleotide polymorphisms are typical for tick-borne encephalitis and West Nile viruses during triple natural mixed infections in Blyth’s reed warbler (Acrocephalus dumetorum)

Wild bird species contribute significantly to the rapid geographic dissemination of tick-borne encephalitis viruses (TBEV) and West Nile virus (WNV), facilitating the establishment of new natural foci of these orthoflaviviruses. However, the impact of TBEV and WNV population variability on shaping these foci, as well as the potential emergence of new human-pathogenic viral variants, remain underexplored. This study aimed to assess the genetic heterogeneity of TBEV (Siberian and Far Eastern genotypes) and WNV, isolated simultaneously from the tissues of a single garden reed warbler (Acrocephalus dumetorum) collected in the suburbs of Tomsk. The methods of viral strain isolation on various cell cultures were used in combination with a whole-genome analysis of isolates through traditional and high-throughput sequencing (NGS) methods. Consensus full-genome nucleotide sequences of the viruses were obtained by Sanger sequencing and compared with those obtained by NGS, with single nucleotide substitutions (single nucleotide variants, SNVs) accounting for 2 % or higher within the population under study. Our findings revealed single nucleotide polymorphisms (SNPs) associated with both synonymous and non-synonymous nucleotide substitutions, primarily located within the non-structural protein genes of TBEV and WNV. Notably, recombination events were not detected in the genomes of isolated orthoflaviviruses. The WNV isolate, Tomsk/bird/2006/A4, and the TBEV isolates, PT12 and PT122, obtained from A. dumetorum, exhibited heterogeneous viral populations, with SNVs ranging in frequency from 1.75 to 19.88 % for WNV and from 2.08 to 23.73 % for TBEV. Most identified SNPs shared similar nucleotide substitutions in the genomes of already known strains of TBEV and WNV, suggesting that these SNVs could play a crucial role in viral adaptation and underscore the genetic and phenotypic diversity of these viruses in nature.

1. Bezrukovа (Gamova) E.G., Pogodina V.V., Levina L.S., Karan L.S., Malenko G.V. The research of different genotypes of TBE strain virus isolated from patients with chronic course of the disease. Medicina v Kuzbasse = Medicine in Kuzbass. 2008;S5:21-28 (in Russian)

2. Borda V., da Silva Francisco Junior R., Carvalho J.B., Morais G.L., Duque Rossi Á., Pezzuto P., Azevedo G.S., … Tanuri A., Stratakis C.A., Aguiar R.S., Cardoso C.C., Vasconcelos A.T.R. Wholeexome sequencing reveals insights into genetic susceptibility to Congenital Zika Syndrome. PLoS Negl Trop Dis. 2021;15(6): e0009507. doi 10.1371/journal.pntd.0009507

3. Chausov E.V., Ternovoi V.A., Protopopova E.V., Konovalova S.N., Kononova J.V., Pershikova N.L., Moskovitina N.S., Romanenko V.N., Ivanova N.V., Bol’shakova N.P., Moskvitin S.S., Korobitsyn I.G., Gashkov S.I., Tiutenkov O.I., Kuranova V.N., Kravchenko L.B., Suchkova N.G., Agulova L.P., Loktev V.B. Genetic diversity of ixodid tick-borne pathogens in Tomsk City and suburbs. Parazitologiya. 2009;43(5):374-388 (in Russian)

4. Current ICTV Taxonomy Release. Taxonomy Browser. Ch. Family: Flaviviridae. Available at: https://ictv.global/report/chapter/flaviviridae/flaviviridae/orthoflavivirus

5. Dai X., Shang G., Lu S., Yang J., Xu J. A new subtype of eastern tick-borne encephalitis virus discovered in Qinghai-Tibet Plateau, China. Emerg Microbes Infect. 2018;7:74. doi 10.1038/s41426-018-0081-6

6. Diallo B., Sissoko D., Loman N.J., Bah H.A., Bah H., Worrell M.C., Conde L.S., … Formenty P., Keïta S., Günther S., Rambaut A., Duraffour S. Resurgence of Ebola virus disease in Guinea linked to a survivor with virus persistence in seminal fluid for more than 500 days. Clin Infect Dis. 2016;63(10):1353-1356. doi 10.1093/cid/ciw601

7. Domingo E., Holland J.J. RNA virus mutations and fitness for survival. Annu Rev Microbiol. 1997;51:151-178. doi 10.1146/annurev.micro.51.1.151

8. Domingo E., Sheldon J., Perales C. Viral quasispecies evolution. Microbiol Mol Biol Rev. 2012;76(2):159-216. doi 10.1128/mmbr.05023-11

9. Domingo E., García-Crespo C., Perales C. Historical perspective on the discovery of the quasispecies concept. Annu Rev Virol. 2021;8(1): 51-72. doi 10.1146/annurev-virology-091919-105900

10. Eigen M., McCaskill J., Schuster P. Molecular quasi-species. J Phys Chem. 1988;92(24):6881-6891. doi 10.1021/j100335a010

11. Holland J.J., De La Torre J.C., Steinhauer D.A. RNA-virus populations as quasispecies. In: Holland J.J. (Ed.) Genetic Diversity of RNA Viruses. Current Topics in Microbiology and Immunology. Vol. 176. Berlin; Heidelberg: Springer, 1992;176:1-20. doi 10.1007/978-3-642-77011-1_1

12. Kaiser J.A., Luo H., Widen S.G., Wood T.G., Huang C.Y., Wang T., Barrett A.D.T. Japanese encephalitis vaccine-specific envelope protein E138K mutation does not attenuate virulence of West Nile virus. NPJ Vaccines. 2019;4:50. doi 10.1038/s41541-019-0146-0

13. Karbowiak G., Biernat B., Werszko J., Rychlik L. The transstadial persistence of tick-borne encephalitis virus in Dermacentor reticulatus ticks in natural conditions. Acta Parasitol. 2016;61(1):201-203. doi 10.1515/ap-2016-0028

14. Kononova Yu.V., Ternovoi V.A., Shchelkanov M.Yu., Protopopova E.V., Zolotykh S.I., Yurlov A.K., Druziaka A.V., Slavskii A.A., Shestopalov A.M., L’vov D.K., Loktev V.B. West Nile virus genotyping among wild birds belonging to ground and tree-brush bird populations on the territories of the Baraba forest-steppe and Kulunda steppe (2003-2004). Voprosy Virusologii = Problems of Virology. 2006;51(4):19-23 (in Russian)

15. Korobitsyn I.G., Moskvitina N.S., Tyutenkov O.Y., Gashkov S.I., Kononova Y.V., Moskvitin S.S., Romanenko V.N., … Konovalova S.N., Tupota N.L., Sementsova A.O., Ternovoi V.A., Loktev V.B. Detection of tick-borne pathogens in wild birds and their ticks in Western Siberia and high level of their mismatch. Folia Parasitol (Praha). 2021;68:024. doi 10.14411/fp.2021.024

16. Kovalev S.Yu., Umpelova T.V., Snitkovskaya T.E., Kilyachina A.S., Romanenko V.V., Kokorev V.S., Glinskikh N.P. Molecular and epidemiological characteristics of tick-borne encephalitis virus in the Sverdlovsk region on the basis of genotype-specific RT-PCR. Voprosy Virusologii = Problems of Virology. 2008;53(2):27-31 (in Russian)

17. Kozlova I.V., Verkhozina M.M., Demina T.V., Dzhioev Yu.P., Doroshchenko E.K., Lisak O.V., Karan L.S., Koliasnikova N.M., Rar V.A., Fomenko N.V., Tkachev S.E., Bogomazova O.L., Borisov V.A., Tuvakov M.K., Zlobin V.I. Combined natural foci of tickborne infections in Baikal region. Epidemiologiya i Vakcinoprofilaktika = Epidemiology and Vaccinal Prevention. 2010;4(53):40-46 (in Russian)

18. Kozlova I.V., Demina T.V., Tkachev S.E., Doroshchenko E.K., Lisak O.V., Verkhozina M.M., Karan L.S., … Savinova Y.S., Chernoivanova O.O., Ruzek D., Tikunova N.V., Zlobin V.I. Characteristics of the Baikal subtype of tick-borne encephalitis virus circulating in Eastern Siberia. Acta Biomed Scientifica. 2018;3(4):53-60. doi 10.29413/ABS.2018-3.4.9

19. Kumar S., Stecher G., Li M., Knyaz C., Tamura K. MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol. 2018;35(6):1547-1549. doi 10.1093/molbev/msy096

20. Law J., Jovel J., Patterson J., Ford G., O’Keefe S., Wang W., Meng B., … Mitchell T., Jordan T., Carpenter E., Mason A.L., Wong G.K. Identification of hepatotropic viruses from plasma using deep sequencing: a next generation diagnostic tool. PLoS One. 2013;8(4):e60595. doi 10.1371/journal.pone.0060595

21. Litov A.G., Deviatkin A.A., Goptar I.A., Dedkov V.G., Gmyl A.P., Markelov M.L., Shipulin G.A., Karganova G.G. Evaluation of the population heterogeneity of TBEV laboratory variants using high-throughput sequencing. J Gen Virol. 2018;99(2):240-245. doi 10.1099/jgv.0.001003

22. Marí Saéz A., Weiss S., Nowak K., Lapeyre V., Zimmermann F., Düx A., Kühl H.S., … Fahr J., Borcher M., Gogarten J.F., Calvignac-Spencer S., Leendertz F.H. Investigating the zoonotic origin of the West African Ebola epidemic. EMBO Mol Med. 2015;7(1):17-23. doi 10.15252/emmm.201404792

23. Mikryukova T.P., Moskvitina N.S., Kononova Y.V., Korobitsyn I.G., Kartashov M.Y., Tyuten Kov O.Y., Protopopova E.V., … Moskvitin S.S., Tupota N.L., Sementsova A.O., Ternovoi V.A., Loktev V.B. Surveillance of tick-borne encephalitis virus in wild birds and ticks in Tomsk city and its suburbs (Western Siberia). Ticks Tick Borne Dis. 2014;5(2):145-151. doi 10.1016/j.ttbdis.2013.10.004

24. Moskvitina N.S., Romanenko V.N., Ternovoi V.A., Ivanova N.V., Protopopova E.V., Kravchenko L.B., Kononova Yu.V., Kuranova V.N., Chausov E.V., Moskvitin S.S., Pershikova N.L., Gashkov S.I., Konovalova S.N., Bolshakova N.P., Loktev V.B. Detection of the West Nile virus and its genetic typing in ixodid ticks (Parasitiformes: Ixodidae) in Tomsk and its suburbs. Parazitologiya. 2008; 42(3):210- 225 (in Russian)

25. Moskvitina N.S., Korobitsyn I.G., Tyuten’kov O.Yu., Gashkov S.I., Kononova Yu.V., Moskvitin S.S., Romanenko V.N., … Konovalova S.N., Tupota N.L., Sementsova A.O., Ternovoi V.A., Loktev V.B. The role of birds in the maintenance of tick borne infections in the Tomsk anthropurgic foci. Biol Bull Russ Acad Sci. 2014;41(4): 387-393. doi 10.1134/S1062359014040086

26. Pogodina V.V., Karan L.S., Kolyasnikova N.M., Gerasimov S.G., Levina L.S., Bochkova N.G., Andayev E.I., Trukhina A.G., Borisova T.I., Sidorova E.A., Nagibina O.A., Malenko G.V., Bezrukova E.G. Polytypic strains in the genofund of tick-borne encephalitis virus. Voprosy Virusologii = Problems of Virology. 2012;57(3):30-37 (in Russian)

27. Postler T.S., Beer M., Blitvich B.J., Bukh J., de Lamballerie X., Drexler J.F., Imrie A., Kapoor A., Karganova G.G., Lemey P., Lohmann V., Simmonds P., Smith D.B., Stapleton J.T., Kuhn J.H. Renaming of the genus Flavivirus to Orthoflavivirus and extension of binomial species names within the family Flaviviridae. Arch Virol. 2023;168(9):224. doi 10.1007/s00705-023-05835-1

28. Pustijanac E., Buršić M., Talapko J., Škrlec I., Meštrović T., Lišnjić D. Tick-borne encephalitis virus: a comprehensive review of transmission, pathogenesis, epidemiology, clinical manifestations, diagnosis, and prevention. Microorganisms. 2023;11(7):1634. doi 10.3390/microorganisms11071634

29. Razumov I.A., Kazachinskaia E.I., Ternovoi V.A., Protopopova E.V., Galkina I.V., Gromashevskii V.L., Prilipov A.G., Kachko A.V., Ivanova A.V., L’vov D.K., Loktev V.B. Neutralizing monoclonal antibodies against Russian strain of the West Nile virus. Viral Immunol. 2005;18(3):558-568. doi 10.1089/vim.2005.18.558

30. Shanshin D.V., Borisevich S.S., Shaprova O.N., Nesmeyanova V.S., Bondar A.A., Porozov Y.B., Khamitov E.M., Kolosova E.A., Shelemba A.A., Ushkalenko N.D., Protopopova E.V., Sergeev A.A., Loktev V.B., Shcherbakov D.N. Phage display revealed the complex structure of the epitope of the monoclonal antibody 10H10. Int J Mol Sci. 2024;25(19):10311. doi 10.3390/ijms251910311

31. Simonin Y. Circulation of West Nile virus and Usutu virus in Europe: overview and challenges. Viruses. 2024;16(4):599. doi 10.3390/v16040599

32. Singh P., Khatib M.N., Ballal S., Kaur M., Nathiya D., Sharma S., Siva Prasad G.V., … Lakhanpal S., Shabil M., Bushi G., Sah S., Serhan H.A. West Nile virus in a changing climate: epidemiology, pathology, advances in diagnosis and treatment, vaccine designing and control strategies, emerging public health challenges – a comprehensive review. Emerg Microbes Infect. 2024;30:2437244. doi 10.1080/22221751.2024.2437244

33. Svyatchenko V.A., Nikonov S.D., Mayorov A.P., Gelfond M.L., Loktev V.B. Antiviral photodynamic therapy: inactivation and inhibition of SARS-CoV-2 in vitro using methylene blue and Radachlorin. Photodiagnosis Photodyn Ther. 2021;33:102-112. doi 10.1016/j.pdpdt.2020.102112

34. Ternovoi V.A., Shchelkanov M.Yu., Shestopalov A.M., Aristova V.A., Protopopova E.V., Gromashevsky V.L., Druzyaka A.V., Slavsky A.A., Zolotykh S.I., Loktev V.B., Lvov D.K. Detection of West Nile virus in birds in the territories of Baraba and Kulunda lowlands (West Siberian migration way) during summer-autumn of 2002. Voprosy Virusologii = Problems of Virology. 2004;49(3):52-56 (in Russian)

35. Ternovoi V.A., Protopopova E.V., Chausov E.V., Novikov D.V., Leonova G.N., Netesov S.V., Loktev V.B. Novel variant of tickborne encephalitis virus, Russia. Emer Infec Dis. 2007;13(10):1574-1578. doi 10.3201/eid1310.070158

36. Ternovoi V.A., Ponomareva E.P., Protopopova E.V., Tupota N.L., Mikryukova T.P., Loktev V.B. Changes in the genome of the TickBorne encephalitis virus during cultivation. Mol Biol. 2024;58(2): 266-278. doi 10.1134/S0026893324020146

37. Thorson A., Formenty P., Lofthouse C., Broutet N. Systematic review of the literature on viral persistence and sexual transmission from recovered Ebola survivors: evidence and recommendations. BMJ Open. 2016;6:e008859. doi 10.1136/bmjopen-2015-008859

38. Worku D.A. Tick-borne encephalitis (TBE): from tick to pathology. J Clin Med. 2023;12(21):6859. doi 10.3390/jcm12216859

39. Zárate S., Hernández-Perez F., Taboada B., Martínez N.E., Alcaráz-Estrada S.L., Del Moral O., Yocupicio-Monroy M. Complete genome of DENV2 isolated from mosquitoes in Mexico. Infect Genet Evol. 2019;71:98-107. doi 10.1016/j.meegid.2019.03.018