Problems of VirologyProblems of VirologyВопросы вирусологии0507-40882411-2097Central Research Institute for Epidemiology1676610.36233/0507-4088-324gkebwvORIGINAL RESEARCHESОРИГИНАЛЬНЫЕ ИССЛЕДОВАНИЯResearch ArticleModeling of mixed infection with Zika and West Nile viruses (Flaviviridae: Orthoflavivirus: Orthoflavivirus zikaense, Orthoflavivirus nilense) in vitro and in vivoМоделирование смешанной инфекции вирусами Зика и Западного Нила (Flaviviridae: Orthoflavivirus: Orthoflavivirus zikaense, Orthoflavivirus nilense) in vitro и in vivohttps://orcid.org/0000-0002-2729-0592SvyatchenkoVictor A.СвятченкоВиктор Александрович
Russian Federation

Candidate of Biological Sciences, Leading Researcher, Department of Molecular Virology of Flaviviruses and Viral Hepatitis

канд. биол. наук, ведущий научный сотрудник отдела молекулярной вирусологии флавивирусов и вирусных гепатитов

svyat@vector.nsc.ruhttps://orcid.org/0000-0002-2782-8364ProtopopovaElena V.ПротопоповаЕлена Викторовна
Russian Federation

Candidate of Biological Sciences, Leading Researcher, Department of Molecular Virology of Flaviviruses and Viral Hepatitis

канд. биол. наук, ведущий научный сотрудник отдела молекулярной вирусологии флавивирусов и вирусных гепатитов

protopopova_ev@vector.nsc.ruhttps://orcid.org/0000-0002-6202-445XLegostaevStanislav S.ЛегостаевСтанислав Сергеевич
Russian Federation

junior researcher of the department of molecular virology of flaviviruses and viral hepatitis

младший научный сотрудник отдела молекулярной вирусологии флавивирусов и вирусных гепатитов

legostaev_ss@vector.nsc.ruhttps://orcid.org/0000-0003-4350-4260MikryukovaTamara P.МикрюковаТамара Петровна
Russian Federation

Candidate of Biological Sciences, Senior Researcher, Department of Molecular Virology of Flaviviruses and Viral Hepatitis

канд. биол. наук, старший научный сотрудник отдела молекулярной вирусологии флавивирусов и вирусных гепатитов

mikryukova_tp@vector.nsc.ruhttps://orcid.org/0000-0003-2577-0434AgafonovAlexander P.АгафоновАлександр Петрович
Russian Federation

Doctor of Biological Sciences, Director General

д-р биол. наук, генеральный директор

agafonov@vector.nsc.ruhttps://orcid.org/0000-0002-0229-321XLoktevValery B.ЛоктевВалерий Борисович
Russian Federation

MD, PhD, DSc, Prof., academician RANS, Chief Researcher, Department of Molecular Virology of Flaviviruses and Viral Hepatitis

д-р биол. наук, профессор, академик РАЕН, главный научный сотрудник отдела молекулярной вирусологии флавивирусов и вирусных гепатитов

loktev@vector.nsc.ruState Research Center of Virology and Biotechnology «Vector»ФБУН «Государственный научный центр вирусологии и биотехнологии «Вектор» Федеральной службы по надзору в сфере защиты прав потребителей и благополучия человека (Роспотребнадзор)1609202570434034823062025Copyright ©; 2025, Svyatchenko V.A., Protopopova E.V., Legostaev S.S., Mikryukova T.P., Agafonov A.P., Loktev V.B.Copyright ©; 2025, Святченко В.А., Протопопова Е.В., Легостаев С.С., Микрюкова Т.П., Агафонов А.П., Локтев В.Б.2025Svyatchenko V.A., Protopopova E.V., Legostaev S.S., Mikryukova T.P., Agafonov A.P., Loktev V.B.Святченко В.А., Протопопова Е.В., Легостаев С.С., Микрюкова Т.П., Агафонов А.П., Локтев В.Б.https://creativecommons.org/licenses/by/4.0https://virusjour.crie.ru/jour/article/view/16766

Introduction. Mosquito-borne human diseases caused by Zika virus and West Nile virus (WNV) are widespread across multiple continents and cause major outbreaks. Their ranges overlap and the possibility of mixed infections is obvious. The information of such mixed infections is limited.

The aim of the study is to investigate the features of mixed infection of WNV and Zika virus in vitro and in vivo in order to assess their possible interference and/or enhancement of viral infection.

Materials and methods. The study used West Nile virus and Zika virus strains Vlg27924 and MR766, respectively. The infectious activity of viruses during mono- and co-infection was determined on Vero E6 cell culture using RT-PCR, as well as on BALB/c mice using various administration schemes.

Results. In vitro studies of co-infection with WNV and Zika virus showed that co-infection leads to interference, with the degree of competitive inhibition of replication being more pronounced for Zika virus, reaching 1000 times or more when compared to mono-infection. During simultaneous infection in mice, Zika virus does not affect the development of lethal infection caused by WNV. However, preliminary (4 and 20 days) infection with a sublethal dose of Zika virus reliably protects animals from subsequent administration of 10 and 100 LD50 WNV, respectively. In pre-infected and co-infected animals with Zika virus, the development of WNV-specific viral neutralizing antibodies was recorded in higher titers than in WNV monoinfected animals.

Conclusion. The presence of in vitro interference between the studied orthoflaviviruses was shown, most pronounced in relation to the Zika virus. No significant effect was observed with simultaneous co-infection in vivo. However, pre-infection of mice with Zika virus provides protection to animals from lethal WNV infection due to the induction of high levels of antibodies that specifically neutralize its infectious activity.

Введение. Заболевания человека, вызываемые вирусами Западного Нила (ВЗН) и Зика и передающиеся через укус комара, широко распространены на разных континентах и обусловливают крупные вспышки. Ареалы этих вирусов перекрываются, что создает возможность возникновения микст-инфекций. Знания о подобных микст-инфекциях ограничены.

Цель исследования. Провести изучение особенностей протекания микст-инфекции ВЗН и вируса Зика in vitro и in vivo с целью оценки их возможной интерференции и/или усиления инфекции.

Материалы и методы. В работе использовали штаммы Влг27924 и MR766 ВЗН и вируса Зика соответственно. Инфекционную активность вирусов при моно- и коинфицировании определяли на культуре клеток Vero E6 с помощью полимеразной цепной реакции с обратной транскрипцией, а также на мышах линии BALB/c.

Результаты. При исследовании коинфицирования ВЗН и вирусом Зика in vitro показано, что совместное инфицирование приводит к интерференции, при этом степень конкурентного ингибирования репликации более выражена в отношении вируса Зика (в 1000 раз и более) при сравнении с моноинфекцией. При одновременном инфицировании мышей вирус Зика не влияет на развитие летальной инфекции, вызванной ВЗН. Однако предварительное (за 4 и 20 сут) инфицирование сублетальной дозой вируса Зика достоверно защищает животных от последующего введения 10 и 100 ЛД50 ВЗН. У предварительно инфицированных вирусом Зика коинфицированных животных зарегистрировано появление специфичных к ВЗН вируснейтрализующих антител в более высоком титре, чем у моноинфицированных ВЗН особей.

Заключение. Показано наличие интерференции in vitro между исследованными ортофлавивирусами, наиболее выраженной в отношении вируса Зика. При одновременном инфицировании мышей вирус Зика не влияет на течение и исход инфекционного процесса, вызванного ВЗН. Предварительное инфицирование мышей вирусом Зика обеспечивает защиту животных от летальной инфекции ВЗН, праймируя индукцию более высокого уровня вируснейтрализующих антител.

Zika virusWest Nile virusco-infectionviral interferenceвирус Зикавирус Западного НилакоинфекцияинтерференцияMinistry of Science and Higher Education of the Russian FederationМинистерство науки и высшего образования Российской Федерации075-15-2025-526Postler T.S., Beer M., Blitvich B.J., Bukh J., de Lamballerie X., Drexler J.F., et al. Renaming of the genus Flavivirus to Orthoflavivirus and extension of binomial species names within the family Flaviviridae. Arch. Virol. 2023; 168(9): 224. https://doi.org/10.1007/s00705-023-05835-1Baker R.E., Mahmud A.S., Miller I.F., Rajeev M., Rasambainarivo F., Rice B.L., et al. Infectious disease in an era of global change. Nat. Rev. Microbiol. 2022; 20(4): 193–05. https://doi.org/10.1038/s41579-021-00639-zPetersen L.R., Brault A.C., Nasci R.S. West Nile virus: review of the literature. JAMA. 2013; 310(3): 308–15. https://doi.org/10.1001/jama.2013.8042van Leur S.W., Heunis T., Munnur D., Sanyal S. Pathogenesis and virulence of flavivirus infections. Virulence. 2021; 12(1): 2814–38. https://doi.org/10.1080/21505594.2021.1996059Brüssow H., Figuerola J. The spread of the mosquito-transmitted West Nile virus in North America and Europe. Microb. Biotechnol. 2025; 18(3): e70120. https://doi.org/10.1111/1751-7915.70120Kariwa H., Murata R., Totani M., Yoshii K., Takashima I. Increased pathogenicity of West Nile virus (WNV) by glycosylation of envelope protein and seroprevalence of WNV in wild birds in Far Eastern Russia. Int. J. Environ. Res. Public Health. 2013; 10(12): 7144–64. https://doi.org/10.3390/ijerph10127144Korobitsyn I.G., Moskvitina N.S., Tyutenkov O.Y., Gashkov S.I., Kononova Y.V., Moskvitin S.S., et al. 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: 2021.024. https://doi.org/10.14411/fp.2021.024Duggal N.K., Langwig K.E., Ebel G.D., Brault A.C. On the fly: interactions between birds, mosquitoes, and environment that have molded West Nile virus genomic structure over two decades. J. Med. Entomol. 2019; 56(6): 1467–74. https://doi.org/10.1093/jme/tjz112Dick G.W.A., Kitchen S.F., Haddow A.J., Zika virus (I). Isolations and serological specificity. Trans. R. Soc. Trop. Med. Hyg. 1952; 46: 509–20. https://doi.org/10.1016/0035-9203(52)90042-4Rabe I.B., Hills S.L., Haussig J.M., Walker A.T., Dos Santos T., San Martin J.L., et al. A review of the recent epidemiology of Zika virus infection. Am. J. Trop. Med. Hyg. 2025; 112(5): 1026–35. https://doi.org/10.4269/ajtmh.24-0420Pielnaa P., Al-Saadawe M., Saro A., Dama M.F., Zhou M., Huang Y., et al. Zika virus-spread, epidemiology, genome, transmission cycle, clinical manifestation, associated challenges, vaccine and antiviral drug development. Virology. 2020; 543: 34–42. https://doi.org/10.1016/j.virol.2020.01.015Rückert C., Weger-Lucarelli J., Garcia-Luna S.M., Young M.C., Byas A.D., Murrieta R.A., et al. Impact of simultaneous exposure to arboviruses on infection and transmission by Aedes aegypti mosquitoes. Nat. Commun. 2017; 8: 15412. https://doi.org/10.1038/ncomms15412Laredo-Tiscareño S.V., Machain-Williams C., Rodríguez-Pérez M.A., Garza-Hernandez J.A., Doria-Cobos G.L., Cetina-Trejo R.C., et al. Arbovirus surveillance near the Mexico–us border: Isolation and sequence analysis of chikungunya virus from patients with dengue-like symptoms in Reynosa, Tamaulipas. Am. J. Trop. Med. Hyg. 2018; 99(1): 191–94. https://doi.org/10.4269/ajtmh.18-0117Cherabuddi K., Iovine N.M., Shah K., White S.K., Paisie T., Salemi M., et al. Zika and Chikungunya virus co-infection in a traveller returning from Colombia, 2016: virus isolation and genetic analysis. JMM Case Rep. 2016; 3(6): e005072. https://doi.org/10.1099/jmmcr.0.005072Iovine N.M., Lednicky J., Cherabuddi K., Crooke H., White S.K., Loeb J.C., et al. Coinfection with Zika and Dengue-2 viruses in a traveler returning from Haiti, 2016: clinical presentation and genetic analysis. Clin. Infect. Dis. 2017; 64(1): 72–5. https://doi.org/10.1093/cid/ciw667Laredo-Tiscareño S.V., Garza-Hernandez J.A., Salazar M.I., De Luna-Santillana E.J., Tangudu C.S., Cetina-Trejo R.C., et al. Surveillance for flaviviruses near the Mexico-U.S. border: co-circulation of Dengue virus serotypes 1, 2, and 3 and West Nile virus in Tamaulipas, Northern Mexico, 2014–2016. Am. J. Trop. Med. Hyg. 2018; 99(5): 1308–17. https://doi.org/10.4269/ajtmh.18-0426Slavov S.N., Gonzaga F.A.C., Pimentel B.M.S., Ramos D.D.A.R., de Araújo W.N., Covas D.T., et al. Zika virus RNA surveillance in blood donors in the Federal District of Brazil during the 2016 outbreak. Hematol. Transfus. Cell Ther. 2020; 42(4): 394–96. https://doi.org/10.1016/j.htct.2019.08.006Vasilakis N., Shell E.J., Fokam E.B., Mason P.W., Hanley K.A., Estes D.M., et al. Potential of ancestral sylvatic dengue-2 viruses to re-emerge. Virology. 2007; 358(2): 402–12. https://doi.org/10.1016/j.virol.2006.08.049Aaskov J., Buzacott K., Thu H.M., Lowry K., Holmes E.C. Long-term transmission of defective RNA viruses in humans and Aedes mosquitoes. Science. 2006; 311(5758): 236–38. https://doi.org/10.1126/science.1115030Paz-Bailey G., Adams L.E., Deen J., Anderson K.B., Katzelnick L.C. Dengue. Lancet. 2024; 403(10427): 667–82. https://doi.org/10.1016/S0140-6736(23)02576-XRodriguez-Morales A.J., Villamil-Gómez W.E., Franco-Paredes C. The arboviral burden of disease caused by co-circulation and co-infection of dengue, chikungunya and Zika in the Americas. Travel Med. Infect. Dis. 2016; 14(3): 177–9. https://doi.org/10.1016/j.tmaid.2016.05.004Svyatchenko 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: 102112. https://doi.org/10.1016/j.pdpdt.2020.102112Toth K., Spencer J.F., Dhar D., Sagartz J.E., Buller R.M., Painter G.R., et al. Hexadecyloxypropyl-cidofovir, CMX001, prevents adenovirus induced mortality in a permissive, immunosuppressed animal model. Proc. Natl. Acad. Sci. USA. 2008; 105(20): 7293–97. https://doi.org/10.1073/pnas.0800200105Svyatchenko V.A., Ternovoi V.A., Lutkovskiy R.Y., Protopopova E.V., Gudymo A.S., Danilchenko N.V., et al. Human adenovirus and influenza A virus exacerbate SARS-CoV-2 infection in animal models. Microorganisms. 2023; 11(1): 180. https://doi.org/10.3390/microorganisms11010180Sanders R., Mason D.J., Foy C.A., Huggett J.F. Evaluation of digital PCR for absolute RNA quantification. PLoS One. 2013; 8(9): e75296. https://doi.org/10.1371/journal.pone.0075296Petrović T., Blazquez A.B., Lupulović D., Lazić G., Escribano-Romero E., Fabijan D., et al. Monitoring West Nile virus (WNV) infection in wild birds in Serbia during 2012: first isolation and characterisation of WNV strains from Serbia. Euro. Surveill. 2013; 18(44): 20622. https://doi.org/10.2807/1560-7917.es2013.18.44.20622DaPalma T., Doonan B.P., Trager N.M., Kasman L.M. A systematic approach to virus-virus interactions. Virus Res. 2010; 149(1): 1–9. https://doi.org/10.1016/j.virusres.2010.01.002Zhu Y., He Z., Qi Z. Virus-host Interactions in early Japanese encephalitis virus infection. Virus Res. 2023; 331: 199120. https://doi.org/10.1016/j.virusres.2023.199120Du Y., Wang C., Zhang Y. Viral coinfections. Viruses. 2022; 14(12): 2645. https://doi.org/10.3390/v14122645Gallichotte E.N., Fitzmeyer E.A., Williams L., Spangler M.C., Bosco-Lauth A.M., Ebel G.D. WNV and SLEV coinfection in avian and mosquito hosts: impact on viremia, antibody responses, and vector competence. J. Virol. 2025; 98(10): e0104124. https://doi.org/10.1128/jvi.01041-24Dejnirattisai W., Supasa P., Wongwiwat W., Rouvinski A., Barba-Spaeth G., Duangchinda T., et al. Dengue virus sero-cross-reactivity drives antibody-dependent enhancement of infection with zika virus. Nat. Immunol. 2016; 17(9): 1102–08. https://doi.org/10.1038/ni.3515Stettler K., Beltramello M., Espinosa D.A., Graham V., Cassotta A., Bianchi S., et al. Specificity, cross-reactivity, and function of antibodies elicited by Zika virus infection. Science. 2016; 353(6301): 823–26. https://doi.org/10.1126/science.aaf8505Lobigs M., Diamond M.S. Feasibility of cross-protective vaccination against flaviviruses of the Japanese encephalitis serocomplex. Expert Rev. Vaccines. 2012; 11(2): 177–87. https://doi.org/10.1586/erv.11.180Sapparapu G., Fernandez E., Kose N., Bin C., Fox J.M., Bombardi R.G., et al. Neutralizing human antibodies prevent Zika virus replication and fetal disease in mice. Nature. 2016; 540(7633): 443–7. https://doi.org/10.1038/nature20564Garg H., Yeh R., Watts D.M., Mehmetoglu-Gurbuz T., Resendes R., Parsons B., et al. Enhancement of Zika virus infection by antibodies from West Nile virus seropositive individuals with no history of clinical infection. BMC Immunol. 2021; 22(1): 5. https://doi.org/10.1186/s12865-020-00389-2