Detection of West Nile Virus in Overwintering Mosquitoes in the Volgograd Region

   Introduction: West Nile fever is a zoonotic, vector-borne viral infection caused by West Nile virus. The possibility of persistence of West Nile virus in overwintering mosquitoes in regions with a temperate climate is of great importance for understanding the mechanisms of pathogen circulation.
   Objective: To detect West Nile virus in mosquitoes during the inter-epizootic period in the Volgograd Region.
   Materials and methods: In 2013–2021, we collected overwintering mosquitoes in different locations of the Volgograd Region using a battery-powered aspirator with a Krishtal’s trap to detect West Nile virus RNA in them using a real-time reverse transcription polymerase chain reaction. An isolate (WNV Volgograd_o16/19) was obtained from sample o16/19 (Cx. pip-
iens, collected on April 2, 2019) with detected West Nile virus RNA using a VERO cell culture. After that, total RNA was isolated from the filtered cell supernatant of that isolate. Metagenomic sequencing of the sample was performed using a high-throughput Illumina MiSeq sequencer, Illumina Inc.
   Results: In total, we collected 4,070 mosquitoes in wintering shelters and tested 157 pools of the insects for West Nile virus RNA. The latter was detected in two pools of Culex pipiens and in one pool of Anopheles maculipennis complex. The phylogenetic analysis showed that the WNV Volgograd_о16/19 strain isolated from the pool of wintering mosquitoes belonged to lineage 2 of West Nile virus. We also established its belonging to the monophyletic clade of West Nile virus strains isolated in the Volgograd, Astrakhan, and Rostov regions in the years 2007 and 2018–2020.
   Conclusions: We were first to detect West Nile virus in overwintering mosquitoes in the Volgograd Region. Our findings confirm the hypothesis that lineage 2 strains of encephalitic West Nile virus persist in mosquitoes during the inter-epizootic period and can be transmitted from mosquito to bird in springtime as one of the mechanisms of forming autochthonous foci in WNV endemic areas of the Russian Federation in the absence of the annual import of this infection.

1. Viktorov D. V., Smelyanskii V. P., Lipnitskii A. V., et al. [West Nile Virus.] Volgograd: Volga-Press Publ.; 2017. (In Russ.)

2. Nasci R. S., Savage H. M., White D. J., et al. West Nile virus in overwintering Culex mosquitoes, New York City, 2000. Emerg Infect Dis. 2001; 7 (4): 742-744. doi: 10.3201/eid0704.010426

3. Andreadis T. G., Armstrong P. M., Bajwa W. I. Studies on hibernating populations of Culex pipiens from a West Nile virus endemic focus in New York City: parity rates and isolation of West Nile virus. J Am Mosq Control Assoc. 2010; 26 (3): 257-264. doi: 10.2987/10-6004.1

4. Farajollahi A. , Crans W. J., Bryant P., et al. Detection of West Nile viral RNA from an overwintering pool of Culex pipiens pipiens (Diptera: Culicidae) in New Jersey, 2003. J Med Entomol. 2005; 42 (3): 490-494. doi: 10.1093/jmedent/42.3.490

5. Bugbee L. M., Forte L. R. The discovery of West Nile virus in overwintering Culex pipiens (Diptera: Culicidae) mosquitoes in Lehigh County, Pennsylvania. J Am Mosq Control Assoc. 2004; 20 (3): 326-327.

6. Nelms B. M., Macedo P. A., Kothera L., Savage H. M., Reisen W. K. Overwintering biology of Culex (Diptera: Culicidae) mosquitoes in the Sacramento Valley of California. J Med Entomol. 2013; 50 (4): 773-790. doi: 10.1603/me12280

7. Rudolf I., Betášová L., Blažejová H., et al. West Nile virus in overwintering mosquitoes, central Europe. Parasit Vectors. 2017; 10 (1): 452. doi: 10.1186/s13071-017-2399-7

8. Gutsevich A. V., Monchadskii A. S., Stackelberg A. A. [Culicidae Mosquitoes. Fauna of the USSR. Insecta Diptera.] Leningrad: Nauka Publ.; 1970; 3 (4). (In Russ.)

9. Colpitts T. M., ed. West Nile Virus: Methods and Protocols. New York, NY: Humana Press; 2016. doi: 10.1007/978-1-4939-3670-0

10. Moser L. A., Ramirez-Carvajal L., Puri V., et al. A universal next-generation sequencing protocol to generate noninfectious barcoded cDNA libraries from high-con-tainment RNA viruses. mSystems. 2016; 1 (3): e00039-15. doi: 10.1128/mSystems.00039-15

11. Martin M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet. journal. 2011; 17 (1): 10-12. doi: 10.14806/ej.17.1.200

12. Li H., Handsaker B., Wysoker A., et al; 1000 Genome Project Data Processing Subgroup. The sequence alignment/map format and SAMtools. Bioinformatics. 2009; 25 (16): 2078-2079. doi: 10.1093/bioinformatics/btp352

13. Bankevich A., Nurk S., Antipov D., et al. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol. 2012; 19 (5): 455-477. doi: 10.1089/cmb.2012.0021

14. Katoh K., Standley D. M. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol. 2013; 30 (4): 772-780. doi: 10.1093/molbev/mst010

15. 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

16. Letunic I., Bork P. Interactive Tree Of Life (iTOL) v4: recent updates and new developments. Nucleic Acids Res. 2019; 47 (W1): W256-W259. doi: 10.1093/nar/gkz239

17. Fedorova M. V., Borodai N. V. On the necessity and ways to improve entomological monitoring in the epidemiological surveillance for West Nile fever. Meditsinskaya Parazitologiya i Parazitarnye Bolezni. 2017;(2):37-42. (In Russ.)

18. Anderson J. F., Main A. J. Importance of vertical and horizontal transmission of West Nile virus by Culex pipiens in the Northeastern United States. J Infect Dis. 2006; 194 (11): 1577-1579. doi: 10.1086/508754

19. Andreadis T. G. The contribution of Culex pipiens complex mosquitoes to transmission and persistence of West Nile virus in North America. J Am Mosq Control Assoc. 2012; 28 (suppl 4): 137-151. doi: 10.2987/8756-971X-28.4s.137

20. Putintseva E. V., Udovichenko S. K., Boroday N. V., et al. Peculiarities of epidemiological situation on the West Nile fever in the Russian Federation in 2020 and forecast for its development in 2021. Problemy Osobo Opasnykh Infektsiy. 2021; (1): 63-72. (In Russ.) doi: 10.21055/0370-1069-2021-1-63-72