Partial Characterization of Tick-Borne Encephalitis Virus Isolates from Ticks of Southern Ukraine

Abstract

Tick-borne encephalitis (TBE) is the most common tick-borne viral infection in Eurasia; thousands of human cases are annually reported from several European countries. Several tick species are vectors of the tick-borne encephalitis virus (TBEV), while TBE appears to be spreading from the Eurasian continent westward to Europe. Fifteen study sites were chosen from five territories of southern Ukraine, including Odessa, Mykolaiv, Kherson Oblast, the Autonomous Republic of Crimea, and Sevastopol. Tick collection was performed in spring season of three consecutive years (1988-1990) using either flagging technique or direct collection of specimens feeding on cattle. A total of 15,243 tick imagoes and nymphs were collected from nine species, including Dermacentor marginatus, D. reticulatus, Haemaphysalis parva, H. punctata, Hyalomma marginatum, Ixodes ricinus, Rhipicephalus bursa, R. rossicus, and R. sanguineus, pooled in 282 monospecific samples. Supernatant of grinded pool was used for inoculation to suckling mice for virus isolation. Eight TBEV isolates were identified from ticks among six study sites. Ticks showed a minimum infection rate from 0.11% to 0.81%. Phylogenetic analysis of the envelope (E) protein gene of seven isolates, assigned all to the European subtype (TBEV-Eu) showing a maximum identity of 97.17% to the "Pan" TBEV-Eu reference strain. Compared to 104 TBEV-Eu isolates they clustered within the same clade as the Pan reference strain and distinguished from other TBEV-Eu isolates. Amino acid sequence analysis of the South Ukrainian TBEV-Eu isolates revealed the presence of four amino acid substitutions 67 (N), 266 (R), 306 (V), and 407 (R), in the ectodomains II and III and in the stem-anchor region of the E protein gene. This study confirmed TBEV-Eu subtype distribution in the southern region of Ukraine, which eventually overlaps with TBEV-FE (Far Eastern subtype) and TBEV-Sib (Siberian subtype) domains, showing the heterogeneity of TBEV circulating in Ukraine.

Keywords: Ukraine; envelope protein; nucleotide sequencing; phylogenetic analysis; tick-borne encephalitis virus.

FIG. 1.

Identified sites where tick-borne encephalitis virus has been isolated in Ukraine. See Supplementary Table S2 for virus strain characteristics. Credit: Dr. Marc Souris, Laos and Cambodia representative, Institute of Research for Development (IRD-UMR 190).

FIG. 2.

Phylogenetic tree construction (MEGA 7 software) based on the nucleotide sequence of the E protein gene illustrating the presence of all three TBEV subtypes in Ukraine. E gene nucleotide sequences were aligned with Muscle and analyzed by Maximum Likelihood method. The reliability of the tree was tested by bootstrap resampling (1000 replications). In addition, comparison of the Ukrainian strains with other TBEV-Eu strains isolated in Europe and Asia is presented in the Supplementary Figure S1A and B. *Reference strain subtype. E, envelope; TBEV, tick-borne encephalitis virus.