Detection of Far-Eastern subtype of tick-borne encephalitis viral RNA in ticks collected in the Republic of Moldova.

Flavivirus in the family Flaviviridae is a causative agent of tick-borne encephalitis, which is usually transmitted by Ixodes persulcatus and I. ricinus ticks1. Based on the geographical prevalence, the TBEV is classified into three subtypes: European (TBEV-Eur), Siberian (TBEV-Sib), and Far-Eastern (TBEV-FE). The mortality rate for TBEV-Eur and TBEV-Sib subtype infections is relatively low (1–2%) while for TBEV-FE, it is up to 30%2. Recently, TBEV-FE subtype was also detected outside of Far-East Eurasia in regions including, Baltic States, Crimea, Republic of Komi, Ural Mountains and Siberia3-5. Interestingly, TBEV-FE genotype is more prevalent in the urban and suburban areas6-7. It has been hypothesized that wild birds potentially disseminate new and unusual TBEV variants, TBEV-infected ixodid ticks, and possibly other tick-borne infections5. To determine the spread of TBEV-FE to new geographical regions, we studied samples of individual ticks collected from domestic animals and agricultural land in Republic of Moldova between 2010 and 2011 for the presence of TBEV RNA. A total of 189 adult (males and females) I. ricinus, Dermacentor spp and Haemaphysalis spp ticks were collected as described previously by Chausov et al1, 3 from vegetation by flagging (adults) and from domestic animals such as cows, goats and sheep (adults, nymphs and larvae) in eight districts of Republic of Moldova (Ungheni, Chisinau, Vadul-lui-Voda, Strasheni, Comrat, Orhei, Drochia and Glodeni) between 2010 and 2011 (Fig. 1). Live ticks were subjected to morphological identification under light microscope before individual storage at –70°C8. The extraction of viral RNA, reverse transcription, and detection of TBEV cDNA by reverse transcription-polymerase chain reaction (RT-PCR) from frozen ticks were carried out as described previously by Hubálek & Rudolf5; and Iurchenko et al6. RT-PCR products were separated using 1.5% agarose gel and gels were purified using Wizard SV Gel and PCR Clean-Up System kit (Promega, USA) following the manufacturer’s instructions. The sequences were determined by BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems, CIIIA) using ABI 3130×l (Applied Biosystems, CIIIA) according to the manufacturer’s instructions. All samples were independently analyzed in duplicate. All sequence alignments and phylogenetic studies were performed using Vector NTI Suite 10.0 (www.invitrogen.com) and MEGA5, respectively9. Tick species were confirmed by sequencing of PCR amplified fragments generated using the cytochrome oxidase subunit I and 16S RNA gene specific primers10. J Vector Borne Dis 52, December 2015, pp. 334–336