Whole-genome sequencing surveillance of Siberian tick-borne encephalitis virus (TBEV) identifies an additional lineage in Kyrgyzstan.

Tick-borne encephalitis virus (TBEV) is the most prevalent tick-borne viral disease in Europe and Asia. There are three main subtypes of the virus: European, Siberian, and Far Eastern, each of which having distinctive ecology, clinical presentation, and geographic distribution. In recent years, other TBEV subtypes have been described, namely the Himalayan and Baikalian subtypes. Differences in virulence between TBEV subtypes have been described, with the Far Eastern subtype causing the most severe disease in humans. Considering the emergence of new TBEV foci, the genetic characterisation of the virus in endemic regions is crucial to not only better understand its epidemiology, but also to identify possible genetic determinants of virulence, as well as develop accurate diagnostics and therapeutics. In our previous study, we identified TBEV in six localities of the Kyrgyz Republic (Kyrgyzstan), and Ala-Archa National Nature Park as a focus of TBEV transmission. Whilst we were able to retrieve the first partial TBEV sequence from Kyrgyzstan from Ixodes persulcatus ticks, we were unable to retrieve a complete genome sequence at that time. In this study, we have utilised a sequence-independent single-primer amplification (SISPA) protocol and retrieved the complete genome sequence of our previous 2009 TBEV tick sample (strain KY09) producing the third complete TBEV genome from Kyrgyzstan, and the first genome from the region clustering within the Vasilchenko lineage, suggesting a wider distribution for the lineage than was previously thought. We have also developed a tiling amplicon scheme for Siberian TBEV (TBEV-Sib) which produced > 90 % reference coverage at 100x sequencing depths for samples with as little as 1.13×10⁴ RNA copies/ml. Since high viral loads are rare in TBEV clinical samples, the developed protocol adds value to TBEV-Sib endemic regions by offering a novel set of primers to further amplify the viral genome prior to sequencing.