Single nucleotide polymorphisms are typical for tick-borne encephalitis and West Nile viruses during triple natural mixed infections in Blyth’s reed warbler (Acrocephalus dumetorum)

Wild bird species contribute significantly to the rapid geographic dissemination of tick-borne encephalitis viruses (TBEV) and West Nile virus (WNV), facilitating the establishment of new natural foci of these orthoflaviviruses. However, the impact of TBEV and WNV population variability on shaping these foci, as well as the potential emergence of new human-pathogenic viral variants, remain underexplored. This study aimed to assess the genetic heterogeneity of TBEV (Siberian and Far Eastern genotypes) and WNV, isolated simultaneously from the tissues of a single garden reed warbler (Acrocephalus dumetorum) collected in the suburbs of Tomsk. The methods of viral strain isolation on various cell cultures were used in combination with a whole-genome analysis of isolates through traditional and high-throughput sequencing (NGS) methods. Consensus full-genome nucleotide sequences of the viruses were obtained by Sanger sequencing and compared with those obtained by NGS, with single nucleotide substitutions (single nucleotide variants, SNVs) accounting for 2 % or higher within the population under study. Our findings revealed single nucleotide polymorphisms (SNPs) associated with both synonymous and non-synonymous nucleotide substitutions, primarily located within the non-structural protein genes of TBEV and WNV. Notably, recombination events were not detected in the genomes of isolated orthoflaviviruses. The WNV isolate, Tomsk/bird/2006/A4, and the TBEV isolates, PT12 and PT122, obtained from A. dumetorum, exhibited heterogeneous viral populations, with SNVs ranging in frequency from 1.75 to 19.88 % for WNV and from 2.08 to 23.73 % for TBEV. Most identified SNPs shared similar nucleotide substitutions in the genomes of already known strains of TBEV and WNV, suggesting that these SNVs could play a crucial role in viral adaptation and underscore the genetic and phenotypic diversity of these viruses in nature.