Every year, tick-borne encephalitis virus (TBEV) causes severe central nervous system infection in 10 000 to 15 000 people in Europe and Asia. TBEV is maintained in the environment by an enzootic cycle that requires a tick vector and a vertebrate host, and the adaptation of TBEV to vertebrate and invertebrate environments is essential for TBEV persistence in nature. This adaptation is facilitated by the error-prone nature of the virus's RNA-dependent RNA polymerase, which generates genetically distinct virus variants called quasispecies. TBEV shows a focal geographical distribution pattern where each focus represents a TBEV hotspot. Here, we sequenced and characterized two TBEV genomes, JP-296 and JP-554, from questing Ixodes ricinus ticks at a TBEV focus in central Sweden. Phylogenetic analysis showed geographical clustering among the newly sequenced strains and three previously sequenced Scandinavian strains, Toro-2003, Saringe-2009 and Mandal-2009, which originated from the same ancestor. Among these five Scandinavian TBEV strains, only Mandal-2009 showed a large deletion within the 3' non-coding region (NCR), similar to the highly virulent TBEV strain Hypr. Deep sequencing of JP-296, JP-554 and Mandal-2009 revealed significantly high quasispecies diversity for JP-296 and JP-554, with intact 3' NCRs, compared to the low diversity in Mandal-2009, with a truncated 3' NCR. Single-nucleotide polymorphism analysis showed that 40% of the single-nucleotide polymorphisms were common between quasispecies populations of JP-296 and JP-554, indicating a putative mechanism for how TBEV persists and is maintained within its natural foci.

The hypothesis of wide spread reticulate evolution in Tick-Borne Encephalitis virus (TBEV) has recently gained momentum with several publications describing past recombination events involving various TBEV clades. Despite a large body of work, no consensus has yet emerged on TBEV evolutionary dynamics. Understanding the occurrence and frequency of recombination in TBEV bears significant impact on epidemiology, evolution, and vaccination with live vaccines. In this study, we investigated the possibility of detecting recombination events in TBEV by simulating recombinations at several locations on the virus' phylogenetic tree and for different lengths of recombining fragments. We derived estimations of rates of true and false positive for the detection of past recombination events for seven recombination detection algorithms. Our analytical framework can be applied to any investigation dealing with the difficult task of distinguishing genuine recombination signal from background noise. Our results suggest that the problem of false positives associated with low detection P-values in TBEV, is more insidious than generally acknowledged. We reappraised the recombination signals present in the empirical data, and showed that reliable signals could only be obtained in a few cases when highly genetically divergent strains were involved, whereas false positives were common among genetically similar strains. We thus conclude that recombination among wild-type TBEV strains may occur, which has potential implications for vaccination with live vaccines, but that these events are surprisingly rare.