In recent decades, many new flavi-like viruses have been discovered predominantly in different invertebrates and, as was recently shown, some of them may cause disease in humans. The Jingmenvirus (JMV) group holds a special place among flaviviruses and flavi-like viruses because they have a segmented ssRNA(+) genome. We detected Alongshan virus (ALSV), which is a representative of the JMV group, in ten pools of adult Ixodes persulcatus ticks collected in two geographically-separated Russian regions. Three of the ten strains were isolated in the tick cell line IRE/CTVM19. One of the strains persisted in the IRE/CTVM19 cells without cytopathic effect for three years. Most ALSV virions purified from tick cells were spherical with a diameter of approximately 40.5 nm. In addition, we found smaller particles of approximately 13.1 nm in diameter. We obtained full genome sequences of all four segments of two of the isolated ALSV strains, and partial sequences of one segment from the third strain. Phylogenetic analysis on genome segment 2 of the JMV group clustered our novel strains with other ALSV strains. We found evidence for the existence of a novel upstream open reading frame in the glycoprotein-coding segment of ALSV and other members of the JMV group.

The genus Flavivirus includes related, unclassified segmented flavi-like viruses, two segments of which have homology with flavivirus RNA-dependent RNA polymerase NS5 and RNA helicase-protease NS3. This group includes such viruses as Jingmen tick virus, Alongshan virus, Yanggou tick virus and others. We detected the Yanggou tick virus in Dermacentor nuttalli and Dermacentor marginatus ticks in two neighbouring regions of Russia. The virus prevalence ranged from 0.5% to 8.0%. We detected RNA of the Alongshan virus in 44 individuals or pools of various tick species in eight regions of Russia. The virus prevalence ranged from 0.6% to 7.8%. We demonstrated the successful replication of the Yanggou tick virus and Alongshan virus in IRE/CTVM19 and HAE/CTVM8 tick cell lines without a cytopathic effect. According to the phylogenetic analysis, we divided the Alongshan virus into two groups: an Ixodes persulcatus group and an Ixodes ricinus group. In addition, the I. persulcatus group can be divided into European and Asian subgroups. We found amino acid signatures specific to the I. ricinus and I. persulcatus groups and also distinguished between the European and Asian subgroups of the I. persulcatus group.

The most significant processes of arbovirus evolution can be expected to occur in the territories where ticks of different species cohabitate and at the boundaries of virus occurrence, where the probability of the appearance of new virus variants is high due to the possible shift in the main vectors and/or vertebrate hosts. One of the most interesting regions in this regard is the Republic of Tuva. Since most of its territory is covered by mountain ranges and intermountain basins, we were able to study the distribution of vectors and viruses in geographically isolated areas at different altitudes and in various landscapes. From 2008 to 2017, we conducted six expeditions to Tuva and collected 3,077 adult ticks and 24 nymphs. The distribution of tick species was confined to specific landscapes, as follows: Dermacentor nuttalli occurred in steppes, D. silvarum inhabited forest-steppe areas, and Ixodes persulcatus inhabited mixed forests. All three species of ticks were collected on plains and mountain slopes. The range of D. silvarum was shown to be lower than 1300 m above sea level (a.s.l.). Only D. nuttalli and I. persulcatus were collected at higher altitudes. According to our observations, single nymphs of D. nuttalli appear on animals one month before larvae appear. This finding confirms the hypothesis that the immature forms of D. nuttalli are able to overwinter under favourable conditions. We isolated 9 strains and 3 isolates of tick-borne encephalitis virus (TBEV) from I. persulcatus, one strain from D. nuttalli and one strain from D. silvarum. The TBEV strain from D. nuttalli was isolated from the territory inhabited only by Dermacentor ticks. All isolated strains belong to the Siberian subtype of TBEV. TBEV was detected in ticks from all the investigated altitudes. There were no statistically significant differences in the virus prevalence between the Dermacentor and Ixodes ticks. The results of our work provide additional support for the hypothesis of the existence of TBEV foci in areas with an absolute dominance of D. nuttalli.