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.

In this work, we presented data from a two-year study of flavi-, flavi-like, and phenuiviruses circulation in the population of ixodid ticks in the Chelyabinsk region. We isolated three tick-borne encephalitis virus (TBEV) strains from I. persulcatus, which was not detected in the ticks of the genus Dermacentor. The virus prevalence ranged from 0.66% to 2.28%. The Yanggou tick virus (YGTV) is widespread in steppe and forest-steppe zones and is mainly associated with ticks of the genus Dermacentor. We isolated 26 strains from D. reticulatus, D. marginatus, and I. persulcatus ticks in the HAE/CTVM8 tick cell line. The virus prevalence ranged from 1.58% to 4.18% in D. reticulatus, ranged from 0.78% to 3.93% in D. marginatus, and was 0.66% in I. persulcatus. There was combined focus of TBEV and YGTV in the territory of the Chelyabinsk region. The Alongshan virus (ALSV) was found to be associated with I. persulcatus ticks and is spread in forest zone. We detected 12 amplicons and isolated 7 strains of ALSV in tick cells. The virus prevalence ranged from 1.13% to 6.00%. The phlebovirus Gomselga and unclassified phenuivirus Stavropol were associated with I. persulcatus and D. reticulatus ticks, respectively. Virus prevalence of the unclassified phenuivirus Stavropol in the Chelyabinsk region is lower than that in neighbouring regions.

Transport of macromolecules through the nuclear envelope (NE) is mediated by nuclear pore complexes (NPCs) consisting of nucleoporins (Nups). Elys/Mel-28 is the Nup that binds and connects the decondensing chromatin with the reassembled NPCs at the end of mitosis. Whether Elys links chromatin with the NE during interphase is unknown. Here, using DamID-seq, we identified Elys binding sites in Drosophila late embryos and divided them into those associated with nucleoplasmic or with NPC-linked Elys. These Elys binding sites are located within active or inactive chromatin, respectively. Strikingly, Elys knockdown in S2 cells results in peripheral chromatin displacement from the NE, in decondensation of NE-attached chromatin, and in derepression of genes within. It also leads to slightly more compact active chromatin regions. Our findings indicate that NPC-linked Elys, together with the nuclear lamina, anchors peripheral chromatin to the NE, whereas nucleoplasmic Elys decompacts active chromatin.

Tick-Borne Encephalitis Virus (TBEV) is a dangerous arbovirus widely distributed in Northern Eurasia. The area of this pathogen changes over time. At the beginning of the 2000s, the Ixodes tick populations in Karelia increased. At the same time, the area of I. persulcatus, the main vector of the Siberian TBEV subtype, also expanded. Herein, we sequenced 10 viruses isolated from ticks collected in three locations from the Karelia region in 2008–2018. PCR positive samples were passaged in suckling mice or pig embryo kidney cells (PEK). After the second passage in suckling, mice viral RNA was isolated and E-gene fragment was sequenced. Viral sequences were expected to be similar or nearly identical. Instead, there was up to a 4.8% difference in nucleotide sequence, comparable with the most diverse viruses belonging to the Baltic subgroup in Siberian TBEV subtype (Baltic TBEV-Sib). To reveal whether this was systemic or incidental, a comprehensive phylogeographical analysis was conducted. Interestingly, viruses within each geographic region demonstrated comparable diversity to the whole Baltic TBEV-Sib. Moreover, Baltic TBEV-Sib has a distribution area limited by three ecological regions. This means that active virus mixing occurs in the vast geographic area forming one common virus pool. The most plausible explanation is the involvement of flying animals in the TBEV spread.

Cell cultures derived from ticks have become a commonly used tool for the isolation and study of tick-borne pathogens and tick biology. The IRE/CTVM19 cell line, originating from embryos of Ixodes ricinus, is one such line. Previously, reovirus-like particles, as well as sequences with similarity to rhabdoviruses and iflaviruses, were detected in the IRE/CTVM19 cell line, suggesting the presence of multiple persisting viruses. Subsequently, the full genome of an IRE/CTVM19-associated rhabdovirus was recovered from a cell culture during the isolation of the Alongshan virus. In the current work, we used high-throughput sequencing to describe a virome of the IRE/CTVM19 cell line. In addition to the previously detected IRE/CTVM19-associated rhabdovirus, two rhabdoviruses were detected: Chimay rhabdovirus and Norway mononegavirus 1. In the follow-up experiments, we were able to detect both positive and negative RNA strands of the IRE/CTVM19-associated rhabdovirus and Norway mononegavirus 1 in the IRE/CTVM19 cells, suggesting their active replication in the cell line. Passaging attempts in cell lines of mammalian origin failed for all three discovered rhabdoviruses.