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.

Members of the Lipopteninae subfamily are blood-sucking ectoparasites of mammals. The sheep ked (Melophagus ovinus) is a widely distributed ectoparasite of sheep. It can be found in most sheep-rearing areas and can cause skin irritation, restlessness, anemia, weight loss and skin injuries. Various bacteria and some viruses have been detected in M. ovinus; however, the virome of this ked has never been studied using modern approaches. Here, we study the virome of M. ovinus collected in the Republic of Tuva, Russia. In our research, we were able to assemble full genomes for five novel viruses, related to the Rhabdoviridae (Sigmavirus), Iflaviridae, Reoviridae and Solemoviridae families. Four viruses were found in all five of the studied pools, while one virus was found in two pools. Phylogenetically, all of the novel viruses clustered together with various recently described arthropod viruses. All the discovered viruses were tested on their ability to replicate in the mammalian porcine embryo kidney (PEK) cell line. Aksy-Durug Melophagus sigmavirus RNA was detected in the PEK cell line cultural supernate after the first, second and third passages. Such data imply that this virus might be able to replicate in mammalian cells, and thus, can be considered as a possible arbovirus.

Members of the jingmenviruses group have been found in arthropods and mammals on all continents except Australia and Antarctica. Two viruses of this group were isolated from patients with fever after a tick bite. Using a nested RT-PCR assay targeting a jingmenvirus polymerase gene fragment, we screened ticks collected in seven regions of Russia and found that the abundant jingmenvirus-positive were of Ixodes ricinus species, with the prevalence ranging from 19.8% to 34.3%. In all cases, DNase/RNase treatment suggested that the detected molecule was DNA and subsequent next generation sequencing (NGS) proved that the viral polymerase gene was integrated in the I. ricinus genome. The copy number of the integrated polymerase gene was quantified by qPCR relative to the ITS2 gene and estimated as 1.32 copies per cell. At least three different genetic variants of the integrated polymerase gene were found in the territory of Russia. Phylogenetic analysis of the integrated jingmenvirus polymerase gene showed the highest similarity with the sequence of the correspondent gene obtained in Serbia from I. ricinus.