1. IntroductionIn recent decades, a large number of arthropod-borne viruses have been discovered, and their number is growing every year [1,2,3,4,5]. Some of them may be pathogenic for mammals, including humans [3,6,7,8]; the role of others as pathogens has not been established and requires study to understand their pathogenic potential and impact on the epidemiology and epizootology of known pathogens.The genus Flavivirus belongs to the family Flaviviridae, and includes small enveloped viruses with a non-segmented ssRNA(+) genome of about 10 kb in length that encodes one large open reading frame, which codes a single polyprotein that is co- and post-translationally cleaved by viral and cellular proteases into three structural (C, prM, and E) and seven non-structural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins [9]. The representatives of the genus Flavivirus are the cause of severe human diseases with damage to the central nervous system and haemorrhagic fevers [10,11,12,13,14]. Currently, both mosquito-borne viruses (West Nile virus [15,16] and Japanese encephalitis virus [17]) and tick-borne viruses (tick-borne encephalitis virus (TBEV) [11], Omsk haemorrhagic fever virus [10,18], Powassan virus [19], Louping ill virus [20]) are circulating in Russia.In the last decade, flavi-like viruses with a segmented genome have been discovered [4], and these viruses were included in the group of unclassified viruses related to the family Flaviviridae [9]. Segmented flavi-like viruses, such as the Jingmen tick virus (JMTV), Alongshan virus (ALSV), Yanggou tick virus (YGTV), and others, have a segmented ssRNA(+) genome, two segments of which have homology with the well-studied flavivirus RNA-dependent RNA polymerase NS5 and RNA helicase-protease NS3, with the remaining two segments being specific to these viruses [4,21]. It has recently been shown that ALSV VP1a shares 23% identical amino acids and 50% chemically similar amino acids with the yellow fever virus envelope (E) protein over a 190 amino acid length segment [22]. Representatives of this group were detected in different arthropods [1,4,21,23,24,25,26,27], reptiles [28], and mammals [24,27,29,30,31], including humans [6,7,32]. There is a wide circulation of segmented flavi-like viruses, such as ALSV, YGTV, and JMTV, in Russia [23,33,34].The recently updated classification of the family Phenuiviridae includes 20 quite heterogeneous genera [35]. The genome of phenuiviruses is represented by three segments of negative or ambisense ssRNA: the large (L) segment encodes RNA-dependent RNA polymerase; the medium (M) segment encodes glycoproteins (Gn and Gc) and, in some viruses, an accessory non-structural NSm protein; and the small (S) segment encodes a nucleoprotein (N) and non-structural NSs protein [36]. Viruses are hosted by blood-feeding arthropods, such as ticks, mosquitoes, and sandflies. Some of the phenuiviruses belong to the ecological group of arboviruses, are transmitted between mammals through the bites of blood-feeding arthropods, and are associated with human diseases [2,3,8]. Previously, we showed a wide distribution and diversity of representatives of the genera Phlebovirus, Ixovirus, and unclassified phenuiviruses in Russia [37,38].Combined foci of the tick-borne pathogens, which are inhabited by different species of ticks, attract much attention [39,40]. A comprehensive assessment of such foci provides an idea of the functioning of the virus–tick–host system and is necessary for understanding the evolution of ecological systems.The Chelyabinsk region is one of such focus. The region is located in the south of the central part of Russia and borders with Kazakhstan. Three natural zones are distinguished on the Chelyabinsk region territory: mountain forest (mountain taiga, coniferous, deciduous, and mixed forests), forest-steppe, and steppe [41]. The region is inhabited by various species of ticks: Ixodes persulcatus, Ixodes trianguliceps, Ixodes granulatus, Ixodes aprophorus, Dermacentor reticulatus, and Dermacentor marginatus [23,42,43,44], but in our study we considered the most common pasture-questing tick species (nemobionts [45]). The mountain forest zone inhabits only I. persulcatus ticks; forest-steppe zone inhabits D. reticulatus and I. persulcatus ticks in approximately equal proportions; and the steppe zone inhabits mainly D. marginatus and D. reticulatus ticks, as well as a small number of I. persulcatus ticks [43].TBEV is one of the main arboviruses in Russia, being the cause of severe human diseases [11]. The entire territory of the Chelyabinsk region is endemic for tick-borne encephalitis (TBE) [11,44]. TBE incidence is registered in the whole region, but TBEV has been detected in ticks only in mountain forest and forest-steppe zones, where I. persulcatus ticks are located [43,44,46,47].Previously, we tested ticks collected in the Chelyabinsk region in 2014 using primers for the genus Flavivirus [33]. We detected nine positive samples. The nucleotide sequence of the PCR product showed that it was ALSV. In ticks collected in 2015 in the same place that ALSV was previously found, we detected two positive samples using ALSV-specific primers. We isolated four strains from the ticks collected in 2014 and two strains from the ticks collected in 2015 in the IRE/CTVM19 tick cell line [23,33].

In this work, we tested previously unstudied ticks collected in 2015 using pan-flavi and ALSV-specific primers, as well as studying all ticks collected in 2014 and 2015 for the presence of the representatives of the genus Phenuivirus, YGTV, and TBEV. We provide newly obtained data from a two-year study of the circulation of flavi-, flavi-like, and phenuiviruses in the population of ixodid ticks in the Chelyabinsk region.

4. DiscussionThe main widespread arbovirus in Russia is TBEV, which is the cause of severe human disease [11]. Recently, an expansion of the TBEV area to the north has been noted [60]. The virus was detected in countries that were not endemic for TBE at the beginning of the 21st century, such as Norway [61,62], the United Kingdom [63,64,65], and the Netherlands [66,67]. In some endemic countries, new TBEV foci are emerging [68]. However, the expansion of the boundaries of the TBEV area occurs not only in the north but also to the south, wherein the emergence of TBEV in North Africa [69,70] and TBEV circulation in the steppe regions of Russia [51] have been noted.The entire territory of the Chelyabinsk region is endemic for TBE [11,44]. The incidence is registered in the forest zone, forest-steppe, and steppe zones [43,44,46], while TBEV is detected only in forest and forest-steppe areas [47]. We collected and studied 3960 ticks (I. persulcatus—1275, Dermacentor ticks—2685). We isolated three TBEV strains from I. persulcatus ticks collected in the forest zone. All TBEV strains belong to the Zausaev group of the Siberian subtype. It was previously shown that it is mainly representatives of the Zausaev group of the Siberian subtype that circulate in the territory of the Chelyabinsk region [47,71], and all entries from Chelyabinsk region in GenBank belong to the Zausaev group of the Siberian subtype. Isolation of the Vasilchenko group TBEV in the Chelyabinsk region was also described, but there is no entry of this strain in GenBank [71]. We did not detect TBEV RNA in ticks of the genus Dermacentor. This is interesting because it was previously shown that in Russia and Europe, the infection rate of Dermacentor ticks was higher, or at least not lower, than that of Ixodes ticks [51,72,73], and TBEV titre was higher in Dermacentor ticks than in Ixodes ticks in experiments [74,75]. In the steppe zone of the Chelyabinsk region, there are forest belts along the roads, which I. persulcatus ticks inhabit. We believe that these ticks might be the cause of TBE cases in the steppe zone. However, we do not exclude the possibility that ticks of the genus Dermacentor might play some role in TBE morbidity in the steppe region, since it was previously shown that people suffered TBE after a bite by Dermacentor ticks [76]. We have previously shown that TBEV and YGTV can circulate simultaneously in the steppe regions of the Republic of Tuva [23,51]. We did not find TBEV in any place in the steppe zones of the Chelyabinsk region where YGTV occurred. Perhaps this is due to the fact that in the steppe regions of the Republic of Tuva the most common species is the D. nuttalli tick, in contrast to the Chelyabinsk region, where D. reticulatus and D. marginatus ticks inhabit the steppe regions.Segmented flavi-like viruses were included in the group of unclassified viruses, related to the genus Flavivirus [9]. One of such viruses—ALSV—was first detected in China in a patient after a tick bite [7]. Moreover, ALSV was detected in mosquitos from China [7]; in ticks from Finland [25], France [24], and Russia [23,33]; and in mammals from China [30]. ALSV was detected in ticks collected in eight regions of Russia and, according to phylogenetic analysis of ALSV proteins VP1a and VP1b coding in segment 2, strains were divided into the “I. ricinus” and “I. persulcatus” groups, and, furthermore, the “I. persulcatus” group was divided into European and Asian subgroups [23]. In the Chelyabinsk region, we previously detected 11 ALSV-positive pools of I. persulcatus ticks and isolated six strains [23,33]. In this study, we additionally isolated one new strain named Salma15-T22545 from I. persulcatus, which clusters with other strains of ALSV from the Chelyabinsk region isolated from I. persulcatus ticks.YGTV was first detected in D. nuttalli in the Xinjiang Uygur Autonomous Region of China, and sequences were deposited in GenBank in 2018. For the first time, we isolated the two strains of YGTV using tick cell lines of I. ricinus (IRE/CTVM19) and H. anatolicum (HAE/CTVM8) [23]. In the Chelyabinsk region we detected and isolated in the HAE/CTVM8 tick cell line a total of 26 strains of YGTV. Twenty-five strains were isolated from Dermacentor ticks collected in the steppe zone, and only one from I. persulcatus ticks collected in the forest zone. The highest infection rate with YGTV was noted in D. reticulatus ticks. Since D. reticulatus and D. marginatus ticks can be found not only in Russia but also in Asia, Europe, and North Africa [42,77], we can expect the circulation of this virus in other countries.All of the above, as well as the fact that the previously described YGTV strains were detected mainly in ticks of the genus Dermacentor, may indicate that Dermacentor ticks can be considered the main vector of YGTV; as was the case for the Omsk haemorrhagic fever virus [10]. This assumption requires further research because it was shown that JMTV, under laboratory conditions, was not transmitted trans-stadially and was not found in the midgut and salivary glands of Dermacentor silvarum ticks [78]. According to the phylogenetic analysis, all strains from the Chelyabinsk region clustered together with the strains from China and the Republic of Altai. We did not find any relationship in the phylogenetic division of the YGTV strains, as was found for the geographic isolation of JMTV [79] and for the host species of ALSV [23]. This may have been due to the insufficient number of strains isolated in different territories.It was previously shown that the NS5-like protein of ALSV encoding in segment 1 is the most divergent, and it is sometimes integrated in the I. ricinus tick genome [80]. The highest similarity was shown for a glycoprotein-coding segment 2 of ALSV [33]; therefore, in our work we used the sequences of segment 2 proteins VP1a and VP1b. It should be noted that there are more nucleotide substitutions in the VP1b protein than in the VP1a protein, while the number of non-synonymous nucleotide substitutions is higher in the VP1a protein for both ALSV and YGTV. The nucleotide sequence of the VP1a coding sequence contains two regions of high conservation [23] in places where it is overlapping with putative nuORF and VP1b frameshift site. Thus, high conservation of the nucleotide sequence is expected. At the same time, VP1a showed a higher rate of the non-synonymous nucleotide substitutions, which may indicate the potential importance of this region for the virus adaptation. Further study of the functions of these proteins and their structure will answer the question of why they differ so much.We noticed that the nucleotide and amino acid sequences of VP1a and VP1b proteins of ALSV were more divergent than the same sequences of YGTV (Figure 4). The lower YGTV diversity compared to ALSV may be caused by the virus sampling bias because most of the YGTV strains in the analysed group were collected in the Chelyabinsk region. However, we cannot exclude the possibility of this being determined by the different ecologies of Dermacentor and Ixodes ticks [81,82,83,84].In our work, we detected flavi-like viruses using primers for the genus Flavivirus and specific primers. In our previous work, we also detected both YGTV (in the Republic of Tuva) and ALSV (in the Republics of Tuva and Karelia) using pan-flavi primers [23,33]. However, as we see from our work and previous works [23,38], the number of flavi-like viruses detected using pan-flavi primers was lower than using specific ones. Pan-flavi primers were designed to detect representatives of the genus Flavivirus [52], at the time, when segmented flavi-like viruses were not known to exist. Although this system is able to occasionally detect flavi-like viruses, primers do not have enough complementary regions in segment 1 (NS5-like protein) of flavi-like viruses to reliably amplify their cDNA. Thus, virus-specific primers must be used for the reliable detection of the segmented flavi-like viruses and developing pan-flavi-like PCR assay may be necessary for quick and cheap analysis.We found YGTV (strain Gubenka15-T22237 from I. persulcatus) and TBEV (strain Zlatoust15-T22241 from I. persulcatus) in one collection site. This fact, as well as the fact that the previously described ALSV and TBEV can be found in one location [38], may indicate that representatives of the genus Flavivirus and unclassified viruses, related to the genus Flavivirus may form combined foci.According to a recently renewed classification, the Phenuiviridae family includes 20 genera, and viruses from 14 of them were detected in arthropods [35]. However, few viruses are associated with human diseases: sandfly/mosquito-borne phleboviruses (e.g., Naples phlebovirus, Rift Valley fever phlebovirus, Sicilian phlebovirus, and Toscana phlebovirus) and some tick-borne bandaviruses (Dabie bandavirus, Heartland bandavirus, and Bhanja bandavirus). Viruses from the remaining 12 genera of phenuiviruses should also be considered either as viruses of arthropods or as viruses with unknown epidemiologic potential.The Phlebovirus genus mainly includes sandfly/mosquito-borne viruses; however, a small group of tick-borne viruses, such as Mukawa phlebovirus and Kuriyama phlebovirus, were also classified into this genus. The Gomselga virus detected in I. persulcatus ticks cluster with the tick-borne phleboviruses (Figure 6). Although the Mukawa phlebovirus and Kuriyama phlebovirus are phylogenetically close to the pathogenic sandfly/mosquito-borne viruses, there is only some serological evidence indicating the infection of wild animals with Mukawa phlebovirus, but no infectious virus was recovered upon the experimental infection [85]. The unclassified phenuivirus Stavropol is abundant on the territory of the European part of Russia and is highly specific to D. reticulatus ticks [37]. There are no data on the pathogenic properties of the Stavropol virus. Due to the unknown pathogenic potential of the viruses Gomselga and Stavropol, further studies of their distribution, properties, and interactions with other pathogens are needed.The results of the current study are highly consistent with our previous publications regarding the widespread distribution of phenuiviruses and their high tick specificity across the European part of Russia [37,38]. In addition to the Chelyabinsk region, the Gomselga virus was recorded in the Republics of Karelia and Tuva for only I. persulcatus ticks, and its prevalence levels were 0.66–1.56%, 1.8–4.5%, and 2.0%, respectively. Conversely, Stavropol virus was detected in the Kaluga, Krasnodar, Moscow, Stavropol, Ulyanovsk, and Voronezh regions and in the Republic of Tatarstan [37], almost exclusively in D. reticulatus ticks. A previously reported prevalence of the Stavropol virus in the Chelyabinsk region (6.95%) was lower than in other regions of Russia [37], especially in the Ulyanovsk (21.5%) and Tatarstan (31.0%) regions. On the other hand, a relatively high prevalence and high specificity of YGTV to D. reticulatus ticks may imply that there is competition or at least interaction between YGTV and the Stavropol virus. Additional data regarding the prevalence of YGTV in other regions, compared with prevalence of the Stavropol virus, would allow us to speculate about this fact.