Our goal was to extend our knowledge about viral diversity within the non-endemic Hy. marginatum tick since we only know a small fraction of viral diversity globally and this poses a threat to anticipate disease emergence [41]. Some viruses have been shown to have the potential to regulate arbovirus transmission in mosquitoes [17], and we have limited information about tick-specific viruses. The metagenome of our tick sample did not contain CCHFV. The taxonomic classification of the short reads revealed that the most abundant virus was the Volzhskoe tick virus, a member of the Bunyavirales. The order Bunyavirales includes several known pathogens, such as CCHFV, La Crosse virus, and Rift Valley fever virus. We could assemble the M and L segments, but we were unable to identify the S segment in the metagenome. Recent metagenomic analyses have shown that Ixodes ricinus bunyavirus-like virus 1 (IRBV1) and the Croatian strain of Bronnoya virus also lack the S segment [42], which, together with our data, suggests that this may be a characteristic feature of unclassified tick-specific bunyaviruses, as these species are the closest relatives of Volzhskoe tick virus based on our phylogenetic trees. However, it is also possible that these segments may be different enough from known ones that they cannot be identified as viral segments.
The predicted amino acid sequence from the ORF of the L segment of the newly identified Volzhskoe tick virus revealed typical Bunyavirales RdRp regions. In Region III, known as the functional core of the molecule, the necessary six conserved motifs [40] were displayed, indicating that the predicted RdRp should be a functional molecule and further confirming the presence of the Volzhskoe tick virus in the metagenome.
Bunyaviruses have a single ORF in their M segment that encodes a polyprotein. Co- and post-translational processes transform this polyprotein into functional glycoproteins, Gn and Gc, which play a crucial role in recognizing host receptors [22]. On the M segment, we found a single long ORF, which was as expected. Using this ORF, we predicted the amino acid sequence of the translated protein, which showed the organization of the bunyavirus GPC. The number of transmembrane domains in bunyavirus GPCs is variable. In the Volzhskoe tick virus GPC, we found three transmembrane helices: two are located around the middle of the polyprotein, namely at the C-terminus of Gn. The third domain is located at the Gc's C-terminus. The NSm protein was not presented in the sequence of the GPC, although not all bunyaviruses encode non-structural proteins in their M genomic segment [16]. We also detected a signal protein in the N-terminal region of the GPC, a common feature for members of the order Bunyavirales. Proprotein cleavage sites were predicted at positions 263 and 683, which may play a role in polyprotein processing as several enzymes, including signalases, proteases, furin, subtilisin/kexin isozyme-1/site-1 protease (SKI-1/S1P), and convertases, are involved in the proteolytic processing of bunyavirus GPC [43]. These results support the hypothesis that this previously uncharacterized virus is capable of infecting and replicating in tick cells based on the sequenced L and M genomic segments.
The Volzhskoe tick virus showed a close phylogenetic relationship with other unclassified species of the Bunyavirales; the most closely related species were also found in various tick species. These included Ixodes ricinus with European, Ixodes scapularis with North-American and Hy. marginatum with palearctic geographical distribution. The importance of providing sequencing data for these ticks is becoming more relevant in light of disease emergence, which is strongly correlated with the population dynamics of the vector, and one of the main benefits of metagenomic surveillance is that we are able to detect coinfections, leading to a deeper understanding of viral interactions.
In addition to the importance of our metagenomic study and the discovery of tick-specific viruses, it also has some limitations. First, the metagenomic approach did not allow us to further classify the Volzhskoe tick virus because its closest relatives were also unclassified. In the future, we would need to isolate and sequence this virus to determine if it has an S segment. Moreover, we are not able to determine based on the sequence information alone whether the Volzhskoe tick virus can infect vertebrates or if it is a tick-specific virus. Based on the current data, we suspect that the Volzhskoe tick virus is most likely a tick-specific virus (ISV), although very little information is available. Obtaining more genomic and ecological data on the virus and its closest relatives is necessary to understanding its potential pathogenicity to humans or animals. Traditional in vitro experiments could also be useful in tick cocultures as well as the serological screening of known hosts of Hyalomma marginatum.
In conclusion, our high-throughput metagenomic analyses provide the first genomic and phylogenetic data about the presence of Volzhskoe tick virus in Hungary, a recently reported tick-borne Bunyavirales member with unknown pathogenicity and host range. Documentation of this virus was possible with the help of volunteer citizen scientists who provided Hyalomma spp. specimens for molecular and metagenomic analysis. To take preventive measures against ticks and tick-borne pathogens applying the DAMA (Document, Assess, Monitor, Act) protocol we will continue with the tick monitoring project in the future as it has demonstrated the ability of citizen science to serve pathogen discovery and monitoring and the anticipation of emerging infectious diseases.