Phlebovirus sequences detected in ticks collected in Russia: Novel phleboviruses, distinguishing criteria and high tick specificity

Phlebovirus is an abundant and rather heterogeneous genus within the Phenuiviridae family (order Bunyavirales). The genus Phlebovirus is divided into two antigenic complexes, which also correspond to the main vector: sandflies/mosquitoes and ticks. Previously, only sandfly/mosquito-borne phleboviruses were associated with human disease, such as Rift Valley fever virus, Toscana virus, Sicilian and Naples Sandfly fever viruses and others. Until recently, tick-borne phleboviruses were not considered as human pathogens. After the discovery of severe fever with thrombocytopenia syndrome, interest to tick-borne phleboviruses has increased dramatically. In the last decade, many novel phleboviruses have been reported in different regions. Despite this, the diversity, ecology and pathogenicity of these viruses still remain obscure. The aim of this work was to study the diversity of phleboviruses in ticks collected in several regions of Russia. We used pan-phlebovirus RT-PCR assays based on multiple degenerate primers targeting the polymerase gene fragment. Arthropod specimens were collected from 2005 to 2018. A total of 5901 Ixodidae ticks combined into 1116 pools were screened. A total of 160 specific amplicons were produced. In three cases RT-PCR assays amplified two distinct viruses from same tick pools. Direct sequencing of amplicons and subsequent phylogenetic analysis revealed twelve representatives of divergent phlebovirus groups. Based on the distribution of pairwise nucleotide sequence identity values, a cut-off (88%) was suggested to distinguish tick-borne phleboviruses. According to this provisional criterion, two viruses found here could be termed novel, while ten viruses have been described in previous studies. Detected phleboviruses demonstrated almost perfect specificity to a tick species or, at least, a genus. The same pattern was observed for tick-borne phleboviruses found in different studies around the world. Viruses that grouped together on a phylogenetic tree and differed less than this sequence identity threshold suggested above were hosted by ticks from the same genus