Real-Time Polymerase Chain Reaction Systems for Detection and Differentiation of Unclassified Viruses of the Phenuiviridae Family

The family <i>Phenuiviridae</i>, part of the order <i>Hareavirales</i>, includes arboviruses and arthropod-associated viruses, with sandflies, mosquitoes, and ticks as primary vectors. Historically, only sandfly/mosquito-borne phenuiviruses were associated with human diseases, but the emergence of severe fever with thrombocytopenia syndrome (SFTS) has highlighted the potential of tick-borne phenuiviruses as human pathogens. Recent discoveries of new arthropod-associated viruses, some of which remain unclassified, underscore the need for sensitive detection and differentiation methods, particularly in regions where these viruses may co-circulate. This study aimed to develop real-time PCR test systems for identifying and differentiating unclassified viruses within the <i>Phenuiviridae</i> family. In this study, tick suspensions containing phenuiviruses, previously obtained during the screening of ticks from various regions of Russia using pan-phenuivirus primers, were used. Specific primers and probes were designed to differentiate five <i>Phenuiviridae</i> viruses of genera <i>Uukuvirus</i>, <i>Ixovirus</i>, <i>Phlebovirus</i> and one unclassified phenuivirus, and their analytical sensitivity and specificity were evaluated. These PCR-based tools provide a robust method for detecting and classifying uncharacterized phenuiviruses, contributing to improved surveillance and understanding their potential epidemiological and epizootological impacts.