Huge diversity of phleboviruses in ticks from Strandja Nature Park, Bulgaria.

The discovery of the first tick-borne phleboviruses associated with severe disease in humans stimulated studies searching for further previously unknown tick-associated viruses. Novel phleboviruses have subsequently been identified in ticks from the USA, Japan and China and recently also from Europe. Here, we investigated the genetic diversity of tick-borne phleboviruses originating from Strandja Nature Park, Bulgaria, a unique primary forest with evergreen plants that was not affected by the last ice ages in the Pleistocene and Holocene. We found a high genetic diversity of 12 phleboviral sequences in 1542 ticks. The sequences formed five distinct groups and clustered with other tick-borne phleboviruses recently identified in Europe. Although isolation experiments of the detected viruses in cell culture failed, viral RNA copy numbers were stable up to 42 days post infection (dpi) in the supernatant of tick cells whereas they disappeared 14 dpi in that of VeroE6/7 cells. In summary, nearly all tick-associated phleboviruses known to occur in Europe have been detected in one geographic region. Our data show that primary ecosystems in temperate regions are also rich in viral diversity and that this is not only true for tropical regions.