Extending the temporal window of arbovirus evolutionary analysis through the recovery of a century-old bandavirus.

Arboviruses evolve under unique ecological constraints imposed by their dual replication cycles in vertebrate and arthropod hosts. This dual-host requirement results in markedly low substitution rates, which complicate molecular clock calibration, particularly when temporal sampling spans only narrow time windows. For RNA arboviruses, wide sampling windows are especially rare due to the intrinsic instability of RNA. Here, we demonstrate that ethanol-preserved museum specimens can help overcome these temporal limitations. We successfully recovered the coding-complete genome of a bandavirus, a negative-sense segmented RNA virus that clusters with the highly pathogenic human severe fever with thrombocytopenia syndrome virus. The virus was detected in a Common pipistrelle (Pipistrellus pipistrellus) bat collected in northern Germany in 1919, making it one of the oldest sequenced mammalian RNA viruses, only comparable to historic measles and influenza A viruses from 1912 and 1918. Screening 1086 contemporary bat samples revealed strains of the same virus species in nine Common pipistrelle bats (2010-2018) and one Serotine bat (Eptesicus serotinus, 1999) from Germany and the Netherlands. Coding-complete genomes indicate frequent genome segment reassortment and widespread circulation of reassortants of this understudied virus species (Bandavirus zwieselense). We detected an exceptionally low substitution rate (< 6.88 × 10-5 substitutions/site/year) between the RdRp coding sequence of the ancient genome and its nearest modern counterpart. Additionally, functional assays demonstrated that the virus's non-structural (NSs) protein effectively inhibits interferon induction in human HEK-293T cells. Our findings highlight the feasibility and scientific value of extracting and analysing ancient viral RNA from ethanol-preserved museum specimens to substantially enhance our understanding of RNA arbovirus evolution.