Novel 20th century rotavirus alphagastroenteritidis strains identified in formalin-fixed and ethanol-preserved museum specimens.

Archival specimens held in biorepositories (e.g. natural history collections) offer rare temporal snapshots of global biodiversity. These collections not only preserve species morphology and aspects of ecology, but increasingly provide access to historical molecular data, including insights into wildlife disease. As several pandemics have originated from animal viruses spilling over into the human population (i.e. SARS-CoV-2/COVID-19, 2009 H1N1 influenza, and HIV/AIDS), characterizing the diversity of viruses circulating in wildlife populations is essential for proactive pandemic preparedness. Yet, current surveillance remains biased toward contemporary viruses of economic importance. One solution to bridging spatiotemporal gaps in wildlife virus knowledge is retrospective screening of vouchered wildlife specimens. However, molecular analysis of specimens has been hindered by formalin fixation, which degrades and cross-links nucleic acids. Here, we demonstrate that formalin-fixed vouchered wildlife specimens retain both host and viral RNA fragments after being stored for up to 60 years. We recovered fragments of divergent strains of Rotavirus alphagastroenteritidis from two Australian species of order Chiroptera; Nyctophilus geoffroyi (lesser long-eared bat) and Rhinolophus megaphyllus (smaller horseshoe bat), representing the first characterization of R. alphagastroenteritidis (RVA) in Australian bats, and the oldest identification of the virus to date worldwide. Concurrently, we sequenced endogenous host RNA, providing a proof-of-concept for dual host-virus transcript recovery from vouchered specimens. This study highlights the role biorepositories can play in reconstructing unbiased historical viral landscapes from specimens, irrespective of the host disease status, and enabling spatiotemporal host-virus insight to advance both biodiversity science and global pandemic preparedness.