Releasing RNA from formalin: novel 20th century rotavirus alphagastroenteritidis strains identified in Australian microbat voucher 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), characterising 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, such efforts have been hindered by formalin fixation of specimens, 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 sixty years. We recovered fragments of divergent strains of Rotavirus alphagastroenteritidis from two Australian microbat species; Nyctophilus geoffroyi (lesser long-eared bat) and Rhinolophus megaphyllus (smaller horseshoe bat), representing the first characterisation of Rotavirus alphagastroenteritidis in Australian bats, and the oldest identification of the virus to date. 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 historical viral landscapes and enabling spatiotemporal host-virus insight to advance both biodiversity science and global pandemic preparedness.