Pan-Viral Sequencing Surveillance Identifies Mammalian Orthoreovirus 2 in United States Wastewater

Mammalian orthoreoviruses (MRVs) are segmented, double-stranded RNA viruses that infect a broad range of mammalian hosts, including humans. Although MRVs have been detected in wastewater in parts of Southeast Asia, they have not previously been reported in U.S. wastewater. Using the VirCapSeq-VERT pan-viral sequencing assay, we identified MRV type 2 (MRV-2) in a wastewater sample collected in March 2024 from the United States Air Force Academy. Complete genome sequences were recovered for all 10 segments. Phylogenetic analyses showed that the virus clustered most closely with an MRV isolate recovered from a big brown bat (Eptesicus fuscus) in Pennsylvania, with additional genomic similarity to a second bat-derived isolate from Nebraska. The MRV sequence signal declined rapidly in subsequent wastewater samples and was nearly undetectable two weeks later. These findings represent the first reported detection of MRV in U.S. wastewater and demonstrate the utility of pan-viral wastewater surveillance for identifying uncommon viruses with potential public health relevance. Continued genomic and epidemiologic surveillance will improve understanding of MRV circulation and zoonotic transmission in North America.