Characterization of a Novel Orthoreovirus Isolated from Dead Stranded Harbor Seals in Washington State, USA
From October 2007 to July 2008, 24 harbor seals (Phoca vitulina) stranded dead along the shores of Puget Sound, Washington State, USA. The carcasses were necropsied and selected samples were processed for histopathology and ancillary diagnostic testing including virus isolation. Isolates displaying identical cytopathic effects (CPE) in VeroSLAMtag cells were recovered from brain, lymph node, lung, spleen, heart, skin, kidney and abdominal cavity fluid from 5/24 seals. The cultures displayed CPE between 18–33 days post infection (DPI) that included cells first becoming granular and refractile before rounding up in clusters and finally detaching from the plastic surface of the flask. A mesenteric lymph node isolate was expanded in VeroSLAMtag cells and processed for transmission electron microscopy (TEM). The TEM revealed arrays of unenveloped, spherical virus particles (approximately 70 nm in diameter) within the cytoplasm of affected cells, consistent with members of the family Reoviridae.1 The same isolate was processed for viral discovery of RNA and DNA viruses using a next-generation sequencing (NGS) approach on an Illumina MiSeq platform. The resulting NGS data produced the complete coding sequences of the segmented double-stranded RNA genome of a novel reovirus. Formal phylogenetic and genetic analyses revealed the isolate represents a new serotype of the species Mammalian orthoreovirus with the genus Orthoreovirus (hereafter referred to mammalian orthreovirus 5; MVR-5).1,2,3,4,5,6 The MVR-5 is the first fully characterized reovirus from diseased harbor seals. A study conducted in 1989 reported the presence of reovirus-like virus particles in a single sample from an emaciated harbor seal pup in Washington State, USA; however, the virus was neither isolated nor formally characterized.7 Herein, MVR-5 was isolated from five males, four weaned pups and one adult. MVR-5 was isolated from brain in one case, lymph node in another case, and multiple organs in the other three cases, indicating the ability of the virus to induce systemic infection. Reoviruses are known to induce significant pathology in a number of organ systems in humans and animals.8 In 2005, a novel fusogenic (i.e., syncytium-forming) Avian orthoreovirus (SSRV) was isolated in VeroSLAMtag cells 3–5 days DPI from multiple tissues of an aborted Stellar sea lion (Eumetopias jubatus) fetus in British Columbia, Canada.9 In this study, we demonstrated a novel serotype of Mammalian orthoreovirus circulating among harbor seals in the Washington State Inland population and capable of infecting multiple organs. The role of MOR-5 in disease remains unknown as the histopathology of these cases is pending. We plan to develop a specific MVR-5 in situ hybridization probe in an effort to link MVR-5 RNA to observed microscopic lesions. Further research is needed to determine MVR-5 epidemiology, pathogenicity, and its potential impact on harbor seal populations.
We would like to thank the Department of Fisheries and Oceans Canada (DFO) for supporting this study. Harbor seals and samples were collected under the NOAA Marine Mammal Stranding Agreement held by the Seattle Seal Sitters Stranding Network, the 109(h) Authority held by Washington Department of Fish and Wildlife.
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