Piscine reovirus (PRV) is a new member of the family Reoviridae and has been linked to heart and skeletal muscle inflammation in farmed Atlantic salmon (Salmo salar L.). Recent sequence-based evidence suggests PRV is about equally related to both genus Orthoreovirus and genus Aquareovirus. Sequence similarities have also suggested PRV might encode a fusion-associated small transmembrane (FAST) protein, which in turn suggests PRV might be the prototype of a new Reoviridae genus with syncytium-inducing potential. In previous support of that designation has been the absence of identifiable PRV-encoded homologs of either the virion outer-clamp protein of ortho- and aquareoviruses or the virion outer-fiber protein of most orthoreoviruses. In the current report, we provide experimental evidence that the putative p13 FAST protein of PRV lacks the defining feature of the FAST protein family, the ability to induce syncytium formation. Instead, p13 is the first example of a cytosolic, integral membrane protein encoded by ortho- or aquareoviruses, and it induces cytotoxicity in the absence of cell-cell fusion. Sequence analysis also identifies signature motifs of the outer-clamp and fiber proteins of other reoviruses in two of the predicted PRV gene products. Based on these findings, we conclude PRV does not encode a FAST protein and is therefore unlikely to be a new fusogenic reovirus. The presence of a novel integral membrane protein and two previously unrecognized, essential outer-capsid proteins has important implications for the biology, evolution, and taxonomic classification of this virus. J Gen Virol. 2013 Jan Key T, Read J, Nibert ML, Duncan R. Dalhousie University