Bioinformatics of Recent Aqua- and Orthoreovirus Isolates from Fish: Evolutionary Gain or Loss of FAST and Fiber Proteins and Taxonomic Implications

Duncan R (2013) Bioinformatics of Recent Aqua- and Orthoreovirus Isolates from Fish: Evolutionary Gain or Loss of FAST and Fiber Proteins and Taxonomic Implications. PLoS ONE 8(7): e68607. doi:10.1371/journal.pone.0068607 Bioinformatics of Recent Aqua- and Orthoreovirus Isolates from Fish: Evolutionary Gain or Loss of FAST and Fiber Proteins and Taxonomic Implications Max L. Nibert 0 Roy Duncan 0 Earl G Brown, University of Ottawa, Canada 0 1 Department of Microbiology and Immunobiology, Harvard Medical School , Boston , Massachusetts, United States of America, 2 Department of Microbiology and Immunology, Department of Biochemistry and Molecular Biology, and Department of Pediatrics, Dalhousie University , Halifax, Nova Scotia , Canada Family Reoviridae, subfamily Spinareovirinae, includes nine current genera. Two of these genera, Aquareovirus and Orthoreovirus, comprise members that are closely related and consistently share nine homologous proteins. Orthoreoviruses have 10 dsRNA genome segments and infect reptiles, birds, and mammals, whereas aquareoviruses have 11 dsRNA genome segments and infect fish. Recently, the first 10-segmented fish reovirus, piscine reovirus (PRV), has been identified and shown to be phylogenetically divergent from the 11-segmented viruses constituting genus Aquareovirus. We have recently extended results for PRV by showing that it does not encode a fusion-associated small transmembrane (FAST) protein, but does encode an outer-fiber protein containing a long N-terminal region of predicted a-helical coiled coil. Three recently characterized 11-segmented fish reoviruses, obtained from grass carp in China and sequenced in full, are also divergent from the viruses now constituting genus Aquareovirus, though not to the same extent as PRV. In the current study, we reexamined the sequences of these three recent isolates of grass carp reovirus (GCRV)-HZ08, GD108, and 104-for further clues to their evolution relative to other aqua- and orthoreoviruses. Structure-based fiber motifs in their encoded outer-fiber proteins were characterized, and other bioinformatics analyses provided evidence against the presence of a FAST protein among their encoded nonstructural proteins. Phylogenetic comparisons showed the combination of more distally branching, approved Aquareovirus and Orthoreovirus members, plus more basally branching isolates GCRV104, GCRV-HZ08/ GD108, and PRV, constituting a larger, monophyletic taxon not suitably recognized by the current taxonomic hierarchy. Phylogenetics also suggested that the last common ancestor of all these viruses was a fiber-encoding, nonfusogenic virus and that the FAST protein family arose from at least two separate gain-of-function events. In addition, an apparent evolutionary correlation was found between the gain or loss of NS-FAST and outer-fiber proteins among more distally branching members of this taxon. - Funding: Funding provided by Canadian Institutes of Health Research grant numbers MOP-13723 and MOP-100584, and the Natural Sciences and Engineering Research Council of Canada grant number OGP1083745. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Family Reoviridae, subfamily Spinareovirinae (turreted reoviruses) includes nine approved genera, two of whichAquareovirus and Orthoreoviruscomprise members that are closely related and consistently share nine homologous proteins. Members of the five approved species in Orthoreovirus have 10 dsRNA genome segments and infect reptiles, birds, and mammals; members of the seven approved species in Aquareovirus have 11 dsRNA genome segments and infect fish and putatively shellfish [1]. Despite these differences in segment number and host range, ortho- and aquareoviruses share homologous proteins encoded by nine of their 10 or 11 genome segments [25] as well as highly similar particle structures [69]. Seven of their nine homologous proteins are structural, i.e., assembled into virions (core RNA-dependent RNA polymerase [RdRp], core nucleoside triphosphate phosphohydrolase [NTPase], core shell, core turret, core clamp, outer shell, and outer clamp), and the other two are non-structural (NS) proteins required for replication and assembly inside cells (NS factory and NS RNA-binding [RNAb]) (Tables 1, 2, and S1). Ortho- and aquareoviruses are thus likely to have shared a common viral ancestor from which these nine genome segments and their encoded proteins were inherited [2]. Ortho- and aquareovirus proteins that are not consistently homologous across the two genera include two proteins of clear biological significance. One is the outer-fiber protein present in most orthoreoviruses, which anchors atop the core-turret protein at the icosahedral fivefold axes of virions [6,10,11] and mediates attachment to cell-surface receptors [1216]. The other is the NS fusion-associated small transmembrane (FAST) protein of aquareoviruses and most orthoreoviruses [17,18], which promotes cellto-cell spread by fostering syncytium formation and release of progeny virions via syncytium-induced cytopathic effects [19,20] (Tables 1, 2, and S1). In members of approved Orthoreovirus species, Length (and size rank) of encoding genome segments for representative strains of Aquareovirus and Orthoreovirus species:a aRepresentative strains are Aquareovirus A, strain Scophthalmus maximus reovirus (AqRV-A); Aquareovirus C, strain Golden shiner reovirus (AqRV-C); Aquareovirus G, strain AGCRV-PB01-155 (AqRV-G); tentative Aquareovirus species, strain GCRV-HZ08; tentative Aquareovirus species, strain GCRV104; tentative Orthoreovirus species, strain Reovirus Salmo/GP-2010/NOR (PRV); Mammalian orthoreovirus, strain Type 1 Lang (MRV); Avian orthoreovirus, strain 176 (ARV); Baboon orthoreovirus strain Baboon reovirus (BRV); and tentative Orthoreovirus species, strain Broome virus (BroV). See Table S1 for GenBank accession numbers. bThese proteins are consistently homologous across both genera. doi:10.1371/journal.pone.0068607.t001 the single genome segment not shared by aquareoviruses is the one that encodes either the outer-fiber protein (in Mammalian orthoreovirus isolates [MRVs]) or the NS-FAST protein (in the Baboon orthoreovirus isolate [BRV]), or both (in Avian orthoreovirus, Nelson Bay orthoreovirus, and Reptilian orthoreovirus isolates [ARVs, NBVs, and RRVs, respectively]) [2123]. Another NS protein is also encoded on this segment in members of approved Orthoreovirus species except RRVs. In members of approved and fully sequenced Aquareovirus species (Aquareovirus A, Aquareovirus C, and Aquareovirus G isolates [AqRVs-A, -C, and -G, respectively]), the two genome segments not shared by orthoreoviruses encode three different NS proteins including the FAST protein [2,4,5,24,25] (Tables 1, 2, (...truncated)