Complete Characterisation of the American Grass Carp Reovirus Genome (Genus Aquareovirus: Family Reoviridae) Reveals an Evolutio

Home , Aquareovirus, Coltivirus, Kadipiro virus, Liao ning virus, Mimoreovirus, Seadornavirus

View metadata, citation and similar papers at core.ac.uk brought to you by CORE

provided by Elsevier - Publisher Connector

Available online at www.sciencedirect.com

Virology 373 (2008) 310–321 www.elsevier.com/locate/yviro

Complete characterisation of the American grass carp reovirus genome (genus Aquareovirus: family Reoviridae) reveals an evolutionary link between aquareoviruses and coltiviruses☆

Fauziah Mohd Jaafar a, Andrew E. Goodwin b, Mourad Belhouchet a, Gwenn Merry b, Qin Fang d, Jean-François Cantaloube c, Philippe Biagini c, Philippe de Micco c, ⁎ Peter P.C. Mertens a, Houssam Attoui a,

a Department of Arbovirology, Institute for Animal Health, Pirbright, Woking, Surrey, GU24 0NF, UK b University of Arkansas at Pine Bluff, Aquaculture/Fisheries Center, 1200 N. University Dr., Mail Slot 4912, Pine Bluff, AR 71601, USA c Unité de Virologie Moléculaire, Etablissement Français du sang Alpes-Méditerranée, 149 Boulevard Baille, 13005 Marseille, France d State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, 430071, China Received 3 September 2007; returned to author for revision 27 November 2007; accepted 6 December 2007 Available online 14 January 2008

Abstract

An aquareovirus was isolated from several fish species in the USA (including healthy golden shiners) that is not closely related to members of species Aquareovirus A, B and C. The virus, which is atypical (does not cause syncytia in cell cultures at neutral pH), was implicated in a winter die- off of grass carp fingerlings and has therefore been called ‘American grass carp reovirus’ (AGCRV). Complete nucleotide sequence analysis of the AGCRV genome and comparisons to the other aquareoviruses showed that it is closely related to golden ide reovirus (GIRV) (N92% amino acid [aa] identity in VP5(NTPase) and VP2(Pol)). However, comparisons with grass carp reovirus (Aquareovirus C) and chum salmon reovirus (Aquareovirus A) showed only 22% to 76% aa identity in different viral proteins. These findings have formed the basis for the recognition of AGCRV and GIRV as members of a new Aquareovirus species ‘Aquareovirus G’ by ICTV. Further sequence comparisons to other members of the family Reoviridae suggest that there has been an ‘evolutionary jump,’ involving a change in the number of genome segments, between the aquareoviruses (11 segments) and coltiviruses (12 segments). Segment 7 of AGRCV encodes two proteins, from two distinct ORFs, which are homologues of two Coltivirus proteins encoded by genome segments 9 and 12. A similar model has previously been reported for the rotaviruses and seadornaviruses. © 2007 Elsevier Inc. All rights reserved.

Keywords: Aquareovirus; Coltivirus; Reoviridae; dsRNA virus; Grass carp reovirus; Golden shiner reovirus; Golden ide reovirus; American grass carp reovirus; Evolution

Introduction linear segments of dsRNA, packaged as exactly one copy of each genome segment per virus particle. The reoviruses that can infect The family Reoviridae contains fifteen recognised genera of aquatic species include members of the genera Aquareovirus viruses (Mertens et al., 2005; Attoui et al., 2006a). These and Mimoreovirus, both of which have eleven segmented ‘reoviruses’ have genomes that are composed of 9, 10, 11 or 12 genomes (Samal et al., 2005; Attoui et al., 2006a), and members of the genus Cardoreovirus, which have twelve-segmented genomes (Mari and Bonami, 1998; Zhang et al., 2004). ☆ Accession numbers: The sequences determined in this study have Genbank accession numbers EF589098-EF589110. Aquareovirus particles physically resemble those of other ⁎ Corresponding author. Fax: +44 14832322448. ‘turreted’ reoviruses by cryo-electron microscopy (in particular E-mail address: [email protected] (H. Attoui). the mammalian orthoreoviruses (MRV)). They are icosahedral

0042-6822/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.virol.2007.12.006 F. Mohd Jaafar et al. / Virology 373 (2008) 310–321 311 in symmetry and are composed of a turreted central ‘core’ (containing the virus genome) surrounded by a double-layered capsid (Shaw et al., 1996). Aquareovirus structure displays marked similarity to the intermediate subviral particles of mammalian othoreovirus, suggesting a close evolutionary relationship. However, aquareovirus particles lack the hemagglutinin spike (sigma-1 protein) of the orthoreoviruses (Shaw et al., 1996). Aquareoviruses were first isolated in the 1970s from North- American cyprinids and were initially referred to as ‘reovirus- like’ or ‘rotavirus-like’ aquatic viruses (Winton et al., 1987; Plumb et al., 1979). They have subsequently been found in a wide variety of aquatic animals, including molluscs, finfish and crustaceans. Although these viruses are often isolated from apparently healthy individuals, they can also cause significant clinical signs and even severe disease (Fang et al., 1989). Six Aquareovirus species have been recognised by the International Committee for the Taxonomy of Viruses (ICTV) (Aquareovirus A(AQRV-A)toAquareovirus F (AQRV-F)), although several other viruses have also been isolated, which may represent additional species (Samaletal.,2005;Mertens et al., 2005). The ICTV has recognised certain criteria for the demarcation of Aquareovirus species (Mertens et al., 2005). Members of a single species can be identified by (i) their ability Fig. 1. Electropherotype of the AGCRV genome. (A) Electron micrograph of the to reassort during dual infections; (ii) RNA sequence analyses trypsintreated sucrose-purified AGCRV particles. The particles show turrets (viruses within different species should have ‘low’ levels of which are typical of turreted reoviruses, including aquareoviruses. The bar represents 50 nm. (B) The left lane represents the electropherotype of AGCRV sequence homology between cognate genome segments, for (AQRV-G). It is compared in the right lane to the electropherotype of GCRV example, genome segment 10, which encodes major outer (AQRV-A). capsid protein VP7, shows nucleotide (nt) sequence variations of N45% and amino acid (aa) variations of N64%, between viruses from two different species); (iii) serological comparisons, using polyclonal or monoclonal antibodies (higher (96 to 99% amino acid identity) (Attoui et al., 2002a). GSRV is a levels of serological cross reactions between the members of significant pathogen of farmed grass carp and fathead minnows the same virus species); (iv) analysis of “electropherotype” by but has also been isolated from wild ‘creek chub’ in the USA agarose gel electrophoresis (AGE) (viruses within the same (Goodwin et al., 2006). species will normally show a uniform electropherotype); and We report the full-genome sequence of American grass carp (v) identification of shared ‘conserved terminal regions’ of the reovirus (AGCRV), a member of new Aquareovirus species genome segments (although some closely related species can (Aquareovirus G) isolated in the USA between 2001 and 2004, also have identical terminal sequences on at least some from grass carp and golden shiner. This virus was implicated in segments). a winter die-off of grass carp fingerlings on a commercial farm Aquareoviruses can be grown in fish cell lines, usually in Arkansas in the USA during 2005. Phylogenetic analyses producing large syncytia as a characteristic cytopathic effect. indicate that golden ide reovirus (GIRV, isolated in Germany by They represent a serious threat to fish breeding and typical Neukirch et al., 1999) represents a second isolate of Aquareo- clinical signs of infection include haemorrhages, which can be virus G. severe (Fang et al., 1989). For example grass carp reovirus The sequence analyses described here permit to hypothesise (GCRV — a strain of AQRV-C) causes an important disease in that there has been an evolutionary “jump” that involves a Asia, characterised by severe haemorrhage and up to 80% change in the number of genome segments, between the mortality in fingerling and yearling grass carp (Fang et al., aquareoviruses (11 segmented) and members of the genus 1989). Striped bass reovirus (SBRV, species AQRV-A: the type Coltivirus (12 segmented arboviruses — family Reoviridae). species of the genus Aquareovirus) was isolated from a Genome segment 7 of the aquareoviruses encodes two distinct moribund striped bass (Morone saxatilis) that was also infected proteins, NS31 and NS16, from separate open reading frames with bacteria (Samal et al., 1990). Golden shiner reovirus arranged in series. Our model presents these as homologues to (GSRV) is a strain of Aquareovirus C that was originally isolated two proteins from Colorado tick fever virus (VP9 and VP12), in 1977 from a moribund golden shiner bait fish (Notemigonus which are encoded by genome segments 9 and 12, respectively. crysoleucas)(Plumb et al., 1979). Although GSRV is associated The mechanisms that may have been involved in this with losses of bait fish in the USA (Plumb et al., 1979; McEntire evolutionary change (e.g., segment duplication followed by et al., 2003), it is nearly identical to a Chinese isolate of GCRV selective deletions) are discussed. 312

Table 1 Lengths of dsRNA segments 1 to 11, encoded putative proteins, 5′ and 3′ non-coding regions of GCRV, CHSRV and AGCRV .Mh afre l iooy33(08 310 (2008) 373 Virology / al. et Jaafar Mohd F. – 321

Segment 5 is the only full-length gene obtained from the golden ide reovirus. Highly conserved terminal sequences are shown in upper case letters. aTheoretical molecular mass in Daltons; Part: partial sequence. N.D: not determined. Segment 5 of AGCRV and GIRV, which have similar organisations, are both boxed. F. Mohd Jaafar et al. / Virology 373 (2008) 310–321 313

Results and discussion on a commercial farm, it was named ‘American grass carp reovirus’ (AGCRV). Virus propagation, titration and electron microscopy The AGCRV genome segments were analysed by AGE and separated into 7 distinct bands (Fig. 1B). Five of the genome AGCRV was propagated in FHM cells causing the cells to segments (segments 4, 5, 6, 10 and 11) migrate individually, round-up and detach from the plastic flask. Upon the while bands 1 and 5 contain 3 co-migrating segments each acidification of the culture medium (pH 5.5, by adding 0.01% (segments 1–3 and segments 7–9, respectively). The sequences citric acid), the virus caused progressive cell fusion. The viral of genome segments 1 to 11 of AGCRV were determined and 7 titre determined at 7 days pot-infection was ∼10 TCID50/ml. have been deposited in the Genbank database under accession The deoxycholate-treated or the Freon 113/Vertrel XF- numbers EF589098–EF589108. The lengths of individual treated virus did not show any drop of infectivity. Virus titres of genome segments and of the proteins they encode are given in 7 10 TCID50 were still detected after treatment. Table 1. The trypsin-treated and sucrose-purified virus from infected culture supernatant showed turrets (Fig. 1A) which are typical Comparison of the non-coding regions of AQRV genome of the turreted members of family Reoviridae, including the segments aquareoviruses. Analysis of 3′ and 5′ non-coding regions (NCRs) showed The AGCRV genome sequence that all of the segments share 6 conserved nucleotides at their 5′ ends and 5 conserved nucleotides at their 3′ ends (5′- U U U The purified genomes of the four isolates were analysed on a GUUUUA /a—— /a /aUCAUC-3′). These are identical to 1.2% agarose gel. The isolates showed an identical migration those of GIRV genome segment 5 (5′-GUUUUAU—— pattern (electropherotype) after agarose gel electrophoresis UUUCAUC-3′)(Attoui et al., 2002a). They are also identical U (AGE) (data not shown), indicating that they belong to the to those of CHSRV (AQRV-A) (5′-GUUUUA /G A same species (based on the Aquareovirus species parameters), —— /UUCAUC-3′) but show two differences at positions 4 although there could be slight sequence variations between the and6(AtoUandUtoAchanges)intheupstreamconserved isolates. Isolate PB01–155 was selected as a representative of terminus when compared to those of GCRV/GSRV (AQRV-C) U A the group of the 4 isolates with identical electropherotypes, for (5′-GUUAUU /G—— /UUCAUC-3′). As with other aqua- further sequence analyses. Since this virus was isolated from reoviruses, the first and last nucleotides of each segment of grass carp and was implicated in a winter die-off of fingerlings AGCRV are inverted complements (Table 1).

Fig. 2. Neighbour-joining phylogenetic trees. (A) Neighbour-joining phylogenetic tree built with the amino acid sequence of the major outer capsid protein VP7 (segment 10) of aquareoviruses. The bootstrap values are shown at the nodes. Panel B illustrates the phylogenetic relationships that exist between the various members of the Reoviridae, based on the sequence of their putative RNA-dependent RNA polymerase. Scale bars at lower left of the trees indicate the number of substitutions per site. 314 F. Mohd Jaafar et al. / Virology 373 (2008) 310–321

The genome segments of most member viruses of the family aquareoviruses, the amino acid identities between homologous Reoviridae that have been analysed all have their first and last proteins are comprised between 71 and 98% (Attoui et al., 2002a). mono- di- or tri-nucleotides as inverted complements (Anzola The genome sequence of AGCRV was compared to available et al., 1987; Chen and Patton, 1998; Mertens and Sangar, 1985; sequences of chum salmon reovirus (CHSRV: AQRV-A), coho Patton, 2001; Potgieter et al., 2002, Xu et al., 1989; Attoui et al., salmon reovirus (CSRV: AQRV-B), grass carp reovirus and 1998, 2000a,b, 2001a,b, 2002a,b, 2005a,b, 2006a,b). Con- golden shiner reovirus (GCRV and GSRV: AQRV-C). Overall served terminal sequences have been reported for all members identity values ranging from 22 to 76% were detected between of the Reoviridae (Mertens et al., 2005 — www.iah.bbsrc.ac.uk/ homologous proteins (Supplemental Table A). However, when dsRNA_virus_proteins/CPV-RNA-Termin.htm). It has been AGCRV was compared to GIRV (as yet unclassified), much suggested that the complementary nature of sequences in the 5′ higher levels of amino acid identity were detected (98% in the and 3′ NCRs could hold each RNA transcript in a circular form viral polymerase (VP2) and 92% with the NTPase (VP5), (a panhandle configuration), either by itself or via interactions encoded by segment 2 and segment 5 of GIRV respectively). with protein components, particularly the replication complex These values are consistent with GIRVand AGCRV belonging to (Anzola et al., 1987; Theron and Nel, 1997; Chen and Patton, the same ‘novel’ virus species. A formal proposal to recognise this 1998; Patton, 2001). Conserved sequences at the 5′ and 3′ ends species as ‘Aquareovirus G’ (AQRV-G) has been made to ICTV. may also act as a part of the ‘sorting’ signals during genome Comparison of the electropherotypes of AQRV-G isolates to assembly and packaging, bringing a single copy of each genome those of Channel catfish reovirus (AQRV-D) (Hedrick et al., segment into the nascent viral capsid (Anzola et al., 1987; Xu et 1984), Turbot reovirus (AQRV-E) and chum salmon reovirus al., 1989). PSR (AQRV-F) (Subramanian et al., 1997), confirmed the distinct nature of AQRV-G. Comparison of AGCRV to the members of other Aquareovirus Most AGCRV genome segments have a single long ORF that species spans almost their entire length (Table 1). Two different versions of segment 7 exist within the genus Aquareovirus. Aquareoviruses are currently classified within 6 species One version exists in AQRV-A, which contains only a single (Aquareovirus A to Aquareovirus F). Within a single species of large ORF (encoding NS49). The other version was initially

Table 2 Comparison of the function roles of AGCRV and MRV genome segments AGCRV MRV Segment [protein] Function a Segment [protein (role)] AA identity to cognate AGCRV protein Segment 1 [VP1] Capping enzyme (Cap) L2 [lambda2(Cap) 27% Segment 2 [VP2] Polymerase (Pol) L1 [lambda3(Pol)] 41% Segment 3 [VP3] Helicase, NTPase L3 [lambda1(Hel)] 32% (T2 protein) (Hel) Segment 4 [NS73] Non-structural, possibly M3 [mu-NS (Vib)]: mu-Ns interacts 25% involved in the formation with sigma-Ns of viral inclusion bodies in the viral inclusion bodies (Vibs) with NS38 Segment 5 [VP5] NTPase M1 [mu-2(associates with microtubules)] 25% Segment 6 [VP4] Outer capsid M2 [mu-1, mu-1c 25% (incomplete T13 layer)] Segment 7 [NS31], [NS16] Non-structural S1 [sigma-1 22% (Sigma-1) b; 23% (cell attachment, (Sigma-1S) type specific antigen) and sigma-1S] [segment 7 of both AGCRV and MRV is bi-cistronic] Segment 8 [VP6] Core protein S2 [sigma2] 23% Segment 9 [NS38] Non-structural, possibly the S3 [sigma-NS(Vib)]: sigma-NS interacts 22% primary determinant of viral with mu-NS for the formation of VIBs inclusion bodies Segment 10 [VP7] Cell attachment, S4 [sigma3 (dsRNA binding)] 30% [Similar hydrophobicity profiles serotype control and common aa motifs between VP7 of aquareoviruses and sigma3 of MRV (Attoui et al., 2002a)] Segment 11 [NS26] Non-structural No equivalent Within family Reoviridae, amino acid identity values higher than 20% are considered as significant, indicating clear homology between proteins. Non-homologous proteins of family Reoviridae share less than 9% amino acid identity. a Attoui et al. (2002a). Information on aquareovirus proteins is available at www.iah.bbsrc.ac.uk/dsRNA_virus_proteins. b Sigma 1 is the viral hemagglutinin. Aquareovirus particles lack the hemagglutinin spike observed in orthoreoviruses (Shaw et al., 1996). F. Mohd Jaafar et al. / Virology 373 (2008) 310–321 315

Fig. 3. Alignment and hydrophobicity profiles of the VP12 of coltiviruses and the NS16 of aquareoviruses (identical or highly similar residues are shown in red). (A) Alignment of the full-length amino acid sequence of the VP12 (accession number U53227) protein of CTFV (Colorado tick fever virus) and EYAV (Eyach virus), and a comparison to that of NS16 of AGCRV (American grass carp reovirus), GCRV (grass carp reovirus) and GSRV (golden shiner reovirus). Identical or similar residues (between proteins of aquareoviruses and coltiviruses) in the alignments are shown in red. Similar residues are defined as those that preserve the chemical character or the structural character of the amino acid. (B) The grey plot represents the hydrophobicity profile of VP12 of CTFV. The black plot represents the hydrophobicity profile of NS16 of AGCRV. The blue plot represents the hydrophobicity profile of the p14 protein of reptilian orthoreovirus. The thick red bar indicates the trans-membrane domain (residues 36–68) of NS16 (48 to 76) of the VP12, were predicted by Winpep software program (Hennig, 2001). The p14 of reptilian orthoreovirus also contains a trans-membrane domain (positions 39–57). The aquareovirus NS16, CTFV VP12 and reptilian orthoreovirus P14 all contain a proline-rich motif towards the COOH terminus of the proteins. identified in AQRV-C (GCRV and GSRV), which contains two (identified as NS31 — based on molecular mass) also shows separate ORFs (encoding NS16 and NS31). Segment 7 of 45% amino acid identity to NS31 of AQRV-C (GSRV and AGCRV was found to contain two distinct ORFs (separated by GCRV) and 24% identity to the carboxy terminal two thirds of a 17-bp NCR), indicating that this segment encodes two distinct the NS49 protein of AQRV-A (CHSRV). Comparisons of NS16 proteins (NS16 and NS31). and NS31 proteins encoded by the two distinct ORFs of The cryo-electron microscopy analyses of GCRV showed segment 7 from AGCRV and AQRV-C therefore identified a that expression products of segment 7 are non-structural single homologue among the proteins of AQRV-A (NS49, proteins and were not identified in the purified particles (Fang encoded by the single large ORF of segment 7 of CHSRV). et al., 2005). The upstream ORF of AGCRV segment 7 (bases Available data from AQRV-A do not provide any evidence of 15 to 423) encodes a protein (identified as NS16 — based on its any post-translational modification of NS49 (particularly theoretical molecular mass), which not only shows 64% aa cleavage). Preliminary expression work for the viral dsRNA identity with NS16 of AQRV-C (GSRV and GCRV), but also segments showed that NS16 and NS31 are both translated from 29% identity with the amino terminal third of NS49, translated the RNA template (data not shown). from the single ORF segment 7 from AQRV-A (CHSRV). The Partial nucleotide sequences (451 bp, including the 5′ non- protein encoded by the downstream ORF of AGCRV segment 7 coding region — accession numbers EF589109 and EF589110) 316 F. Mohd Jaafar et al. / Virology 373 (2008) 310–321 were generated for segment 2 (the polymerase gene) from isolates VP7 amino acid sequences, shows that although AGCRV PB04–151 and PB04–123, respectively. These sequences show clusters separately from other aquareoviruses (Fig. 2A). 99.3% and 99.1% nt identity with AGCRVand 99.8% with each other (with 100% aa identities in each case). This supports Comparison of AGCRV to orthoreoviruses, coltiviruses and previous indications (from electropherotype analysis) that the 4 other reoviruses isolates (PB02–24, PB04–123, PB04–151, PB01–155) all belong to the same virus species. Previous studies of the genus Seadornavirus (12 segmented Comparisons of the outer capsid proteins of different species dsRNA genome) have shown that the isolates of species Banna within a single genus of reoviruses can show very high levels of virus, Kadipiro virus and Liao ning virus show amino acid variation (Mertens et al., 2005). For example amino acid identities of 18% to 42% (maximum) between their homo- sequence identity values of only 23% have been observed logous proteins (Attoui et al., 2000a, 2005b, 2006b). between members of different species within the genus Orbi- Comparative studies of the family Reoviridae have shown virus, 18% between species of Seadornavirus and 19% between that amino acid identity levels of ≥20% are often detected species of Orthoreovirus (Attoui et al., 2001a,b, 2006a,b). between proteins that are clearly homologous (based on their Aquareovirus segment 10 encodes the major outer capsid structural or functional roles) from the members of different protein VP7. VP7 of American grass carp reovirus was com- genera of reoviruses. Such levels of identity are therefore pared with those from different isolates of striped bass reovirus, regarded as significant, and an indication of common ancestry chum salmon reovirus and threadfin reovirus (AQRV-A), coho within the family as a whole (Attoui et al., 2002a,b, 2005a,b; salmon reovirus (AQRV-B), grass carp reovirus and golden Mertens et al., 2005). Non-homologous proteins of closely shiner reovirus (AQRV-C). The results showed 14 to 99.6% aa related members of the family Reoviridae share usually less identity overall, with N79% aa identity within a single species. than 9% amino acid identity. Amino acid identities of 79.2 to 86.6% were detected between All of the AGCRV genome segments appear to have the different isolates of AQRV-A: 95.8–99.6% within AQRV-C: homologues in the genome of MRV, showing remarkable levels but only 14 to 16% between AQRV-A and C, and 18.1 to of amino acid sequence identity in the proteins they encode 18.5%, between AQRV-B and C. Comparisons of segment 10 (amino acid identity levels of N22%, up to 41% in the from American grass carp reovirus with AQRV-A, B or C, gave polymerase — Table 2). In addition, genome segment 7 of identity values in VP7 of only 16 to 18%, 17%, or 24 to 26% both viruses has a bicistronic organisation. Genome segment 10 respectively, confirming other indications that AGCRV repre- encodes VP7 of the aquareoviruses and sigma-3 of MRV, sents a distinct species. A phylogenetic tree, constructed with respectively, which have similar hydrophobicity profiles and

Fig. 4. Diagrammatic representation of the evolutionary model that the ancestral segment 7 followed to give rise to segment 7 of CHSRV (AQRV-A), GCRV (AQRV-C), AGCRV (AQRV-G), MRVand segments 9 and 12 of CTFV.The higher levels of sequence homology detected between the translation products of segment 9 and segment 12 from the coltiviruses, with those from the two ORFs of AQRV-G segment 7, than with the product of the single ORF from AQRV-A, support the hypothesis of insertion of a non-coding region and separation of the long ORF in the ancestral segment into 2 ORFs. The previously published co-speciation hypothesis (Attoui et al., 2002a) suggested that the ancestral virus is a marine virus, from which the mammalian viruses (presented in this diagram) possibly evolved. : non-coding regions. F. Mohd Jaafar et al. / Virology 373 (2008) 310–321 317 common amino acid motifs (Attoui et al., 2002a,b). Only major product), and the ‘read-through’ protein VP9′ (minor Segment 11 of the aquareoviruses appears to have no homologue product) in infected cells and by the expression from a cDNA in MRV. Comparisons of AGCRV and MRV support the copy of segment 9, under a CMV promoter, in mammalian cells previous published data and the conclusion that there is a (Mohd Jaafar et al., 2004). relatively close relationship between the orthoreoviruses and The coding regions, before and after the central stop codon in aquareoviruses indicating that they were derived from a genome segment 9, have similar sizes. A further comparison of common ancestor more recently than some of the other more these coding regions indicates that the portion after the stop distantly related genera of reoviruses (Attoui et al., 2002a). codon is an almost complete duplicate of the entire sequence The comparison of AGCRV proteins to those of members before the stop codon. This is demonstrated by the presence of of the Reoviridae other than orthoreoviruses showed that many conserved amino acid motifs, at the beginning, middle amino acid identities between cognate proteins (identified on and end of VP9, when compared to the amino acid sequence of the bases of their function) are not significant (less than 9%). VP9′ beyond the stop codon (data not shown). The amino acid It has previously been suggested that aquareoviruses and identity between the 2 parts of the VP9′ (i.e., VP9 compared to orthoreoviruses and their respective hosts have evolved by co- VP9′ minus VP9) was 35%. Sequence duplication and speciation (Attoui et al., 2002a). As a model, the ancestral accumulation of point mutations in the duplicated region, homologue of non-structural protein NS31 could have leading to increased genetic diversity and protein length, have acquired new domains by heterologous recombination, thereby previously been reported for other viruses including some providing the orthoreovirus ancestor with mammalian cell members of family Reoviridae (Attoui et al., 2006a). Duplica- attachment domains. Heterologous recombination has pre- tion of the VP9 sequence could have originated by concatemer- viously been identified in some other reoviruses, particularly isation, followed by deletion of the junction region within the between Eyach virus (genus Coltivirus) and its major host, the concatamer. Concatamerisation, involving duplication of the European rabbit (Attoui et al., 2002b). entire sequence within a single genome segment, with or An amino acid alignment of NS16 (encoded by segment 7) without subsequent internal deletions, has previously been from AGCRV, with VP12 (encoded by segment 12) of Colorado reported for orbiviruses (Eaton and Gould, 1987; Anthony, tick fever virus (CTFV) indicates that these proteins are 2007) orthoreoviruses (Desselberger, 1996), rotaviruses (Tian homologues with an amino acid identity level of 30% (aa et al., 1993) and phytoreoviruses (Murao et al., 1996). similarity 41%) and similar hydrophobicity profiles (Figs. 3A Internal similarities were also identified in the amino acid and B). The software program ‘Winpep’ predicts a trans- sequence of VP9 itself, which potentially represent the signature membrane domain in NS16 and VP12 (at aa 36–68 and aa 48 to of another more ancient sequence duplication. For instance, a 76 respectively), and hydrophobic patches (aa 1–20), suggest- sequence of 70 aa (between aa 121 and aa 179) is likely to have ing that they are both membrane proteins. been generated by duplication of the sequence between aa 18 Genome segment 7 of reptilian orthoreovirus also encodes and 79, followed by diversification, resulting in 34% aa identity two proteins from two distinct open reading frames (Corcoran in these regions. and Duncan, 2004; Salsman et al., 2005). The upstream open The VP9 protein encoded by segment 9 of CTFV is signi- reading frame (nt 25–399) encodes a type III fusion protein ficantly longer (∼68 aa) than NS31 encoded by the downstream designated p14, which is responsible for syncytium formation ORF of AGCRV segment 7. The internal similarities within by this virus (Corcoran and Duncan, 2004; Salsman et al., VP9 make it difficult to align its entire amino acid sequence 2005). The p14 protein which also has a trans-membrane with that of NS31 (data not shown). However, the amino and domain (positions 37–57) and a hydrophobic patch between carboxy terminal regions of VP9 show 31% and 35% aa positions 8 and 21 has a polyproline sequence towards its identity, respectively, to a comparable region of NS31 (data not carboxy terminus (aa 99 to 112). Similar sequences were shown). The hydrophobicity profile of these proteins is also identified in NS16 and VP12 (126–138 and 136–146, similar for both their carboxy and amino terminal regions, respectively — Fig. 3). These similarities suggest that NS16 although the additional sequence (duplication) in the middle of and VP12 may also be fusion proteins, which would be VP9 makes comparison of this central region to NS31 compatible with the characteristics of AGCRV and its ability to impossible. induce cell fusion (under mildly acidic conditions). Colorado A diagrammatic representation of the model of evolution of tick fever virus (CTFV, genus Coltivirus) can also induce cell the ancestral segment 7 and its diversification into various forms fusion in Vero or BHK-21 cell cultures after acidification of the (segment 7 of AQRV-A, AQRV-C, AQRV-G, MRV and seg- cell culture media (Attoui, 1998). Previous studies have ment 9 and 12 of CTFV) is shown in Fig. 4. indicated that acidification may be necessary for the induction of syncytia formation in cells infected by many different viruses Comparison of the reovirus polymerases (Castilla et al., 1994; Seth et al., 2003; Forzan et al., 2004). Previous analyses of the nucleotide sequence of Coltivirus Phylogenetic comparisons, using sequences of the viral genome segment 9 have revealed the presence of a “leaky” stop RNA-dependent RNA polymerase (RdRp), can be used to dis- codon (Attoui et al., 2002b). Read-through of this codon was tinguish the genera of turreted and non-turreted reoviruses, as confirmed experimentally by expression studies, which demon- two distinct clusters (Attoui et al., 2006a — Fig. 2B). Previous strated production of both the ‘termination’ protein VP9 (the studies have also indicated that most reoviruses from a single 318 F. Mohd Jaafar et al. / Virology 373 (2008) 310–321 genus will have amino acid identity N30% in the polymerase these segments with partial deletions are viable, including a (Attoui et al., 2002a,b, 2005a). truncated version of CTFV segment 12 (Attoui, 1998). The amino acid sequence of the viral polymerase was The clear monophyletic but genetically very diverse nature compared for representative isolates of nine distinct genera of of the family Reoviridae provides a unique opportunity to reoviruses, showing that they are all clearly distinct (illustrated examine and explore the mechanisms that have been involved in by a radial Neighbour-Joining tree — Fig. 2B). Previous studies the evolution of a complex (multisegmented) RNA virus have indicated that the members of a single Aquareovirus genome over very long periods of time (millions of years). species will show N95% aa sequence identity in the viral Although the levels of sequence identity that can be detected polymerase (Attoui et al., 2002a). AGCRVand GIRV (which are between the homologous proteins of the different reoviruses are both currently unclassified) show 98% aa identity in the often low by the standards of other genetic groups, they are very polymerase, but only 57 to 74% aa identities with the strongly supported by the similar functional and structural polymerases of other aquareoviruses, indicating that they belong motifs or roles of the individual viral proteins. This has made it to the same novel virus species (which is proposed as AQRV-G, possible to clearly identify evolutionary jumps that have and has received initial support from ICTV). resulted in changes in the numbers of genome segments and The highest level of amino acid sequence identity that was the overall size of the genome, between the different species and detected between the polymerase of AGCRVand a member of a genera within the family. Although the number of genome distinct genus of reovirus was 41% to the polymerase of MRV, segments clearly varies between the different reoviruses, supporting previous indications that these genera are closely functional constraints, the overall size of the particle itself and related (derived from a common ancestor, ∼510 MYA — the possible limit of a maximum of 12 genome segments that Attoui et al., 2002a). Although this value of amino acid identity may be imposed by the number of icosahedral vertices of the is higher than that separating genera, the classification of the particle (number of transcriptase complexes) appear to provide aquareoviruses and orthoreoviruses as members of two distinct limits to the ‘evolutionary space’ that these viruses can explore. but closely related genera is based on multiple parameters and not simply their genetic relatedness (Attoui et al., 2002a,b). For Materials and methods example, the aquareoviruses can infect many marine and freshwater species, while the orthoreoviruses primarily infect Isolation, propagation and titration of the viruses mammals, birds and reptiles. The common origins of these viruses, and of their respective hosts, suggest ‘co-speciation’ of Four aquareovirus isolates (PB02–24, PB04–123, PB04– the viruses with their respective hosts (Attoui et al., 2002a). 151 and PB01–155) were made from grass carp and golden shiner. Isolate PB04–151 was contributed by Ron Hedrick Conclusion (Hedrick et al., 1989). Tissue lysates from these fish were inoculated into confluent monolayers of Fathead minnow The topology of the phylogenetic tree constructed using the (FHM) cells in L-15 medium supplemented with 10% foetal viral polymerase amino acid sequences (Fig. 2B) indicates that bovine serum and incubated at 22 °C. Isolate PB04–144 the coltiviruses (12 segmented) have a common phylogenetic produced syncytia in inoculated FHM, while the remaining origin with the aquareoviruses (11 segmented) and orthoreo- isolates caused only rounding of the cells. Isolates were viruses (10 segmented). expanded by growth on FHM cells in Eagles minimum essential A model of genetic ‘jump’, involving a change in the number medium with Hank's salts (EMEM) incubated at 30 °C. of genome segments, has previously been reported between Clarified supernatants from virus-infected cell cultures were rotaviruses (eleven segmented, horizontally transmitted viruses) subjected to ultracentrifugation at 36,000 rpm using an SW41 and the seadornaviruses (12 segmented arboviruses) (Mohd rotor. The pelleted viruses were re-suspended in 200 μlof Jaafar et al., 2005a,b). Comparisons of AGCRV sequence with EMEM and their RNA extracted using RNA Now reagent other reoviruses identified VP12 and VP9 proteins of the (Biogentex), as previously described (Attoui et al., 2000c). The coltiviruses (translated from genome segments 12 and 9, respec- dsRNA segments were separated by 1% agarose gel electro- tively), as homologues of NS16 and NS31 from AGCRV. This phoresis (AGE). Individual RNA bands were cut from the gel suggests that another evolutionary ‘jump’, involving a change in and the dsRNA purified using the RNaid kit (Qbiogen). the number of genome segments, occurred at some time in the The virus (from the supernatant) titre (TCID50) was assayed past between the aquareoviruses (11 genome segments) and the in 96-well microtitre plates containing FHM cells at 7 days post- coltiviruses (12 genome segments). This hypothesis reflects a infection. genetic rearrangement during the evolution of these two genera The virus from the culture supernatant was treated with from a single common ancestor. The mechanism by which this organic solvent including Freon113 and Vertrel XF. Briefly, genetic reorganisation occurred may have involved duplication equal volumes of the suspension and the solvent were mixed by of a genome segment 7 followed by deletions to generate two vortexing for 5 min. The mixture was centrifuged at 2000×g at 8 separate and distinct segments in the ancestral Coltivirus strain. °C for 10 min and the supernatant was recovered and titrated by Deletions in dsRNA genome segments are frequently observed TCID50. as truncated dsRNA segments (Ahmed and Fields, 1981; Lee Aliquots of the virus from the supernatant were treated with et al., 1986; Anzola et al., 1987; Ni and Kemp, 1994). Some of 1% sodium deoxycholate. Briefly, 1 ml of the clarified F. Mohd Jaafar et al. / Virology 373 (2008) 310–321 319 supernatant was treated with an equal volume of 2% sodium segments 1 to 11 of AGCRV with other AQRVs was identified deoxycholate (final concentration 1%). After incubation for 1 h using the local BLAST software program implemented in the at room temperature (Auleltta and Marlowe, 1968), the mixture DNATools package (version 5.2.018, S.W. Rasmussen: Valby was diluted with an equal volume of fresh serum-free EMEM Data Center, Denmark). Analyses of protein hydrophobicity and the infectivity was assessed by titration on FHM cells. profiles (Kyte and Doolittle, 1982) were performed using the Winpep software program (Hennig, 2001). Analysis of protein Virus preparation for electron microscopy secondary structures was performed using the online “NNPRE- DICT” software program: http://alexander.compbio.ucsf.edu/ Infected cells were harvested at 10 days post-infection. The ~nomi/nnpredict.html. Coiled-coils structures were analysed clarified culture supernatant (centrifugation at 2000×g for using the software program COILS available online at http:// 10 min) was concentrated by ultrafiltration (MWCO of 5000; www.ch.embnet.org/cgi-bin/COILS_form_parser. Sartorius). The concentrated material was treated with 10 μg/ml trypsin (Samal et al., 1990) for 30 min at 37 °C and then Acknowledgment incubated with 1.5 M CaCl2 for 30 min at room temperature. The treated material was diluted in Tris–HCl 10 mM, pH 7.5 This work was supported by EU Grant ‘Reo ID’ number and layered over a discontinuous gradient made of 3 ml of 35% QLK2-2000-00143, by Defra and by BBSRC. W/V and 0.5 ml 66% W/W sucrose in Tris–HCl 10 mM. The tubes were centrifuged at 30,000 rpm in an SW40 rotor for Appendix A. Supplementary data 30 min. The material at the interface of the sucrose layers was recovered, diluted in 20 ml of Tris–HCl 10 mM and con- Supplementary data associated with this article can be found, centrated by ultrafiltration once again. in the online version, at doi:10.1016/j.virol.2007.12.006. The virus particles from concentrated material were adsorbed to formvar/carbon coated grids, stained with 2% potassium phosphotungstate for 30 s and dried prior to being examined by References electron microscopy. Ahmed, R., Fields, B.N., 1981. Reassortment of genome segments between Preparation and cloning of the dsRNA reovirus defective interfering particles and infectious virus: construction of temperature-sensitive and attenuated viruses by rescue of mutations from DI – The genome segments of the selected aquareovirus isolates particles. Virology 111, 351 363. ‘ ’ Anthony, S. 2007. Genetic studies of epizootic hemorrhagic disease viruses were copied into cDNA (using a single primer amplification EHDV. PhD dissertation. technique), then cloned and sequenced as previously described Anzola, J.V., Xu, Z., Asamizu, T., Nuss, D.L., 1987. Segment-specific inverted (Attoui et al., 2000c; Lambden et al., 1992). Briefly, a defined 3′- repeats found adjacent to conserved terminal sequences in wound tumour amino blocked oligodeoxyribonucleotide was ligated to both of virus genome and defective interfering RNAs. Proc. Natl. Acad. Sci. U. S. A. – the 3′ ends of the dsRNA segments, using T4 RNA ligase, 84, 8301 8305. Attoui, H. 1998. Etude biologique et moléculaire du genre Coltivirus. PhD followed by reverse transcription and PCR amplification using a dissertation. complementary primer. PCR amplicons were analysed by aga- Attoui, H., Charrel, R., Billoir, F., Cantaloube, J.F., de Micco, P., de Lamballerie, rose gel electrophoresis, ligated into the PGEM-T cloning vector X., 1998. Comparative sequence analysis of American, European and Asian (Promega) and transfected into competent XL-blue Escherichia isolates of viruses in the genus Coltivirus. J. Gen. Virol. 79, 2481–2489. coli. Insert sequences were determined using M13 universal Attoui, H., Billoir, F., Biagini, P., De Micco, P., de Lamballerie, X., 2000a. Complete sequence determination and genetic analysis of Banna virus and primers, the D-rhodamine DNA sequencing kit and an ABI prism Kadipiro virus: proposal for assignment to a new genus Seadornavirus 377 sequence analyser (Perkin Elmer). within the family Reoviridae. J. Gen. Virol. 81, 1507–1515. Attoui, H., Billoir, F., Cantaloube, J.F., Biagini, P., De Micco, P., de Lamballerie, Sequences retrieved from databases X., 2000b. Sequence determination and analysis of the full-length genome of Colorado tick fever virus, the type species of genus Coltivirus family Reoviridae. Biochem. Biophys. Res. Commun. 273, 1121–1125. Aquareovirus sequences and the RdRp sequences for other Attoui, H., Billoir, F., Cantaloube, J.F., Biagini, P., De Micco, P., de Lamballerie, members of family Reoviridae were obtained from the X., 2000c. Strategies for the sequence determination of viral dsRNA international databases (see Supplemental Table B for accession genomes. J. Virol. Methods 89, 147–158. numbers). Attoui, H., Stirling, J.M., Munderloh, U.G., Billoir, F., Brookes, S.M., Burroughs, J.N., de Micco, P., Mertens, P.P.C., de Lamballerie, X., 2001a. Complete sequence characterization of the genome of the St Croix River virus, a new Sequence comparison methods orbivirus isolated from cells of Ixodes scapularis. J. Gen. Virol. 82, 795–804. Attoui, H., Biagini, P., Stirling, J., Mertens, P.P.C., Cantaloube, J., Meyer, F., de Sequence alignments were performed using the Clustal W Micco, A., de Lamballerie, P., 2001b. Sequence characterization of Ndelle software program (Thompson et al., 1994). Sequence identities virus genome segments 1, 5, 7, 8, and 10: evidence for reassignment to the were calculated using MEGA 3.1 (Kumar et al., 2004). genus Orthoreovirus, family Reoviridae. Biochem. Biophys. Res. Commun. 287, 583–588. Neighbour-joining phylogenetic trees (Saitou and Nei, 1987) Attoui, H., Fang, Q., Mohd Jaafar, F., Cantaloube, J.F., Biagini, P., de Micco, P., were constructed using the P-distance or the Poisson correction de Lamballerie, X., 2002a. Common evolutionary origin of aquareoviruses models implemented in MEGA. The genetic relatedness of and orthoreoviruses revealed by genome characterization of golden shiner 320 F. Mohd Jaafar et al. / Virology 373 (2008) 310–321

reovirus, grass carp reovirus, striped bass reovirus and golden ide reovirus Mari, J., Bonami, J.R., 1998. W2 virus infection of the crustacean Carcinus (genus Aquareovirus, family Reoviridae). J. Gen. Virol. 83, 1941–1951. mediterraneus: a reovirus disease. J. Gen. Virol. 69, 561–571. Attoui, H., Mohd Jaafar, F., Biagini, P., Cantaloube, J.F., de Micco, P., Murphy, McEntire, M.E., Iwanowicz, L.R., Goodwin, A.E., 2003. Molecular, physical, F.A., de Lamballerie, X., 2002b. Genus Coltivirus family Reoviridae: and clinical evidence that golden shiner virus and grass carp reovirus are genomic and morphologic characterization of Old World and New World variants of the same virus. J. Aquat. Anim. Health 15, 257–263. viruses. Arch. Virol. 147, 533–561. Mertens, P.P., Sangar, D.V., 1985. Analysis of the terminal sequences of the Attoui, H., Mohd Jaafar, F., Belhouchet, M., Biagini, P., Cantaloube, J.F., de genome segments of four orbiviruses. Virology 140, 55–67. Micco, P., de Lamballerie, X., 2005a. Expansion of family Reoviridae to Mertens, P.P.C., Attoui, H., Duncan, R., Dermody, T.S., 2005. Reoviridae. In: include nine-segmented dsRNA viruses: isolation and characterization of a Fauquet, C.M., Mayo, M.A., Maniloff, J., Desselberger, U., Ball, L.A. new virus designated Aedes pseudoscutellaris reovirus assigned to a proposed (Eds.), Virus Taxonomy. Eighth Report of the International Committee on genus Dinovernavirus. Virology 343, 212–223. Taxonomy of Viruses. Elsevier/Academic Press, London, pp. 447–454. Attoui, H., Mohd Jaafar, F., de Lamballerie, X., Mertens, P.P.C., 2005b. Sea- Mohd Jaafar, F., Attoui, H., de Micco, P., de Lamballerie, X., 2004. Termination dornavirus, Reoviridae. In: Fauquet, C.M., Mayo, M.A., Maniloff, J., and read-through proteins encoded by genome segment 9 of Colorado tick Desselberger, U., Ball, L.A. (Eds.), Virus Taxonomy. Eighth Report of the fever virus. J. Gen. Virol. 85, 2237–2244. International Committee on Taxonomy of Viruses. Elsevier/Academic Press, Mohd Jaafar, F., Attoui, H., Mertens, P., de Micco, P., de Lamballerie, X., 2005a. London, pp. 504–510. Structural organisation of a human encephalitic isolate of Banna virus Attoui, H., Mohd Jaafar, F., Belhouchet, M., de Micco, P., de Lamballerie, X., (genus Seadornavirus, family Reoviridae). J. Gen. Virol. 86, 1141–1146. Brussaard, C.P., 2006a. Micromonas pusilla reovirus: a new member of the Mohd Jaafar, F., Attoui, H., Bahar, M.W., Siebold, C., Sutton, G., Mertens, P.P. family Reoviridae assigned to a novel proposed genus (Mimoreovirus). C., de Micco, P., Stuart, D.I., de Lamballerie, X., 2005b. The structure and J. Gen. Virol. 87, 1375–1383. function of the outer coat protein VP9 of Banna virus. Structure 13, 17–29. Attoui, H., Mohd Jaafar, F., Belhouchet, M., Tao, S., Chen, B., Liang, G., Tesh, Murao, K., Uyeda, I., Ando, Y., Kimura, I., Cabauatan, P.Q., Koganezawa, H., R., de Micco, P., de Lamballerie, X., 2006b. Liao ning virus, a new Chinese 1996. Genomic rearrangement in genome segment 12 of rice dwarf seadornavirus which replicates in transformed and embryonic mammalian phytoreovirus. Virology 216, 238–240. cells. J. Gen. Virol. 87, 199–208. Neukirch, M., Haas, L., Lehmann, H., Von Messeling, V., 1999. Preliminary Auleltta, A.E., Marlowe, M.L., 1968. Effect of sodium deoxycholate on rubella characterization of a reovirus isolated from golden ide (Leciscus idus virus. Appl. Microbiol. 16, 1224. melanotus). Dis. Aquat. Org. 35, 159–164. Castilla, V., Mersich, S.E., Candurra, N.A., Damonte, E.B., 1994. The entry of Ni, Y., Kemp, M.C., 1994. Subgenomic S1 segments are packaged by avian Junin virus into Vero cells. Arch. Virol. 136, 363–374. reovirus defective interfering particles having an S1 segment deletion. Virus Chen, D., Patton, J.T., 1998. Rotavirus RNA replication requires a single- Res. 32, 329–342. stranded 3′ end for efficient minus-strand synthesis. J. Virol. 72, 7387–7396. Patton, J.T., 2001. Rotavirus RNA replication and gene expression. Novartis Corcoran, J.A., Duncan, R., 2004. Reptilian reovirus utilizes a small type III Found. Symp. 238, 64–77. protein with an external myristylated amino terminus to mediate cell–cell Plumb, J.A., Bowser, P.R., Grizzlz, J.M., Mitchell, A.J., 1979. Fish viruses: fusion. J. Virol. 78, 4342–4351. a double-stranded RNA virus icosahedral virus from a North American Desselberger, U., 1996. Genome rearrangements of rotaviruses. Arch. Virol., cyprinid. J. Fish. Res. Board Can. 36, 1390–1394. Suppl. 12, 37–51. Potgieter, A.C., Steele, A.D., van Dijk, A.A., 2002. Cloning of complete Eaton, B.T., Gould, A.R., 1987. Isolation and characterization of orbivirus genome sets of six dsRNA viruses using an improved cloning method for genotypic variants. Virus Res. 6, 363–382. large dsRNA genes. J. Gen. Virol. 83, 2215–2223. Fang, Q., Ke, L.H., Cai, Y.Q., 1989. Growth characterization and high titre Saitou, N., Nei, M., 1987. The neighbor-joining method: a new method for culture of GCHV. Virologica Sinica 3, 315–319. reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406–425. Fang, Q., Shah, S., Liang, Y., Zhou, Z.H., 2005. 3D reconstruction and capsid protein Salsman, J., Top, D., Boutilier, J., Duncan, R., 2005. Extensive syncytium characterization of grass carp reovirus. Sci. China C Life Sci. 48, 593–600. formation mediated by the reovirus FAST proteins triggers apoptosis- Forzan, M., Wirblich, C., Roy, P., 2004. A capsid protein of nonenveloped induced membrane instability. J. Virol. 79, 8090–8100. bluetongue virus exhibits membrane fusion activity. Proc. Natl. Acad. Sci. Samal, S.K., Dopazo, C.P., McPhillips, T.H., Baya, A., Mohanty, S.B., Hetrick, U. S. A. 101, 2100–2105. F.M., 1990. Molecular characterization of a rotaviruslike virus isolated from Goodwin, A.E., Nayak, D.K., Bakal, R.S., 2006. Natural infections of wild striped bass Morone saxatilis. J. Virol. 64, 5235–5240. creek chubs (Semotilus atromaculatus) and cultured fathead minnows Samal, S.K., Attoui, H., Mohd Jaafar, F., Mertens, P.P.C., 2005. Aquareovirus, (Pimephales promelas) by Chinese grass carp reovirus (golden shiner virus). Reoviridae. In: Fauquet, C.M., Mayo, M.A., Maniloff, J., Desselberger, U., Ball, J. Aquat. Anim. Health 18, 35–38. L.A. (Eds.), Virus Taxonomy. Eighth Report of the International Committee on Hedrick, R.P., Rosemark, R., Aronstein, D., Winton, J.R., Mcdowell, T., Taxonomy of Viruses. Elsevier/Academic Press, London, pp. 511–516. Amend, D.F., 1984. Characteristics of a new reovirus from Channel catfish Seth, S., Vincent, A., Compans, R.W., 2003. Activation of fusion by the SER (Ictalurus punctatus). J. Gen. Virol. 65, 1527–1534. virus F protein: a low-pH-dependent paramyxovirus entry process. J. Virol. Hedrick, R.P., Groff, J.M., McDowell, T., Wingfield, W.H., 1989. Character- 77, 6520–6527. istics of reoviruses isolated from cyprinid fishes in California, USA. In: Shaw, A.L., Samal, S.K., Subramanian, K., Prasad, B.V.V., 1996. The structure Ahne, W., Kurstak, E. (Eds.), Viruses of Lower Vertebrates. Springer-Verlag, of aquareovirus shows how the different geometries of the two layers of the Berlin, pp. 241–251. capsid are reconciled to provide symmetrical interactions and stabilization. Hennig, L., 2001. WinPep 2.11: novel software for PC-based analyses of amino Structure 4, 957–967. acid sequences. Prep. Biochem. Biotechnol. 31, 201–207. Subramanian, K., Hetrick, F.M., Samal, S.K., 1997. Identification of a new Kumar, S., Tamura, K., Nei, M., 2004. MEGA3: integrated software for genogroup of aquareovirus by RNA–RNA hybridization. J. Gen. Virol. 78, molecular evolutionary genetics analysis and sequence alignment. Brief. 1385–1388. Bioinform. 5, 150–163. Theron, J., Nel, L.H., 1997. Stable protein–RNA interaction involves the Kyte, J., Doolittle, R., 1982. A simple method for displaying the hydropathic terminal domains of bluetongue virus mRNA but not the terminally character of a protein. J. Mol. Biol. 157, 105–132. conserved sequences. Virology 229, 134–142. Lambden, P., Cooke, S.J., Caul, E.O., Clarke, I.N., 1992. Cloning of Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. CLUSTAL W: improving noncultivable human rotavirus by single primer amplification. J. Virol. 66, the sensitivity of progressive multiple sequence alignment through sequence 1817–1822. weighting, position-specific gap penalties and weight matrix choice. Nucleic Lee, M., Pietras, D.F., Nemeroff, M.E., Corstanje, B.J., Field, L.J., Bruenn, J.A., Acids Res. 22, 4673–4680. 1986. Conserved regions in defective interfering viral double-stranded RNAs Tian, Y., Tarlow, O., Ballard, A., Desselberger, U., Mccrae, M.A., 1993. from a yeast virus. J. Virol. 58, 402–407. Genomic concatemerization/deletion in rotaviruses: a new mechanism for F. Mohd Jaafar et al. / Virology 373 (2008) 310–321 321

generating rapid genetic change of potential epidemiological importance. Xu, Z., Anzola, J.V., Nalin, C.M., Nuss, D.L., 1989. The 3′-terminal sequence of J. Virol. 67, 6625–6632. wound tumor virus transcripts can influence conformational and functional Winton, J.R., Lannan, C.N., Fryer, J.L., Hedrick, R.P., Meyers, T.R., Plumb, J.A., properties associated with the 5′ terminus. Virology 170, 511–522. Yamamoto, T., 1987. Morphological and biochemical properties of four Zhang, S., Shi, Z., Zhang, J., Bonami, J.R., 2004. Purification and characteriza- members of a novel group or reoviruses isolated from aquatic animals. J. Gen. tion of a new reovirus from the Chinese mitten crab, Eriocheir sinensis. Virol. 68, 353–364. J. Fish Dis. 27, 687–692.