Unité des Virus Emergents EA3292, Université de la Méditerranée, Faculté de Médecine de Marseille, EFS Alpes-Méditerranée, 13005 Marseille, France1
Hydrobiology Institute, Wuhan Institute of Virology, CAS, Wuchang, 430071, Wuhan, Hubei, China2
Maladies virales émergentes et systèmes dinformation UR 034, Institut de Recherche pour le Développement, Marseille, France3
Author for correspondence: Houssam Attoui. Fax +33 4 91 32 44 95. e-mail virophdm{at}lac.gulliver.fr
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Abstract |
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Introduction |
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Aquareoviruses have been isolated from a wide variety of aquatic animals, including molluscs, finfish and crustaceans. In the past, they have been referred to as reovirus-like or rotavirus-like aquatic viruses. Like members of the genus Rotavirus, their genomes are composed of 11 segments of dsRNA. The genome is contained in a core surrounded by a double-layered icosahedral capsid that physically resembles capsids of mammalian orthoreoviruses (MRV), as demonstrated by cryoelectron microscopy (Shaw et al., 1996 ).
Aquareoviruses grow in fish cell lines and produce large syncytia that represent the typical cytopathic effect of their replication. In fishes, their typical pathogenic effect is haemorrhage, which represents a serious threat to fish breeding. As an example, Grass carp reovirus (GCRV) provokes severe haemorrhage in fingerling and yearling grass carp, leading to 80% mortality (Fang et al., 1989 ).
Previously, partial genome sequences have been deposited in databases for Aquareovirus A, Aquareovirus B and GCRV. In this paper, we report the full-length genome sequences of Golden shiner reovirus (GSRV, Aquareovirus C) and GCRV (Aquareovirus C). We also have characterized genome segments 2, 3, 4, 8 and 10 of SBRV (Aquareovirus A) and segments 2 and 5 of golden ide reovirus (GIRV, unclassified). These molecular data provide the opportunity for the first robust analysis of the genetic relationships between aquareoviruses A, B and C and unclassified viruses. In addition, the analysis of complete genome sequences from three different aquareoviruses allowed the reassessment of the evolutionary relationship between viruses belonging to the genera Aquareovirus and Orthoreovirus.
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Methods |
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Grass carp reovirus (GCRV).
GCRV is now considered to belong to Aquareovirus C. Four isolates are now identified (Fang et al., 1989 ) as GCRV-873 (prototype strain), GCRV-875, GCRV-876 and GCRV-991. The virus was isolated from the freshwater grass carp fish (Ctenopharyngodon idellus, family Cyprinidae) in the Peoples Republic of China (Chen & Jiang, 1984
; Ke et al., 1990
) and provokes severe haemorrhage in fingerling and yearling grass carp (Fang et al., 1989
). GCRV was propagated in Ctenopharyngodon idellus kidney (CIK) cells. The cells were incubated for 3 days at 28 °C. Other experimental conditions were as above.
Golden ide reovirus (GIRV).
GIRV is an unclassified aquareovirus. The virus was isolated in Germany (Neukirch et al., 1999 ) from the freshwater golden ide fish (Leuciscus idus melanotus). It differs from other aquareoviruses in its distinct cytopathic effect in cell culture (absence of syncytia and formation of focal aggregates of round cells) and by its sensitivity to chloroform. This virus was provided by Dr M. Neukirch. GIRV was grown in FHM cells at 20 °C. Other experimental conditions were as above.
Striped bass reovirus (SBRV).
SBRV is a member of the species Aquareovirus A. The virus was isolated in the USA from the salt-water striped bass fish (Morone saxatilis, family Centrarchidae) with haemorrhagic lesions of the skin (Subramanian et al., 1994 ). This virus was provided by Dr S. K. Samal as a suspension grown in a Chinook salmon embryo cell line (CHSE-214). The virus was studied without further propagation.
Preparation of virus dsRNA.
Clarified supernatants from virus-infected cell cultures were subjected to ultracentrifugation. The pelleted viruses were resuspended in 200 µl EMEM and RNA was extracted with RNA-Now reagent (Biogentex) as described previously (Attoui et al., 2000 ). The dsRNA segments were separated on a 10% polyacrylamide gel, excised and purified using the RNaid kit (Bio-101).
Cloning and sequence determination of genome segments of Aquareovirus C: GCRV and GSRV.
Segments 1, 2 and 3 of GCRV-873 were sequenced in a previous study (Fang et al., 2000 ; accession nos AF260511AF260513). Cloning of segments 411 of GCRV-873 and segments 111 of GSRV was achieved using the single-primer amplification technique (Attoui et al., 2000
), and this completed the genome characterization for both viruses.
Partial sequence determination of genes 8 and 10 of GCRV: isolates GCRV-875, GCRV-876 and GCRV-991.
dsRNAs from GCRV-875, GCRV-876 and GCRV-991 were heat-denatured at 99 °C for 1 min in the presence of 15% DMSO and reverse-transcribed under standard conditions using Superscript-II (Invitrogen) and a random hexaprimer mixture.
Primers were designed from the sequence of segments 8 and 10 of GCRV-873 and are reported in Table 1. PCR was performed under standard conditions using Taq polymerase (Invitrogen) at an annealing temperature of 55 °C. The PCR products were sequenced directly using the corresponding PCR primers, a D-Rhodamine DNA sequencing kit and an ABI Prism-377 sequence analyser (Perkin Elmer).
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Sequences retrieved from databases.
The nearly complete genome of Chum salmon reovirus (CSRV, Aquareovirus A) was retrieved from GenBank. Unpublished sequences of segments 111 of CSRV were deposited by S. Rao, G. R. Carner, W. Chen and J. R. Winton under accession numbers AF418294AF418304. A partial sequence of genome segment 10 of Coho salmon reovirus CSR (Aquareovirus B) was retrieved from GenBank (accession number CSU90430).
Sequence analysis methods.
Sequence alignments were performed using CLUSTAL W (Thompson et al., 1994 ) and the local-BLAST program implemented in the DNATools package (version 5.01.661, S. W. Rasmussen). Phylogenetic analysis was carried out by the neighbour-joining method (Saitou & Nei, 1987
) and the p-distance determination algorithm in the MEGA program (Kumar et al., 1993
). Sequence relatedness is reported as percentage identity. Tree drawing was performed with the help of the program TreeView (Page, 1996
). Comparisons of GSRV and GCRV sequence data with those available from nucleotide and protein databases were performed by using the NCBI program gapped BLAST (http://www3.ncbi.nlm.gov/blast).
Hydropathy profiles were analysed by using amino acid sequence hydropathy values determined by the method of Kyte & Doolittle (1982) implemented in Microsoft Excel. Sequences aligned with CLUSTAL W were exported while all alignment-generated gaps were maintained (gap hydropathy value=0). This permitted the comparison of positional hydropathy profiles for amino acid sequences of unequal lengths.
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Results |
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Sequence determination of GIRV (unclassified) and SBRV (Aquareovirus A)
Primers ReAqSeg2S2/AquaSeg2R amplified a 645 bp sequence of segment 2 of SBRV. Sequencing of this product allowed the design of an SBRV sequence-specific reverse primer designated SBRseg2rev. This primer, together with primer GSVseg2S, allowed PCR amplification of an overlapping sequence of 1491 bp. The final sequence obtained from SBRV segment 2 was 2026 bp long (accession no. AF450318).
Primers ReAqSeg2S2/AquaSeg2R also amplified a 645 bp sequence of segment 2 of GIRV. Sequencing of this product allowed the design of a GIRV sequence-specific reverse primer designated GIRVseg2rev. This primer, together with primer ReAqSeg2S1, allowed PCR amplification of an overlapping sequence of 836 bp. The final sequence of GIRV segment 2 was 1360 bp long (accession no. AF450323).
Segments 3, 4, 8 and 10 of SBRV and segment 5 of GIRV were separated and cloned by single-primer amplification. PCR amplification of cDNA from segment 3 using primer B (Attoui et al., 2000 ) has generated an amplicon that corresponded to a specific sequence of 1186 bp (accession no. AF450319). PCR from segment 4 resulted in a 1116 bp sequence (accession no. AF450320) located at the 3' terminus. Segments 8 and 10 were cloned as full-length sequences and were found to be respectively 1317 bp long (accession no. AF450321) and 987 bp long (accession no. AF450322). Segment 5 of GIRV was cloned as a full-length product and was found to be 2238 bp long (accession no. AF450324).
Sequence analysis of Aquareovirus C viruses GCRV and GSRV
Comparison of the genome electrophoretypes.
The genomes of GCRV and GSRV showed identical electrophoretypes on a 1·2% agarose gel. Slight variations were detected in the PAGE profiles. This could be explained by sequence variations, as observed with other viruses belonging to a single species within the family Reoviridae (Mertens et al., 2000 ).
Comparison of GSRV with GCRV.
The comparison of genome segments 111 of GCRV and GSRV showed high degrees of identity (nucleotide sequences, 90·5697·68%; amino acid sequences, 9699·75%). Cognate segments in the genomes of the two viruses had the same electrophoretic mobility. The detailed nucleotide and amino acid sequence identity values between cognate genes of these two viruses are shown in Table 3.
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The orthoreovirus genome segment 7 (segment S1) is also bicistronic, encoding the outer capsid cell-attachment protein and a basic protein of unknown function. Based on their similar organization, it is possible that genome segments 7 of Aquareovirus C and MRV are equivalent.
By analogy to Aquareovirus A and B, segment 10 of Aquareovirus C encodes outer capsid proteins. This is also true for segment 10 of MRV. Analysis of the hydropathy plot of the proteins encoded by this segment of GSRV or GCRV and MRV showed similar hydropathy profiles in the amino-terminal part of the protein, with four domains in the order hydrophobichydrophilichydrophobichydrophilic (Fig. 2). Alignment of these protein sequences also showed numerous similar motifs (positions 2127, GRLTLYT/GRVSIYS; 4555, CGRYTICAFCL/CGGAVVCMHCL; 128131, IVEL/LVEL; 248255, DDGHQARSA/DFGHFGLSH) with respect to the MRV sequence. Accordingly, it is likely that segments 10 of Aquareovirus C and MRV are equivalent. If this is true, segment 11 of Aquareovirus C would have no equivalent in the MRV genome.
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Comparison of sequences of segments 8 and 10 among GCRV isolates.
Sequence comparison of amplicons from different virus isolates showed that, for segments 8 and 10, nucleotide and amino acid sequence identities were nearly 100%.
Sequence analysis of Aquareovirus A viruses SBRV and CSRV
Comparison of SBRV to CSRV.
Sequences from segments 2, 3, 4, 8 and 10 of SBRV were characterized in this study. Sequence comparison of these segments to their cognates in CSRV revealed nucleotide sequence identities between 70·77 and 78·65% and amino acid sequence identities between 71·2 and 95·87% (the highest values being those of polymerase sequences; Table 3).
Comparison of Aquareovirus A with Aquareovirus C.
The nucleotide and amino acid sequence identities between SBRV or CSRV and GSRV or GCRV respectively ranged from 49·45 to 63·89% and from 20·45 to 65·53%. All segments of Aquareovirus C genomes have cognates in the genomes of Aquareovirus A viruses. The correspondence reflects the order of the electrophoretic mobility of the genomes perfectly, as shown in Fig. 1.
Three obvious differences between Aquareovirus A and Aquareovirus C genomes were noticed, the first being the monocistronic character of segment 7 of CSRV, while those of GCRV and GSRV are bicistronic. Remarkably, both proteins NS16 and NS31 from Aquareovirus C (encoded by the two distinct ORFs of segment 7) showed similarity to the protein encoded by segment 7 of CSRV (NS49, encoded by a unique ORF) (sequence identities shown in Table 3). The second difference is the lack of similarity between the NS38 protein (segment 9) of CSRV and the
NS protein (segment 9) of MRV, while NS38 of GCRV and GSRV showed obvious similarity to this protein. The third difference is that segment 11 of CSRV is tricistronic (ORF1, nt 25435; ORF2, nt 89553; ORF3, nt 375731). Only the proteins encoded by ORF1 (NS13) and ORF3 (NS15) showed similarity to the NS26 protein (segment 11, monocistronic) of Aquareovirus C.
Comparison of SBRV and CSRV with MRV.
BLAST comparison of the genomes of SBRV and CSRV with a local Reoviridae sequence database revealed similarity to the genome of MRV. Values of calculated amino acid sequence identity between CSRV or SBRV and MRV ranged between 18 and 40%.
Similarly to Aquareovirus C species, segments 1, 2, 3, 4, 5, 6 and 8 of CSRV exhibited similarity to segments 1, 2, 3, 4, 5, 6 and 8 in the order given in Fig. 1. The relationships between segments 7, 10 and 11 of CSRV and those of MRV were comparable to those between Aquareovirus C and MRV. As mentioned above, and in contrast to Aquareovirus C, the NS38 protein (segment 9) of CSRV did not exhibit similarity to the
NS protein (segment 9) of MRV.
Analysis of NCRs of CSRV and SBRV segments.
Segments 111 of CSRV and the full-length segments 8 and 10 of SBRV were found to have conserved terminal sequences. All positive strands of the sequenced dsRNA segments had the motif 5' GUUUUAU/G 3' in common at the 5' end and the motif 5' A/UUCAUC 3' in common at the 3' end. Again, the first and last nucleotides of each segment are inverted complements.
Comparison of terminal sequences of the NCRs of Aquareovirus A and Aquareovirus C.
The conserved terminal sequences of Aquareovirus A are highly similar to those of Aquareovirus C, with one obvious difference. In the 5' motif, the adenine base is located at position 4 (5' GUUAUUU/G 3') from the terminus for Aquareovirus C and at position 6 for Aquareovirus A (5' GUUUUAU/G 3').
Sequence analysis of GIRV segments 2 and 5
Comparison of GIRV with Aquareovirus A and Aquareovirus C.
The nucleotide and amino acid sequence identities between GIRV and Aquareovirus A viruses respectively ranged from 48·24 to 62·97% and from 33·89 to 64·98%. The nucleotide and amino acid sequence identities between GIRV and Aquareovirus C viruses respectively ranged from 59·24 to 71·69% and from 56·73 to 81·34%.
Comparison of GIRV with MRV.
BLAST comparison of GIRV genome with the local Reoviridae database showed similarity to the genome of MRV. Values of amino acid sequence identities between GIRV and MRV were 26% in segment 5 and 41% in segment 2. These values are comparable to those between Aquareovirus A or Aquareovirus C and MRV.
Analysis of NCRs of GIRV segments: comparison with Aquareovirus A and Aquareovirus C.
Only segment 5 of GIRV was sequenced fully. The 5'- and 3'-terminal nucleotides of the positive strand of segment 5 were identical to those of Aquareovirus A.
Global analysis of the sequenced genomes of aquareoviruses
Sequence comparison of the putative RNA-dependent RNA polymerases of GCRV, GSRV, SBRV and GIRV and other members of the family Reoviridae.
The amino acid sequences of the polymerases of GCRV, GSRV, SBRV and GIRV were compared with the sequences of putative RNA-dependent RNA polymerases of representative viruses from nine genera of the family Reoviridae: Seadornavirus (12 segments), species Banna virus (isolate BAV-In6423; accession no. AF133430) and Kadipiro virus (isolate KDV-Ja7075; AF133429); Coltivirus (12 segments), species Colorado tick fever virus (isolate CTFV-Fl; AF134529); Orthoreovirus (10 segments), species Mammalian orthoreovirus serotypes 1 (MRV-1; M24734), 2 (MRV-2; M31057) and 3 (MRV-3; M31058) and Ndelle virus (NDEV; AF368033); Orbivirus (10 segments), species African horse sickness virus serotype 9 (AHSV-9; U94887), Bluetongue virus serotypes 2 (BTV-2; L20508), 10 (BTV-10; X12819), 11 (BTV-11; L20445), 13 (BTV-13; L20446) and 17 (BTV-17; L20447) and Palyam virus isolate CHUV (Baa76549); Rotavirus (11 segments), species Rotavirus A (RV-A) strains BoRV-A/RF (J04346), BoRV-A/UK (X55444), SiRV-A/SA11b (X16830), SiRV-A/SA11 (AF015955), PoRV-A/Go (M32805) and AvRV-A (Baa24146), Rotavirus B strain Hu/MuRV-B/IDIR (M97203) and Rotavirus C strain PoRV-C/Co (M74216); Fijivirus (10 segments), species Nilaparvata lugens reovirus strain NLRV-Iz (D49693); Phytoreovirus (12 segments), species Rice dwarf virus isolates RDV-Ch (U73201), RDV-H (D10222) and RDV-A (D90198); Oryzavirus (10 segments), species Rice ragged stunt virus strain RRSV-Th (U66714); and Cypovirus (10 segments), species Bombyx mori cytoplasmic polyhedrosis virus-1 strain Bm-1 CPV (AF323781).
It was found that all members of a single genus exhibited amino acid sequence identities of over 30% (Fig. 3a). The only exception was Rotavirus B, which was only 22% identical to other rotaviruses. Between members of the genera Aquareovirus and Orthoreovirus, the amino acid sequence identity ranged from 40 to 42%. This value is therefore comparable to the amino acid sequence identity observed between members of a single genus. The results of this analysis are illustrated by a radial neighbour-joining tree (Fig. 3b
).
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Analysis of the G+C contents of the genomes of Aquareovirus A, Aquareovirus B, Aquareovirus C and GIRV.
The G+C contents of the genomes of GCRV and GSRV ranged between 53 and 60 mol%; the highest value was calculated from segments 4 and 11. The G+C content of the genomes of GSRV and SBRV ranged between 53 and 57 mol%; the highest value was calculated from segment 4. The G+C content of segment 2 of GIRV was 52 mol% and that of segment 5 was 54 mol%. The G+C content of segment 10 of CSR was found to be 56 mol%.
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Discussion |
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One of the first important findings of the current study pertains to the taxonomy of unclassified isolates. Firstly, the genome of GCRV was sequenced completely and genetic analyses have shown that it is almost identical to that of GSRV (Aquareovirus C): (i) the genomes of the two viruses are of comparable sizes (GCRV, 23695 bp; GSRV, 23696 bp); (ii) cognate segments of the viruses are of comparable sizes; (iii) each genome segment is flanked by identical 5' and 3' conserved NCRs; (iv) nucleotide and amino acid sequence identities between the two viruses are very high (respectively 9097% and 96100%) and the nucleotide sequence variation is mainly a function of the third codon position. These findings show clearly that GSRV and GCRV should be considered as isolates of the same species. Consequently, the hitherto unclassified GCRV belongs to the species Aquareovirus C.
Secondly, segments 2 and 5 of the unclassified GIRV were sequenced. Assessment of the genetic relationship of GIRV to the species Aquareovirus B, D, E and F was impossible because of the absence of sequence data from these species, but sequence comparison permitted us to exclude GIRV from Aquareovirus A and Aquareovirus C.
Besides the study of members of the genus Aquareovirus, the current study sheds new light on the relationship between orthoreoviruses and aquareoviruses. It is a general rule within the family Reoviridae, that the genetic relatedness of viruses belonging to different genera is very low. In the polymerase gene, the only one that allows sequence comparison between different genera, analysis of amino acid sequence identity frequency distribution (Fig. 3a) shows that viruses belonging to different genera have low amino acid sequence identity (<20%), denoting a very distant phylogenetic relationship. This is more evident in other genes for which comparison is practically impossible. The situation of orthoreoviruses and aquareoviruses is therefore a remarkable exception: (i) in the polymerase gene, the amino acid sequence identity between members of the two genera is up to 42% (a value usually observed between members of a single genus); (ii) a clear genetic relationship can be observed between members of the two genera in seven other genes (see Fig. 1
) where amino acid sequence identities range between 17 and 42%; (iii) the T2 protein (the innermost shell of the capsid) of members of the family Reoviridae is a protein that is structurally conserved between viruses belonging to different genera; however, it is not possible to perform a significant sequence comparison. This protein is identified as the
1 protein in the orthoreovirus genome. The equivalent aquareovirus protein, VP3, shows significantly high (37%) amino acid sequence identity to
1. This means that the genetic relationship between orthoreoviruses and aquareoviruses is comparable to that between viruses such as St Croix River virus and Bluetongue virus (belonging to the genus Orbivirus) or Kadipiro virus and Banna virus (both belonging to the genus Seadornavirus). Homology was also identified in the conservation of terminal sequences. For instance, the 5' and 3' conserved terminal sequences of GCRV segments are 5' GUUAUU 3' and 5' A/UUCAUC 3', compared to 5' GCUAUU 3' and 5' A/UUCAUC 3' in genome segments 4, 7, 8 and 10 of MRV.
Altogether, these data are undoubtedly an indication of the common evolutionary origin of these viruses. Fig. 4 shows a simplified scheme of the evolution of the main hosts of orthoreoviruses and aquareoviruses. The common ancestor of fish and the group reptiles+birds+mammals existed around 510 million years (My) ago. It has been proposed that the molecular evolutionary rate of genomes of related dsRNA viruses is 10-8 to 10-9 mutations/nucleotide/year, which is equivalent to that of dsDNA (Attoui et al., 2002
). If this is applied to the polymerases of orthoreoviruses and aquareoviruses, it appears that divergence between these two groups occurred 49520 My ago. Despite this imprecision in the evaluation of divergence, it cannot be excluded that orthoreoviruses appeared following the emergence of the evolutionary group that eventually gave rise to reptiles, birds and mammals.
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Concerning the first question, it should be noted that the ICTV defines taxa as members of a polythetic class. Therefore, despite the unusual genetic relatedness between orthoreoviruses and aquareoviruses, there are strong arguments that justify their classification within two separate genera: (i) as reported above, the viruses originate from distinct econiches; (ii) aquareoviruses have genomes composed of 11 segments and orthoreovirus genomes are composed of 10 segments of dsRNA; (iii) the G+C content of orthoreoviruses ranges between 44 and 48 mol%, while that of aquareoviruses ranges between 52 and 60 mol%; (iv) orthoreoviruses are non-syncytializing viruses, in contrast to the majority of aquareoviruses; and (v) there is no antigenic relationship between the two groups.
Accordingly, the authors consider that the maintenance of the classification of these viruses in two different genera is justified and that orthoreoviruses and aquareoviruses constitute an interesting, but isolated, example of two genera that undoubtedly originate from a common evolutionary ancestor.
Concerning quantitative taxonomy using polymerase sequences, analysis of amino acid sequence identity frequency distribution (Fig. 3a) shows that all members of a single genus have amino acid sequence identities of >30%. There is, however, one exception, Rotavirus B, which is only 22% identical to other rotaviruses. Similarly, it can be observed that all viruses belonging to different genera have amino acid sequence identities of <30%, the only exceptions being orthoreoviruses and aquareoviruses, as discussed above. Therefore, the only criterion that remains indisputable is the assignment of two viruses to different genera if their polymerase amino acid sequence identity is <20%. Clearly, this is a modest contribution to phylogenetic classification. However, it is important to pursue the sequence characterization of representative members of the family Reoviridae to allow better definition of the quantitative basis of species delineation in the different genera and to try to improve the genetic criteria for the definition of genera.
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Acknowledgments |
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Footnotes |
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References |
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Received 16 January 2002;
accepted 24 March 2002.