Muscovy duck reovirus {sigma}C protein is atypically encoded by the smallest genome segment

Gaëlle Kuntz-Simonb,1, Ghislaine Le Gall-Reculé1, Claire de Boisséson2 and Véronique Jestin1

French Agency for Food Safety (AFSSA), Poultry and Swine Research Laboratory, Avian and Rabbit Virology, Immunology and Parasitology Unit1 and Viral Genetics and Biosafety Unit2, Zoopôle Les Croix, BP 53, 22440 Ploufragan, France

Author for correspondence: Véronique Jestin. Fax +33 2 96 01 62 63. e-mail v.jestin{at}ploufragan.afssa.fr


   Abstract
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Although muscovy duck reovirus (DRV) shares properties with the reovirus isolated from chicken, commonly named avian reovirus (ARV), the two virus species are antigenically different. Similar to the DRV {sigma}B-encoded gene (1201 bp long) previously identified, the three other double-stranded RNA small genome segments of DRV have been cloned and sequenced. They were 1325, 1191 and 1124 bp long, respectively, and contained conserved terminal sequences common to ARVs. They coded for single expression products, except the smallest (S4), which contained two overlapping open reading frames (ORF1 and ORF2). BLAST analyses revealed that the proteins encoded by the 1325 and 1191 bp genes shared high identity levels with ARV {sigma}A and {sigma}NS, respectively, and to a lesser extent with other orthoreovirus counterparts. No homology was found for the S4 ORF1-encoded p10 protein. The 29·4 kDa product encoded by S4 ORF2 appeared to be 25% identical to ARV S1 ORF3-encoded {sigma}C, a cell-attachment oligomer inducing type-specific neutralizing antibodies. Introduction of large gaps in the N-terminal part of the DRV protein was necessary to improve DRV and ARV {sigma}C amino acid sequence alignments. However, a leucine zipper motif was conserved and secondary structure analyses predicted a three-stranded {alpha}-helical coiled-coil feature at this amino portion. Thus, despite extensive sequence divergence, DRV {sigma}C was suggested to be structurally and probably functionally related to ARV {sigma}C. This work provides evidence for the diversity of the polycistronic S class genes of reoviruses isolated from birds and raises the question of the relative classification of DRV in the Orthoreovirus genus.


   Introduction
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Abstract
Introduction
Methods
Results
Discussion
References
 
Members of the genus Orthoreovirus, family Reoviridae, share common physico-chemical properties and morphological characteristics (Nibert et al., 1996 ). These include a double-stranded RNA (dsRNA) genome consisting of 10 segments packaged into a non-enveloped icosahedral double-capsid shell. Although they also share similar electropherotypes and protein-coding pattern compositions, orthoreoviruses are not entirely homogeneous (Duncan, 1999 ). They can be subdivided based on distinct biological properties, most notably their host range and the unusual ability of certain members to induce cell–cell fusion. Thus, according to the Seventh Report of the International Committee on Taxonomy of Viruses, the Orthoreovirus genus consists of four species that separate into three distinct subgroups (Van Regenmortel et al., 2000 ). The non-fusogenic mammalian orthoreovirus (MRV) species represents subgroup I. The fusogenic reoviruses separate into subgroup II, containing the avian reovirus (ARV)-type species and the Nelson Bay virus (NBV) isolated from a flying fox, and subgroup III occupied by the baboon reovirus (BRV). Two isolates from snakes, sharing the syncytium-inducing properties of subgroups II and III but unclassified to date, are defined as tentative species in the genus and form subgroup IV.

In addition to their fusogenic properties, ARVs differ from the prototype MRV on the basis of their natural pathogenicity and the lack of haemagglutinating activity (Kawamura et al., 1965 ). Their genomic dsRNA segments can be divided, on the basis of electrophoretic mobility, into three size classes: large (L1–L3), medium (M1–M3) and small (S1–S4) (Gouvea & Schnitzer, 1982 ; Rerik et al., 1990 ; Wu et al., 1994 ). They code for at least 11 primary translation products separated into {lambda}, µ and {sigma} classes (Varela & Benavente, 1994 ; Shmulevitz & Duncan, 2000 ). ARV proteins have been less well characterized than their MRV counterparts and most of the reported studies concern {sigma} proteins and S class-encoded genes. S1 is the only polycistronic gene among ARV small dsRNA genome segments. It contains three sequential overlapping open reading frames (ORFs), encoding predicted polypeptides of 10, 17 and 35 kDa, respectively (Kool & Holmes, 1994 ; Vakharia et al., 1996 ). The p10 protein was recently described to be a non-structural transmembrane protein responsible for the fusogenic property of the virus (Shmulevitz & Duncan, 2000 ). The second ORF has not been shown to be functional to date. However, as this p17-encoded ORF is truncated in the ARV S1133 vaccine strain S1 segment, it has been suggested to play a role in pathogenicity (Shapouri et al., 1995 ). The 3'-terminal ORF encodes the minor outer capsid component. This protein is structurally and functionally related to the MRV protein {sigma}1 (Shapouri et al., 1996 ; Lee & Gilmore, 1998 ). Expressed and located at the vertices of the spikes, {sigma}C is a homo-trimer displaying cell-binding activity and inducing type-specific neutralizing antibodies (Wickramasinghe et al., 1993 ; Grande et al., 2000 ). S2 encodes the major core protein, {sigma}A, a dsRNA-binding polypeptide suggested to play a role in resistance of ARV to interferon (Martinez-Costas et al., 2000 ; Yin et al., 2000 ). S3 encodes the major outer capsid protein, {sigma}B, structurally related to MRV {sigma}3 (Yin et al., 1997 ). {sigma}B reacts with an anti-reovirus polyclonal serum and is one of the group-specific neutralization antigens (Wickramasinghe et al., 1993 ). Finally, S4 encodes the small non-structural protein, {sigma}NS, which binds to single-stranded RNA in a sequence non-specific manner and may play an important role in the earliest stages of particle assembly (Yin & Lee, 1998 ; Becker et al., 2001 ).

These previous investigations on ARV genes and proteins have mainly been concerned with chicken reovirus. However, reoviruses have been recovered from a variety of other domestic and wild birds. These include turkey (Lozano et al., 1989 ), goose (Hlinak et al., 1998 ), pheasant (Mutlu et al., 1998 ), pigeon (Vindevogel et al., 1982 ), quail (Ritter et al., 1986 ) and psittacine birds (Conzo et al., 2001 ). Reoviruses have also been isolated from duck species, such as Pekin (Jones & Guneratne, 1984 ), mallard and muscovy ducks (Gaudry et al., 1972 ; McFerran et al., 1976 ).

Muscovy duck (Cairina moschata) reovirus (DRV) shares common properties with other avian reoviruses, such as a double-layered capsid with polygonal capsomeres, syncytium formation in cell culture and inability to haemagglutinate (Malkinson et al., 1981 ). However, DRV pathogenicity differs from that of ARV (Malkinson et al., 1981 ; Marius-Jestin et al., 1988 ) and, in contrast to turkey reovirus, which was found to be antigenically related to the chicken reovirus (Nersessian et al., 1989 ), cross-neutralization tests demonstrated that DRV is antigenically different from ARV (Heffels-Redmann et al., 1992 ). Similarly, all the strains isolated in our laboratory from muscovy ducks with symptoms of disease were grouped into one serotype with no cross-reactivity to ARV S1133 (V. Jestin, unpublished results).

We previously reported that DRV {sigma}B and its encoded gene are related to ARV counterparts (Le Gall-Reculé et al., 1999 ). In order to extend our knowledge of DRV, we characterized the other three DRV S class genome segments. The homologies we found between putative DRV {sigma} class proteins and orthoreovirus counterparts have allowed us to establish DRV gene coding assignments. Based on the determination of paired identities between homologous genes and proteins, and together with the results of comparative sequence-based structural predictions, we have established the phylogenetic relationships between DRV and other Orthoreovirus genus members.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Virus strains and purification of dsRNA.
DRV strains 89330 and 89026 have been previously described (Le Gall-Reculé et al., 1999 ). ARV-S1133 is the commercially available chicken reovirus vaccine strain (Van der Heide et al., 1974 ). DRVs and ARVs were propagated on the chorio-allantoic membrane of specific pathogen-free muscovy duck or hen eggs, respectively. Virions were extracted by treatment with 1,1,2-trichlorotrifluoroethane and concentrated through a 40% sucrose cushion.

Semi-purified virions were incubated for 2 h at 37 °C in an extraction buffer (100 mM Tris–HCl, pH 8·0, 10 mM EDTA, 1 mM DTT, 1000 U/ml RNase inhibitor) containing 1% (w/v) SDS and 200 µg/ml proteinase K. Nucleic acids were extracted twice with phenol, recovered by ethanol precipitation and suspended in 1 mM EDTA. Viral dsRNA segments were further purified by LiCl fractionation precipitation (Diaz-Ruiz & Kaper, 1978 ) and fractionated by 10% PAGE run in TBE buffer (89 mM Tris–HCl, 89 mM boric acid, 2 mM EDTA, pH 8·3) for 16 h at 14 mA. Genomic dsRNA segments were visualized under UV light after ethidium bromide treatment.

{blacksquare} cDNA synthesis, cloning and sequencing.
Double-stranded RNA segments were individually excised from the gel and purified using the RNaid Kit with SPIN (Bio 101), according to the manufacturer’s instructions. cDNA syntheses from DRV-89026 S1 and S3 segments and ligation of PCR-amplified products into pUC18 vector were as previously described (Le Gall-Reculé et al., 1999 ). In the case of DRV-89330 S3 and S4 segments, cDNA synthesis was performed with modifications. Oligonucleotide primer P1 was ligated to both 3' ends of purified dsRNA segments by T4 RNA ligase in the presence of 10% DMSO overnight at 4 °C. P1-tailed dsRNAs were purified by spin-column chromatography and ethanol precipitated. Double-strands were denatured at 95 °C for 5 min in the presence of primer P2 (complementary to P1) and 14% DMSO. cDNA synthesis was carried out by SuperScript II RNase H- reverse transcriptase (Gibco BRL) for 1 h at 42 °C. After RNA degradation by ribonuclease H, complementary cDNA strands were annealed and repaired (Lambden et al., 1992 ). cDNAs were amplified by PCR using AmpliTaq DNA polymerase (Perkin Elmer) and primer P2, consisting of a denaturation step at 94 °C for 2 min, followed by 30 cycles of 30 s at 94 °C, 30 s at 54 °C and 90 s at 72 °C, and a final elongation step at 72 °C for 7 min. Amplified DNA products were ligated into pMOSBlue vector (Amersham Pharmacia Biotech).

Cloned genes were sequenced using universal primers, U19 primer (5' GTTTTCCCAGTCACGACGT 3') in the case of pMOSBlue recombinant plasmids, plus DRV genome-specific primers selected for chromosome walking strategy sequencing. For each gene, a consensus sequence was deduced from alignments of 3–12 recombinant plasmid sequences. To confirm the consensus sequences, reverse transcription (RT) was performed on total genomic dsRNA using specific primers and PCR was carried out using AmpliTaqGold polymerase (Perkin Elmer).

{blacksquare} Northern blot analysis.
Specific probes from cloned viral cDNA were labelled with alkaline phosphatase (AlkPhos Direct kit, Amersham). For Northern blot hybridization experiments, purified DRV dsRNA segments were fractionated by 10% PAGE, transferred on to a nylon membrane (Hybond-N+, Amersham) in 0·2 M NaOH and hybridized to S gene-specific labelled probes using the AlkPhos Direct kit, according to the manufacturer’s instructions. A chemiluminescent signal was generated by reaction with CDP-Star reagent (Amersham) and detected by autoradiography.

{blacksquare} Sequence accession numbers.
DRV-89026 {sigma}B-encoding segment has been previously sequenced (accession number AJ006476) (Le Gall-Reculé et al., 1999 ). Accession numbers for DRV genome segments sequenced in this study are shown in Table 1. Sequences of other orthoreovirus genome segments were obtained from the DDBJ/EMBL/GenBank (Table 1). The BRV S4 ORF2 gene sequence was kindly provided by R. Duncan.


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Table 1. Accession numbers for DRV and orthoreovirus genome segments

 
{blacksquare} Computer-based sequence analysis.
Nucleotide and deduced amino acid sequences were obtained and analysed using Infobiogen web site software (http://www.infobiogen.fr). Comparisons of sequences with those available in databases were performed using the NCBI BLAST program (http://www.ncbi.nlm.nih.gov/BLAST/). Binary and multiple sequence alignments were generated by ALIGN (Myers & Miller, 1988 ) and CLUSTAL W (Thompson et al., 1994 ) software, respectively. Searches for biologically relevant sites and signatures were performed by scanning the sequences against the PROSITE database. Distributional and periodicity analyses were evaluated using the Statistical Analysis of Protein Sequences (SAPS) program (Brendel et al., 1992 ) available on the ISREC server (http://www.isrec.isb-sib.ch/software/SAPS_form.html). The Multicoil program (Wolf et al., 1997 ) was used to predict the location of coiled-coil regions in amino acid sequences and to classify the prediction as dimeric or trimeric. Physico-chemical profiles were obtained using the Network Protein Sequence (NPS) @nalysis web site (http://pbil.ibcp.fr/NPSA). The Multivariate Linear Regression Combination (MLRC) tool (Guermeur et al., 1999 ) available on this site was used for secondary structure predictions. Membrane-spanning regions were predicted using the TMpred program (Hofmann & Stoffel, 1993 ).

{blacksquare} Phylogenetic analyses.
Multiple alignments of homologous protein sequences, obtained by CLUSTAL W, were used as input for phylogenetic analysis by PHYLIP (Phylogeny Interference Package Version 3.573c), available on the Pasteur Institut web site (http://bioweb.pasteur.fr/seqanal/phylogeny). Maximum parsimony analysis was performed using PROTPARS after bootstrapping by SEQBOOT. Bootstrap values were given for 100 random samplings of each alignment. Based on all samplings, the most probable unrooted tree was calculated by CONSENSE and displayed as a rectangular cladogram using for outgroup the most distant virus from DRV. Cladograms were drawn using TREEVIEW 6.1 (Page, 1996 ).


   Results
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Analysis of the electrophoretic migration pattern of DRV dsRNA genome segments
Genomic dsRNA segments from DRV-89330 and DRV-89026 strains were separated by PAGE (Fig. 1, lanes 2 and 3). Both migration patterns revealed a genome composed of 10 dsRNA segments divided into three size classes: large (L1–L3), medium (M1–M3) and small (S1–S4), the numbering reflecting the relative position in the gel. Differences in electrophoretic mobility between the DRV-89026 and DRV-89330 strains were mostly found concerning the S class segments, indicating a possible genetic polymorphism. For comparison, the chicken reovirus vaccine strain, ARV-S1133, was analysed on the same gel (Fig. 1, lane 1). Whereas DRV and ARV L and M genes migrated to similar positions in the gel, differences were clearly evident in the mobility of the S segments. Indeed, no DRV segment was found at the ARV-S1133 S1 gene position that migrates more closely to the M segments than to the other S genes. Atypically, as compared with ARV, the four DRV S genes migrated altogether, at positions similar to S2, S3 and S4 genes of ARV-S1133.



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Fig. 1. Comparison of the electrophoretic mobility of DRV and ARV genome segments. Purified dsRNA genome segments of ARV-S1133 (1·5 µg, lane 1), DRV-89330 (1 µg, lane 2) and DRV-89026 (1 µg, lane 3) were resolved by 10% PAGE and visualized by ethidium bromide staining. The locations of the large (L), medium (M) and small (S) size class segments are indicated, along with the numbering scheme of the S class genome segments.

 
Characterization of DRV S class genome segments
In order to identify DRV small genome segments, we individually gel-purified the dsRNA present in the S class bands, either from DRV-89026 and/or DRV-89330. cDNA was synthesized and amplification products were selected, ligated into cloning vector and sequenced. Four genes were characterized. They were 1325 (DRV-89026), 1201 (DRV-89330), 1191 (DRV-89026) and 1124 (DRV-89330) bp long, respectively (Table 2). As mentioned above, the DRV-89026 1201 bp gene had been cloned in a previous study (Le Gall-Reculé et al., 1999 ). Restriction probes specific for each gene were synthesized and hybridized to genomic dsRNA in Northern blotting experiments to determine precisely the electrophoretic mobility of the genes. In the case of DRV-89026, S1–S4 hybridized to the 1325, 1201, 1191 and 1124 bp genes, respectively (Table 2). The same results were obtained for DRV-89330, except that S2 and S3 hybridized to the 1191 and 1201 bp genes, respectively (Table 2).


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Table 2. S class genome segments encoding the {sigma} class proteins of muscovy duck reovirus

 
The four genes exhibited a conserved cDNA heptanucleotide sequence (GCTTTTT) at the 5' terminus. At the 3' end, the octanucleotide TATTCATC was conserved among three of them. The 3' terminus of the 1191 bp gene varied by a single nucleotide, here underlined, TACTCATC. These conserved 5'- and 3'-terminal sequences indicated that the cDNA clones represented full-length replicas of the genomic dsRNA segments. A 6–8-nucleotide-long inverted repeat sequence, unique to each gene, was identified adjacent to the conserved terminal sequences (data not shown). These inverted repeat sequences were also found in ARV genes (Ni et al., 1996 ). Defined as segment-specific signals, they ensure proper sorting of the individual genome segments, allowing only one copy of each gene to be package into each virion.

Amino acid sequences were deduced from cDNA sequences of the DRV genome segments. The three larger S genes contained a single ORF, each encoding a single predicted expression product (Table 2). By contrast, analysis of the 1124 bp gene sequence revealed that S4 was bicistronic, containing two sequential overlapping ORFs (ORF1 and ORF2) (Table 2). ORF1 extended from position 63 to 350, encoding a putative polypeptide of 10·8 kDa (95 residues), which we named p10. ORF2 started at position 280 and terminated at position 1089, encoding a protein of 29·4 kDa (269 residues). DRV-89026 S4 segment was amplified from total genomic dsRNA by RT–PCR using primers up36 (5' TCGCCGCTGAACTGACTA 3') and lo1088 (5' CGCCGCCACTACCTAAAA 3') specified from the DRV-89330 S4 segment. The 1070 bp amplification product showed a striking degree of identity (99·23%) to its DRV-89330 counterpart and contained two ORFs similar to DRV-89330 ORF1 and ORF2.

Amino acid sequences of DRV small gene expression products were used for BLAST analysis. Inferred homology with ARV proteins allowed us to establish the genome segment coding assignment of DRV strains (Table 2). As previously reported, the 1201 bp gene has been found to encode the major outer capsid protein {sigma}B (Le Gall-Reculé et al., 1999 ). Similarly, the DRV-89330 1201 bp gene encoded a protein 367 amino acids long (40·8 kDa) and 98·4% identical to DRV-89026 {sigma}B. BLAST searching revealed that the DRV-89026 1325 and 1191 bp genes encoded proteins sharing high homology (around 90%) with the ARV major core protein ({sigma}A) and non-structural ({sigma}NS) proteins, respectively. By contrast, no homologue of p10, the S4 ORF1 expression product, was found in databases. Finally, BLAST searching revealed that the S4 ORF2 expression product showed homology to the ARV minor outer capsid protein ({sigma}C), although with only a weak similarity (around 30%). It should be noted that no difference was observed between the DRV-89026 and DRV-89330 p10 proteins. A single amino acid differs between both 29 kDa products at position 82 (S->F).

Structural analysis of DRV {sigma}C protein
From the BLAST analysis reported above, {sigma}C was found to be the DRV {sigma} class protein displaying the highest level of sequence divergence compared with orthoreovirus counterparts. Comparison of amino acid sequences of the DRV and ARV homologous {sigma}C proteins showed that insertion of large gaps (10, 13 and 38 alignment positions long) at the N-terminal part of the DRV {sigma}C protein was necessary to optimize the alignment (Fig. 2). In the remaining C-terminal regions, only a few gaps one alignment position long were inserted in the ARV sequences to maintain an optimal alignment.



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Fig. 2. DRV {sigma}C protein deduced amino acid sequences (strains 89330 and 89026) and their alignments with homologous minor outer capsid proteins (Som4, Sommerville 4). Amino acid positions for each individual sequence are numbered on the right. Identical amino acids in all the aligned sequences are indicated by an asterisk (*). Amino acids that are identical in at least 50% of the sequences are indicated by a plus (+). Grey shading indicates the leucine zipper patterns. The putative fusion motif found in ARV-Ram1 is boxed with a dotted line. The first and last residues of the major heptad repeat patterns are boxed with a bold line. Open circles over the aligned sequences indicate the location of conserved proline and cysteine residues and filled circles indicate the location of conserved hydrophobic residues.

 
Examination of the DRV {sigma}C protein for structural and functional motifs predicted that it possessed several potential phosphorylation and myristoylation sites (data not shown) but no N-glycosylation site, in comparison with the chicken equivalent (Shapouri et al., 1995 ). DRV {sigma}C contained a leucine zipper motif at positions 45–66 (Figs 2 and 3). This motif has also been reported within ARV (Fig. 2) and MRV counterparts, and would be involved in oligomerization of the protein (Belli & Samuel, 1993 ; Vakharia et al., 1996 ). Based on MultiCoil analyses, DRV {sigma}C should contain a three-stranded coiled coil, assuming it was an homo-trimer (Fig. 3). On the other hand, a gap has been introduced at a position previously described to correspond to a putative fusion motif within ARV {sigma}C (residues 13–24) (Fig. 2) (Theophilos et al., 1995 ). However, although involvement of the ARV {sigma}C in the virus-induced cell fusion process has been reported (Meanger et al., 1999 ), direct evidence is still lacking and has been denied by others (Shmulevitz & Duncan, 2000 ).



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Fig. 3. Structural patterns, secondary structure predictions and physico-chemical profile of the DRV {sigma}C protein. The top panel indicates the regions exhibiting heptad repeat patterns of apolar residues (grey boxes), the location of the leucine zipper pattern (dotted box) and represents the MultiCoil score trimer probability graph (dashed line). The central panel is a linear representation of the MLRC prediction results (vertical longest lanes: positions of residues predicted to be in {alpha}-helical configuration; vertical smallest lanes: positions of residues predicted to be in extended strand configuration; other residues: in random coil configuration). The bottom panel represents a hydropathy profile of the DRV {sigma}C protein according to the algorithm of Kyte and Doolitle, averaged over a window of seven residues (hydrophobic below the line).

 
The DRV {sigma}C amino acid sequence also showed that it was a slightly acidic protein (pI=6·08), with a mainly hydrophilic portion in the middle part and a mainly hydrophobic N-terminus (Fig. 3). According to secondary structure predictions, DRV {sigma}C could have the following features: 41·26% of the residues in the form of {alpha}-helices, 14·87% in the form of extended strands and 43·87% in random coils. The {alpha}-helices are not uniformly distributed and about 63% of them are in the N-terminal half of the molecule (Fig. 3). SAPS analysis revealed a heptapeptide repeat (a-b-c-d-e-f-g) of hydrophobic residues between amino acids 42 and 103 (Figs 2 and 3). This pattern, representing approximately a quarter of the molecule and containing most of the {alpha}-helices, is typical of an {alpha}-helical coiled-coil structure where the residues at positions a and d are primarily hydrophobic and form the interface between the {alpha}-helices. Another heptad repeat was detected by SAPS in the middle part of the protein, at positions 135–183 (Figs 2 and 3). However, only the most N-terminal part of this region contained a high percentage of {alpha}-helices, leading us to suppose that only a few residues would be in a coiled-coil configuration. In contrast to the N-terminus, the C-terminal part of the DRV {sigma}C protein was a mixture of {alpha}-helices, {beta}-sheets and random coils, showing no dominant structural pattern. It did not contain a consistent repeat pattern of conserved apolar residues but was characterized by a large number of conserved aromatic residues, which other regions did not contain (Fig. 2). Another striking feature of the sequence was the presence of two proline residues at amino acid positions 36 and 104, flanking the first heptad repeat (Fig. 2). It should be noted that proline 104 was a conserved residue as compared to the ARV {sigma}C sequence, together with proline 118 and proline 206. These three conserved proline residues, together with a conserved cysteine residue at position 121 (Fig. 2), may play an important role in protein structure, bordering well-defined morphologic regions.

Altogether, these features suggest that DRV {sigma}C could be organized as a fibrous tail at the N-terminus and a globular-like structure, the head, at the C-terminal portion. The most carboxy-terminal sequences contributing to the tail would form a short coiled coil followed by a cross-{beta} region. This domain could form a distinct structural region, the neck.

Paired identities between homologous S class genes and {sigma} class proteins of DRV and other orthoreoviruses
The nucleotide and predicted amino acid sequences of all the DRV small genome segments were aligned with homologous genes and proteins (data not shown). The aligned sequences were then used in pairwise comparisons and the percentage of nucleotide and amino acid sequence identities were determined (Table 3).


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Table 3. Percentage identities of homologous genome segments and encoded proteins of DRV-89026 and other orthoreoviruses

 
DRV {sigma}NS- and {sigma}A-encoded genes exhibited more than 77% identity with ARV homologous segments, resulting in amino acid identities in the range of 88–90% (Table 3). Indeed, amino acid sequences were essentially contiguous and required only a few small gaps to maintain the alignments (data not shown). DRV-89026 {sigma}A-encoded segment (S1) was also found to be 60% identical to the NBV S2 segment, and DRV {sigma}A and {sigma}NS proteins shared 50–60% identity with their NBV counterparts (Table 3). By contrast, sequence comparisons of these DRV proteins with MRV or BRV homologues revealed extensive divergence, with amino acid identities that ranged from 25 to 31% (Table 3). Alignments of DRV-89026 {sigma}B protein with ARV and MRV counterparts have been previously reported and required the insertion of numerous gaps (Le Gall-Reculé et al., 1999 ). In this study, we also aligned DRV-89026 and DRV-89330 {sigma}B proteins with NBV and BRV counterparts, as well as the nucleotide sequences of encoding genes (data not shown). Thus, DRV {sigma}B was only 60–61% identical to ARV {sigma}B and extensive divergence was observed when aligned with NBV, MRV and BRV homologues (32%, 24% and 24%, respectively) (Table 3). Finally, alignment of DRV and ARV {sigma}C proteins (Fig. 2) showed that both proteins possessed an overall sequence identity of only 21–25%, with an obvious clustering of conserved residues in the C-proximal domain (Table 3). The highest scores have been obtained with American strains (ARV-176, ARV-1733 and ARV-S1133) compared with Australian strains (RAM-1 and Sommerville-4).

Phylogenetic relationships of DRV and other orthoreoviruses
The evolutionary relationships between DRV and the other Orthoreovirus genus members was determined by phylogenetic analysis. The aligned sequences of {sigma} class proteins found homologous by BLAST (Table 3) were used as input for maximum parsimony analyses using bootstrapping. Based on the unrooted trees established for the major core (Fig. 4A), the non-structural (Fig. 4B) and the major outer capsid (Fig. 4C) proteins, this analysis supported the classification of DRV into the previously defined Orthoreovirus genus subgroup II, together with ARV and NBV. The greater extent of amino acid conservation between DRV and ARV for these proteins led us to classify DRV closer to ARV than to NBV. No homology having been found between DRV {sigma}C and the other orthoreovirus counterparts except ARV {sigma}C, phylogenetic analysis was restricted to DRV and ARV in the case of the minor outer capsid protein. The cladogram confirmed that DRV {sigma}C was more related to ARV American strains than to Australian strains (Fig. 4D).



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Fig. 4. Phylogenetic trees of DRV and other orthoreoviruses. Phylogenetic trees were generated using the aligned amino acid sequences of homologous {sigma} class proteins. Maximum parsimony analysis with bootstrapping was performed using the PHYLIP package and the resulting rectangular cladograms represent the most probable unrooted consensus tree for each of the four {sigma} class proteins: (A) major core proteins; (B) non-structural proteins; (C) major outer capsid proteins; (D) minor outer capsid proteins. The bootstrap values are given for each internal edge. The Orthoreovirus genus subgroups I, II and III, defined according to the Seventh Report of the ICTV, are indicated on the right-hand side of each cladogram.

 

   Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Electrophoretic analysis of DRV dsRNA genomes has revealed the general migration pattern of orthoreovirus genes into three size classes. However, whereas DRV L and M class genes migrated to similar positions as ARV counterparts in a polyacrylamide gel, we clearly showed great differences between both species with regard to the mobility of the S segments. These differences have not been shown by others, probably due to different PAGE experimental conditions (Heffels-Redmann et al., 1992 ). The cloning and sequencing of the four DRV small genome segments led us to determine their coding assignment and confirmed the divergence between DRV and ARV at the molecular level. Indeed, we showed that the DRV {sigma}C protein is atypically encoded by S4, the smallest genome segment, and not by S1, as usually described for ARV, NBV and MRV. DRV S4 is the single polycistronic DRV S class gene. It contains two ORFs, similar to the MRV S1 gene (Cashdollar et al., 1985 ) but different from the ARV and NBV S1 genes, which contain three ORFs each (Shmulevitz & Duncan, 2000 ). From length comparison of the ORFs, it could be hypothesized that DRV S4 represented a truncated equivalent of ARV S1 gene, which missed the ARV S1 ORF2 equivalent and thus failed to express the ARV p17 counterpart.

As observed for all other orthoreoviruses, DRV {sigma}C is encoded by the major ORF of the polycistronic S class genome segment, located at the 3' end. However, DRV S4 ORF2 (810 nt) is smaller than ARV S1 ORF3 (977 nt), and DRV {sigma}C was predicted to be shorter than ARV {sigma}C (29·4 kDa and 34·9 kDa, respectively). This expected size has been confirmed by expressing recombinant DRV-89330 {sigma}C protein in insect cells (G. Kuntz-Simon and others, unpublished results). To date, only BRV has been previously suggested to possess an S4 segment equivalent to S1 of other species (Duncan, 1999 ). As for DRV S4, BRV S4 gene was shown to be bicistronic, the second ORF encoding a 16 kDa protein (R. Duncan, personal communication). However, we did not detect any relevant homology between DRV {sigma}C and BRV p16. BLAST analyses also failed to reveal any homology between DRV ORF1-encoded p10 and ARV p10. Moreover, by analysing the DRV p10 amino acid sequence, we did not find any potential transmembrane domain, a secondary structure described to be a common feature of identified orthoreovirus fusion-associated small transmembrane proteins (Shmulevitz & Duncan, 2000 ). Further experiments are necessary to determine whether DRV p10 does or does not play a role in cell–cell fusion.

By comparing DRV and ARV {sigma}C amino acid sequences, we revealed that the shorter size of DRV {sigma}C depends on the introduction of gaps at the N-terminal part. However, despite the missing sequence portions, computer-based analyses predicted that DRV {sigma}C had a structure related to that suggested for ARV {sigma}C (Shapouri et al., 1995 ), itself related to that demonstrated for MRV {sigma}1 (Nibert et al., 1990 ; Fraser et al., 1990 ). The fibrous tail at the N-terminal part is shorter than the ARV equivalent but similarly organized as a three-stranded {alpha}-helical coiled coil. Further experiments are required to demonstrate the oligomerization of this protein. However, the conservation of essential structural domains among other highly diverging genogroups of orthoreoviruses indicates that DRV {sigma}C could have a similar localization in the virion and a similar function in virus–host cell interaction. As such, we have demonstrated the ability of baculovirus-expressed DRV {sigma}C protein to induce serum-neutralizing antibodies and protection against experimental reovirus infection in ducks (G. Kuntz-Simon and others, unpublished results).

In contrast with {sigma}C, BLAST analyses and pairwise comparisons revealed that the three other DRV {sigma} class-encoded genes and proteins, {sigma}A, {sigma}B and {sigma}NS, were highly related to ARV, and to a lesser extent to other orthoreovirus counterparts. Sequence analyses also suggested a high similarity at the secondary structure level (data not shown). We have previously shown that DRV {sigma}B has an antigenic character (Le Gall-Reculé et al., 1999 ), but further experiments are necessary to determine whether DRV {sigma}A, {sigma}NS and {sigma}B are functionally related to ARV counterparts. It should be noted that a shift in S2 and S3 segment assignment was observed between the two DRV strains we used in this study. We showed that {sigma}B-encoded genes of both strains are 1201 bp long and 98·4% identical. Moreover, they migrated at the same position in the electrophoresis gel. In contrast, the {sigma}NS-encoded gene seemed to be retained in the case of DRV-89330. This retention could be due to the larger size of DRV-89330 {sigma}NS-encoded gene, as compared with DRV-89026 counterpart, and/or to a modification at the secondary structure level. Cloning and sequencing of the DRV-89330 S2 segment, as well as electrophoresis under denaturing conditions, would permit us to answer these questions. Polymorphism in the electrophoretic mobility of individual segments was previously described by comparing various strains from other single orthoreovirus species (Gouvea & Schnitzer, 1982 ; Dermody et al., 1990 ; Wu et al., 1994 ). However, this polymorphism was essentially described for considerable heterogeneity with regard to the migration of the minor outer capsid protein-encoded gene and not for a non-structural-encoded gene as here. This is all the more surprising as we found the DRV-89330 and DRV-89026 {sigma}C-encoded genes to be quite similar (99·2%) and no polymorphism in electrophoretic mobility was observed between S4 segments of both strains.

The data we obtained about molecular organization of DRV small genome segments raised the question about the relative classification of DRV in the Orthoreovirus genus. Examination of cDNA sequences revealed that the four DRV S class genes contained conserved octa- and pentanucleotides at the 5' and 3' end, respectively, both common to ARVs. The 5' GCT triplet is also present in NBV and MRV genome segments but absent from BRV (Duncan, 1999 ). On the other hand, the 3' TCATC sequence is common in all orthoreoviruses. This pentanucleotide, distinct from the 3'-terminal sequences of other genera in the Reoviridae family, has been described to represent a signature for orthoreoviruses (Duncan, 1999 ). These terminal sequences supported the classification of DRV as an orthoreovirus. Moreover, they led us to hypothesize that genetic reassortment could occur between DRV and ARV genome segments. Thus, DRV and ARV, which markedly differ in pathogenic properties (Marius-Jestin et al., 1988 ), protein profiles (Heffels-Redmann et al., 1992 ) and electropherotypes (this study), could be selected to generate reassortants. In conjunction with biochemical and immunological analysis, this genetic approach could be used to study the functional roles of individual genes or proteins, notably the role of p17 in ARV pathogenesis.

In addition to the presence of conserved terminal sequences, and in conjunction with previous biophysical comparisons (Malkinson et al., 1981 ) and antigenic relationships, the phylogenetic relationships we established between DRV and other orthoreoviruses supported the classification of DRV into Orthoreovirus genus subgroup II. The fact that the tree topologies were identical for the three most conserved {sigma} class proteins indicated that natural genome reassortment did not contribute to the recent evolution of these orthoreoviruses. However, based on the divergent antigenic relationships between DRV and ARV (Heffels-Redman et al., 1992 ), and providing evidence for the diversity of the polycistronic S class genes of reoviruses isolated from birds (this study), we suggest that DRV segregates into a specified genogroup among avian reoviruses. For comparison, it would be interesting to obtain data on turkey reovirus {sigma}C protein, which has been shown to be antigenically related to ARV. Nevertheless, the derivation of duck and chicken reoviruses from a common ancestor is still unclear. Jeurissen & Janse (1998) reported the very low cross-reactivity of antibodies specific for chicken with duck tissues, contrary to the situation observed with turkey or quail cells. They related these results to the phylogenetic distance between the types of birds examined, since ducks belonging to the Anseriformes represent ‘primitive’ birds, whereas the three other species belong to the Galliformes and represent more ‘advanced’ birds. In evolutionary terms, the duck is more closely related to amphibious reptiles such as turtles. Therefore, it should be interesting to compare the DRV small genome sequences to those of reoviruses isolated from reptiles (Lamirande et al., 1999 ; Vieler et al., 1994 ; Ahne et al., 1987 ).


   Acknowledgments
 
We are grateful to Marie-Odile Le Bras for performing reovirus multiplication and to Martine Cherbonnel for helping in some laboratory experiments. We thank Roy Duncan (Dalhousie University, Halifax, Canada) for having kindly provided the BRV S4 ORF2 sequence. This work has been supported by a grant from the FEDER.


   Footnotes
 
b Present address: AFSSA–Ploufragan, Swine Virology and Immunology Unit, BP 53, 22440 Ploufragan, France.


   References
Top
Abstract
Introduction
Methods
Results
Discussion
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Received 11 September 2001; accepted 11 January 2002.



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