1 Unité des Virus Emergents: EFS Alpes-Méditerranée and Faculté de Médecine de Marseille, Université de la Méditerranée, 27 Boulevard Jean Moulin, 13005 Marseille cedex 5, France
2 Institute for Animal Health, Pirbright Laboratory, Ash Road, Pirbright, Woking, Surrey GU24 0NF, UK
3 Maladies Virales Émergentes et Systèmes d'Information UR034, Institut de Recherche pour le Développement, Marseille, France
Correspondence
Houssam Attoui
h-attoui-ets-ap{at}gulliver.fr
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for BAV-Ch genome segments 3, 4 and 5 are AY549307AY549309.
A supplementary table showing details of the RdRp sequences used in phylogenetic analysis is available in JGV Online.
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INTRODUCTION |
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The BAV genome clearly identifies the virus as a member of the family Reoviridae, a large family of viruses containing 10, 11 or 12 segments of dsRNA, which currently includes a total of 12 distinct genera (Mertens, 2004; Mertens & Diprose, 2004
; Mertens et al., 2004
). The genomes of BAV and Kadipiro virus (KDV) have been sequenced (Attoui et al., 2000
) and these data identify them as distinct species within the new genus Seadornavirus (type species BAV), family Reoviridae (Attoui et al., 2000
, 2004a
). However, until now, the morphology and biochemistry of the seadornaviruses have not been studied extensively.
Reoviruses (a term used here to indicate any member of the family Reoviridae) have been isolated from a wide range of mammals, birds, reptiles, fish, crustaceans, marine protists, insects, ticks, arachnids, plants and fungi and include a total of 75 virus species, with 30 further tentative species reported to date (Brussaard et al., 2004
; Mertens et al., 2004
). Reovirus particles have icosahedral symmetry with a diameter of approximately 6085 nm. They are usually regarded as non-enveloped, although some can acquire a transient membrane envelope during morphogenesis or cell exit (Murphy et al., 1968
; Martin et al., 1998
; Mertens et al., 2000
; Owens et al., 2004
). The morphology of some reoviruses has been studied intensively by X-ray crystallography and cryo-electron microscopy (Prasad et al., 1988
; Yeager et al., 1990
, 1994
; Grimes et al., 1998
; Gouet et al., 1999
; Hill et al., 1999
; Reinisch et al., 2000
; Diprose et al., 2001
; Nason et al., 2004
) and they can contain one, two or three concentric protein layers, identified here as subcore, core and outer capsid, respectively. The inner-capsid layers and proteins are involved primarily in virus assembly and replication, and show a remarkable degree of structural conservation between different genera, exemplified by the subcore shell, which is constructed from 120 molecules of a single protein (Grimes et al., 1998
; Reinisch et al., 2000
; Mertens, 2004
). In contrast, the outer-capsid proteins, which are involved in virus transmission, cell attachment and penetration, show greater variation, reflecting differences in the targeted host species, as well as responses to immune selective pressure by neutralizing antibodies.
The reoviruses can be subdivided into two groups. The spiked or turreted viruses have 12 icosahedrally arranged projections (called turrets or spikes) situated on the surface of the icosahedral core particle, one at each of the fivefold axes (e.g. orthoreoviruses or cypoviruses) (Baker et al., 1999; Hill et al., 1999
; Nibert & Schiff, 2001
). In contrast, cores of the non-spiked or non-turreted viruses have a protein bilayer structure, with a smooth or bristly surface appearance (e.g. rotaviruses or orbiviruses) (Grimes et al., 1998
; Baker et al., 1999
; Mertens et al., 2000
, 2004
).
We report a biomolecular study of the original Chinese strain of BAV (BAV-Ch), an isolate from the cerebrospinal fluid of a patient with encephalitis. Electron microscopy and electrophoretic analyses were used to identify the individual virus structural proteins and their location within the virus particle. The copy numbers of each protein present in purified virions were also determined, confirming their individual structural and functional roles. Sequence analysis of the viral genome has been completed, helping to identify homologous proteins in other reoviruses, and a large subset of the viral proteins was expressed for antibody production, identifying two serotypes of BAV.
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METHODS |
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BAV cores were purified from the initial cell pellet. These were lysed in 5 ml deionized water (18 M resistivity units) and run in a Potter homogenizer for 15 strokes, mixed vigorously with an equal volume of Vertrel-XF (Dupont) and centrifuged at 2000 g. The aqueous phase was layered onto either a discontinuous Optiprep (Sigma) gradient [10, 20, 30, 40 and 55 % Optiprep in 100 mM Tris/HCl (pH 7·5)] or a caesium chloride gradient (Burroughs et al., 1994
), then centrifuged at 10 °C for 2 h at 210 000 g. BAV particles were recovered at the interface of the 40 and 55 % layers and subjected to a second round of caesium chloride ultracentifugation. The resulting core particle band was harvested and centrifuged over a sucrose cushion [66 % (w/w) in 100 mM Tris/HCl (pH 7·5), 10 mM MgCl2] at 150 000 g for 2 h, then recovered and dialysed overnight at 25 °C against 100 mM Tris/HCl (pH 8·0), 10 mM MgCl2.
Electron microscopy.
Purified BAV particles were adsorbed onto Formvar/carbon-coated grids, stained with 2 % potassium phosphotungstate or uranyl acetate and examined by using a Philips Morgagni 280 transmission electron microscope. Infected cells recovered at 2430 h p.i were pelleted by centrifugation at 600 g for 10 min and fixed for 1 h in 4 % glutaraldehyde, post-fixed for 30 min in 1 % phosphate-buffered osmium tetroxide, dehydrated in a graded ethanol series and embedded in an AralditeEpon mixture (Mollenhauer, 1964). Thin sections were cut, stained with lead citrate (Rehse-Küpper et al., 1976
) and examined by transmission electron microscopy (TEM).
Sequence analysis of BAV-Ch genome segments 3, 4 and 5.
BAV-Ch dsRNA was extracted from concentrated purified virus by using RNA-Now reagent (Biogentex) and copied into cDNA as described previously (Attoui et al., 1998). The full lengths of segments 3, 4 and 5 were sequenced by using primers designed from segments 3, 4 and 5 of BAV-In6423 (GenBank accession nos AF134515AF134517). The theoretical protein sequence was used for identification of proteins by mass spectrometry (see below). The BAV-Ch sequences were compared with those of BAV-In6423 by using the BLAST program implemented in the DNATools package (version 5.2.018; S. W. Rasmussen, Valby Data Center, Denmark).
Recombinant protein production and animal immunization.
Viral proteins VP7VP12 of BAV-Ch and VP9 of BAV-In6969 were expressed as described previously (Mohd Jaafar et al., 2004). Briefly, segments 712 were cloned in vector pGEX-4T-2 to express glutathione S-transferase (GST)-fused proteins in Escherichia coli BL-21. Proteins were purified by glutathione affinity chromatography.
Mouse immune ascitic fluid (MIAF) against BAV-Ch virions was prepared by four initial intraperitoneal (IP) injections, at 2 week intervals, of 100 p.f.u. inactivated purified virus particles into 9-week-old mice. Similarly, MIAFs against individual recombinant-expressed proteins were also prepared for VP7VP12 inclusive, using 100 µg each protein. MIAF production was induced by injecting 0·5 ml pristane IP and ascitic fluid was recovered 12 days later.
Analysis of virus structural proteins by SDS-PAGE, mass spectrometry and Western immunoblotting.
Percoll-purified whole virus was mixed with an equal volume of denaturation buffer [160 mM Tris/HCl (pH 6·8), 4 mM EDTA, 3·6 % SDS, 60 mM dithiothreitol (DTT), 0·2 % -mercaptoethanol, 0·8 % methionine] and heat-denatured at 95 °C for 3 min. The proteins were analysed by SDS-PAGE using 10 % gels, then stained with 0·05 % Coomassie blue in methanol/acetic acid/water (45 : 10 : 45).
Individual BAV proteins were identified by electrospray ionization mass spectrometry/mass spectrometry (Eurogentec Proteomics). Protein bands were excised from gels and destained by using alternating high and low concentrations of acetonitrile/Tris/HCl (pH 8·1) [250 µl of a mixture of 250 mM Tris/HCl (pH 8·1) and 50 % (v/v) acetonitrile (mg gel)1, followed by washing with 200 µl acetonitrile to dehydrate the gel]. DTT was added (65 mM final concentration) to reduce disulphide bonds, followed by alkylation of reduced cysteine residues by 2·5 % (w/v) iodoacetamide. Bands were digested in gel by using 44 µg porcine trypsin (mg gel)1 (Roche) under non-reducing conditions in 50 mM Tris/HCl (pH 8·1) for 16 h at 35 °C. The resulting peptides were desalted by using ZipTips (Millipore) and analysed by using QStar XL (Applied Biosystems). MS data were acquired within the range of 1002000 m/z. Peptides were also subjected to capillary liquid chromatography and separated on a C18 column using an acetonitrile/formic acid mixture (70 % aqueous acetonitrile containing 0·1 % formic acid) before injection into LCQ-FTICR and QTOF 2 mass spectrometers. MS data were acquired within the range of 502000 m/z. The sequences of at least three peptides (10 aa or longer) were used to identify each protein derived from the purified particles by referring to the viral genome sequence.
The virus structural proteins separated by SDS-PAGE (whole virus purified on Percoll) were electroblotted onto nitrocellulose membranes. The presence of proteins VP7VP12 was tested by Western immunoblotting with the corresponding ascitic fluids produced by using recombinant proteins. The cross-reactivity of VP9 from BAV-Ch and BAV-In6969 was tested by Western immunoblotting.
Radiolabelling and enumeration of the virion structural proteins.
C6/36 cells grown in a 75 cm2 flask were infected with BAV-Ch in Eagle's minimum essential medium (EMEM). After incubation at 27 °C for 6 h under 5 % CO2, the culture medium was replaced with methionine-deficient EMEM containing 50 µCi [35S]methionine ml1. Labelled cells were harvested and dissolved in denaturation buffer. Labelled proteins were analysed by SDS-PAGE as described above. The gel was dried and autoradiographed.
For enumeration of individual structural proteins from BAV-Ch particles, the cells were incubated in the presence of [35S]methionine for 3 days and virus was purified from the supernatant by centrifugation at 150 000 g for 1 h over 35 % sucrose (w/v) in Tris/HCl (100 mM, pH 8·0). The proteins were separated by 10 % SDS-PAGE using a meltable matrix (Protoprep; National Diagnostics). The gel was stained with Coomassie blue and individual protein bands were excised and melted as described by the manufacturer, then added to 5 ml Safe-Emulsifier scintillation fluid in polyethylene scintillation vials (Packard Instruments), mixed vigorously and counted in a Packard 460 liquid scintillation counter. Values for the number of methionine residues in each protein and the amount of label in each protein band were used to calculate the molar ratios of the different structural proteins.
Phylogenetic relationships between seadornaviruses and rotaviruses.
The sequences of the RNA-dependent RNA polymerases (RdRps) of different reoviruses (see Supplementary Table in JGV Online) were used in phylogenetic analyses. The sequences were aligned by using CLUSTAL W (Thompson et al., 1994) and a tree was constructed by using MEGA2 (Kumar et al., 2001
) with P-distance and Poisson correction. The alignment showed that the most conserved region among the polymerases lies within the core domain of the enzyme (located at similar positions in RdRps of different reoviruses) between aa 697 and 835 of BAV. This region, and the whole of the polymerase sequence, were used in phylogenetic comparisons. The sequences of the other seadornavirus proteins were also compared with those of rotaviruses and other reoviruses.
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RESULTS |
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Morphology and morphogenesis of virus particles
BAV-Ch virus particles purified from the supernatant of infected C6/36 cells were visualized by negative staining and electron microscopy (Fig. 1). The intact virion has a three-layered capsid structure, organized as two concentric capsid shells (core and outer capsid). The morphology and mean external diameter (7275 nm) of the intact BAV-Ch virions stained with phosphotungstic acid (Fig. 1a
) are typical of non-turreted reoviruses (Mertens et al., 2004
), having an appearance reminiscent of the rotaviruses, with fibre proteins projecting from the surface (Estes, 2001
). However, the fibres in BAV are much more numerous and appear to be less extended than in rotaviruses. Similar structures were also observed on negatively stained KDV virions (Attoui et al., 2004a
). Some of the unpurified BAV-Ch virus particles pelleted from infected tissue-culture supernatant had an envelope-like structure (data not shown), which may have been generated by budding through the cell membrane, as described for the orbiviruses (Martin et al., 1998
; Owens et al., 2004
).
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Thin sections showed large, electron-dense structures within the cytoplasm of BAV-Ch-infected cells (Fig. 1d), which correspond to the viral inclusion bodies (VIB) that are thought to be the main site of replication and particle assembly of other reoviruses (Brookes et al., 1993
; Estes, 2001
; Nibert & Schiff, 2001
; Mertens & Diprose, 2004
). Particles (
50 nm in diameter) with a smooth surface were detected mainly at the periphery of the VIB, although some particles were also observed within the VIB matrix. Virus particles were also detected within large vacuoles that were dispersed throughout the cytoplasm of the infected cell. These vacuoles contained multiple double-layered vesicles, lined with viral particles (
50 nm in diameter) at their inner surface (Fig. 1d
); it is possible that this reflects some involvement of cellular membrane structures or organelles in virus morphogenesis, transport or replication (as reported previously for the rotaviruses; Jourdan et al., 1997
; Sapin et al., 2002
). Virus entry into cells by endocytosis was suggested by the detection of virus particles in pits at the cell surface. Virions, which were also observed near the cell membrane, appeared to be budding from the cell surface.
Identification of structural and non-structural proteins
Host-cell protein synthesis is shut off 2 h post-BAV infection of C6/36 cells (data not shown), and the shut-off is complete by 6 h p.i. [35S]Methionine added to C6/36 cell cultures at 6 h p.i. was incorporated almost exclusively into 12 protein bands (resolved by SDS-PAGE; Fig. 2) that are thought to represent the different viral proteins (one protein per genome segment). Most of these have apparent molecular masses that agree with the theoretical sizes predicted by sequence analysis of the viral genome. The only exception is VP7, which migrates more slowly than expected.
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Enumeration of BAV structural proteins
[35S]Methionine-labelled BAV-Ch particles were purified and analysed by SDS-PAGE and the ratios of the different structural proteins were calculated (see Methods). VP2 and VP8 are the two most abundant proteins of the BAV core. The lower relative abundance and higher molecular mass of VP2 identifies it as the subcore-shell protein (equivalent to VP3 of Bluetongue virus (BTV) and VP2 of rotavirus; Mertens et al., 2000; Estes, 2001
). In contrast, VP8 is smaller and more abundant, identifying it as the core-surface T13 protein. VP8 and VP2 have a molar ratio of 6·5 in purified BAV-Ch particles, identical to the ratio of 780/120 that was previously detected between the subcore and core-surface proteins of both BTV and rotavirus (Lawton et al., 1997
; Grimes et al., 1998
; Stuart et al., 1998
; Mertens et al., 2000
). On this basis, the numbers of the VP8 and VP2 molecules in the BAV core are assumed to be 780 and 120, respectively, allowing the mean copy number of the other protein components of virus particles or cores to be calculated (Table 2
).
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Comparisons of BAV VP9 (283 aa) with the outer-coat proteins of other non-turreted reoviruses revealed size and sequence similarities (20 % amino acid identity) to the VP8* subunit (241 aa) of simian rotavirus A (strain SA11) VP4 (GenBank accession no. P12976) (Fig. 5a
). Similarities (
26 % amino acid identity) were also detected between BAV VP10 and the VP5* subunit of simian SA11 rotavirus A VP4 (Fig. 5b
), suggesting that VP9 and VP10 may have a collective role similar to that of rotavirus outer-capsid protein VP4.
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The RdRps of rotaviruses and BAV or KDV have 17 and 19 % amino acid identity, respectively, reflecting a similar protein function, but confirming that these viruses belong to two different genera of reoviruses (Mertens et al., 2000; Attoui et al., 2004a
, b
). The phylogenetic tree constructed by using the partial polymerase sequences (Fig. 6
) shows that rotaviruses and seadornaviruses are on adjacent branches, indicating that they share a common, if distant, phylogenetic origin. P-distance and Poisson correction algorithms gave a tree with identical topology (similar to the tree built from the complete polymerase sequences; Attoui et al., 2000
), in which four genera (Rotavirus, Seadornavirus, Phytoreovirus and Orbivirus) are located along the same evolutionary branch (although bootstrap values are low, as observed in all phylogenetic reconstructions of the family Reoviridae to date).
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DISCUSSION |
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Electron microscopy, radiolabelling, SDS-PAGE, mass spectrometry, sequencing and serological comparisons of viral proteins were also used to analyse the virus-particle structure. These studies demonstrate that BAV is a non-turreted virus and has a double-layered core particle (with a smooth outline) that is typical of the non-turreted reoviruses [e.g. the orbiviruses (Grimes et al., 1998) and rotaviruses (Estes, 2001
)]. When viewed by negative staining and electron microscopy, intact BAV-Ch particles have an appearance that is similar to that of the rotaviruses, with fibre proteins projecting from their surface. The mass distribution of BAV proteins in the core and outer coat is at a ratio of approximately 60 : 40 %. This is comparable to the other non-turreted reoviruses, including rotavirus (57 : 43 %) and the orbiviruses (54 : 46 %). In contrast, the protein distribution of the turreted orthoreoviruses is 35 : 65 %.
Thin-section TEM of BAV-infected cells showed intracellular virus factories (VIBs), with virions concentrated at their periphery, that are similar to those generated by other reoviruses (Mertens et al., 2000). However, one distinctive feature of BAV replication is the presence of virions within double-layered vesicles, contained within larger vacuole structures that are dispersed throughout the cytoplasm of infected cells. These structures may be involved in transport of the virus particles to the cell membrane prior to release by budding, or could indicate some involvement of intracellular membranes in virus assembly, as reported previously for the rotaviruses (Jourdan et al., 1997
; Sapin et al., 2002
).
BAV particles were observed budding from the cell surface by thin-section electron microscopy. Considerable numbers of virus particles (approx. 40 % of viral yield) were detected in the culture supernatant as early as 30 h p.i., although cells were not lysed until at least 4 days p.i. Virus particles surrounded by an envelope-like structure were found in material pelleted from the infected-culture supernatant. Orbiviruses can bud from the surface of persistently (non-lytically) infected insect and mammalian cells (Martin et al., 1998; Takamatsu et al., 2003
; Owens et al., 2004
), releasing membrane-enveloped virus particles. Budding of BAV particles without disrupting the cell-surface membrane may reflect its initial non-lytic multiplication in C6/36 cells.
BAV-Ch infection shuts off C6/36 cell protein synthesis, resulting in incorporation of [35S]methionine solely into viral proteins. The electrophoretic migration pattern and rate of polypeptides observed (one per genome segment) were in general agreement with genome sequence analyses. The only exception was VP7, which migrated slightly slower than expected.
Intact BAV-Ch virus particles contain seven structural proteins, five of which are situated in the virus core (VP1, VP2, VP3, VP8 and VP10). Based on their locations in the virus core, molecular masses and molar ratio, VP2 (120 copies) was identified as the putative BAV subcore-shell T2 protein and VP8 (780 copies) was postulated to be the core-surface T13 protein, allowing the numbers of other proteins in the BAV particle to be calculated. A BLAST sequence analysis identified BAV VP1 as the viral RNA polymerase, detecting conserved motifs also found in polymerases of other reoviruses. These include SGEL at positions 714717 [which conforms to the motif SG(E/K/L/R/S)(A/F/K/L/N/P/T)] and GDD at positions 759761, which is a core motif of the enzyme (Mertens et al., 2000). The BAV core is predicted to contain
27 copies of VP1, which is twice that previously observed in the orthoreoviruses, orbiviruses and the rotavirus polymerases. This suggests packaging of two BAV polymerase molecules at each of the 12 icosahedral vertices.
BAV VP3 is the least abundant of the core structural proteins, with approximately seven copies detected per particle. The sequence of BAV and KDV VP3 is similar to that of VP3 of rotaviruses (the guanylyltransferase). BAV VP3 also exhibits guanylyltransferase activity (Mohd Jaafar et al., 2005a). Structural studies of other reoviruses suggest that transcriptase complexes may be situated at each of the fivefold axes of the core particle, possibly as one complex per genome segment (Payne & Mertens, 1983
; Prasad et al., 1996
; Grimes et al., 1998
; Gouet et al., 1999
; Zhang et al., 1999
, 2003
; Diprose et al., 2001
; Pesavento et al., 2001
). We would therefore have predicted at least 12 copies of VP3 per core particle, to allow capping of the nascent mRNAs synthesized at each transcription site.
Earlier sequencing studies demonstrated amino acid identities ranging from 72 to 100 % between the proteins of different BAV isolates (Attoui et al., 2000). However, VP9 showed a maximum of 40 % amino acid identity, identifying two distinct virus genotypes: genotype A, represented by the Chinese isolate, BAV-Ch, and genotype B, represented by several isolates from mosquitoes caught in Indonesia (Attoui et al., 2000
). The outer coat of the BAV virion is composed of
300 copies per particle of VP4 and VP9. The only other outer-coat protein from a reovirus that is known to be incorporated in similar numbers is VP5 of BTV, which exists as 360 copies (present as 120 trimers) per particle (Hewat et al., 1992
; Schoehn et al., 1997
; Stuart et al., 1998
; Mertens et al., 2000
). Native and recombinant VP9 proteins of BAV-Ch (genotype A) and BAV-In6969 (genotype B) failed to cross-react by Western immunoblotting, indicating that VP9 is both antigenically variable and can be used to identify two serotypes, A and B (Mohd Jaafar et al., 2004
).
The location of VP9 on the outermost capsid layer, together with sequence similarities to rotavirus VP8*, suggest an involvement in cell attachment and that it is a likely target for neutralizing antibodies. Indeed, the structure and function of VP9 have recently been analysed by X-ray crystallography (Mohd Jaafar et al., 2005b), demonstrating that it forms trimers and shows structural similarities to the VP8* subunit of rotavirus cell-attachment protein VP4. Recent findings suggest that at least the VP5* subunit of rotavirus VP4 could also form trimers (Dormitzer et al., 2004
). Anti-VP9 antibodies (BAV-Ch VP9) were shown to neutralize virus infectivity, whilst soluble trimeric VP9 protein remarkably increased infectivity of BAV in C6/36 cells (Mohd Jaafar et al., 2005b
). These findings identified the involvement of VP9 in cell attachment and penetration.
BAV VP10 (260 copies per BAV core particle) appears to have no direct equivalent in the cores of other non-turreted reoviruses. However, sequence alignments showed significant sequence similarities with the VP5* subunit of simian rotavirus A outer-coat protein VP4 (Fig. 5
). Antibodies to VP10 do not neutralize virus infection, although when used in immunoblotting of BVA-Ch proteins from infected cells, they identified a band of the expected size (
28 kDa), as well as two other bands at
50 and
70 kDa (Fig. 4d
), indicating that VP10 may form dimers and trimers.
If BAV VP9 and VP10 have a collective role similar to that of the VP8*/VP5* subunits of rotavirus VP4 during the initiation of virus infection, it would suggest that VP10 may be present at the outer surface of the core and could form a stalk-base for trimers of VP9 (outer-coat protein). It may be significant that these proteins are present in similar numbers per BAV particle (300 and
250, respectively). Rotavirus VP5* and VP8* are generated by proteolytic cleavage of VP4, increasing the specific infectivity of the rotavirus particle. The expression of BAV VP9 and VP10 from two separate genome segments also achieves separation of the two protein sequences, which may be functionally important. It suggests that a significant evolutionary jump has occurred between the members of the genera Seadornavirus and Rotavirus, which is reflected in the different numbers of genome segments (12 and 11, respectively).
BAV proteins VP5, VP6, VP7, VP11 and VP12 were detected in infected cells, but were not found in the purified virion and can therefore be regarded as non-structural. Sequence comparisons failed to identify roles for VP5, VP6 and VP11. Previously, VP7 was found to exhibit similarities to certain protein kinases (Attoui et al., 1998, 2000
).
BAV and other seadornaviruses replicate efficiently in both mosquito and mammalian cell lines and are potential emerging BSL3 pathogens that may pose a future threat to human health. In this study, we have completed the BAV-Ch genome sequence analysis and an initial characterization of the virus architecture and organization. A possible evolutionary link between the rotaviruses and seadornaviruses is proposed, based on similar virus-particle morphology and sequence similarities observed in the outer-coat, guanylyltransferase and polymerase genes and proteins between BAV and the rotaviruses.
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ACKNOWLEDGEMENTS |
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Received 2 September 2004;
accepted 14 December 2004.