Laboratoire de Virologie Moléculaire, EFS Alpes-Méditerranée1 and Laboratoire de Virologie Moléculaire, Tropicale et Transfusionnelle2, Unité des Virus Emergents EA3292, Faculté de Médecine de Marseille, 27 Boulevard Jean Moulin, 13005 Marseille cedex 5, France
Institute for Animal Health, Pirbright Laboratory, Ash Road, Pirbright, Woking, Surrey GU24 0NF, UK3
Department of Entomology, University of Minnesota, 219 Hodson Hall, 1980 Folwell Avenue, St Paul, MN 55108, USA4
Author for correspondence: Xavier de Lamballerie. 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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Orbiviruses are transmitted by Culicoides midges, ticks, phlebotomine flies and anopheline and culicine mosquitoes and have genomes consisting of 10 segments of double stranded RNA (dsRNA). The type species of the genus is Bluetongue virus (BTV). BTV, African horsesickness virus (AHSV) and Epizootic haemorrhagic disease virus (EHDV) represent the three economically most important vertebrate pathogen species belonging to this genus. BTV, AHSV and EHDV are transmitted by Culicoides midges (Mertens, 1999 ; Mertens et al., 2000
).
Broadhaven virus (BRDV), a tick-borne orbivirus of the Great Island virus species, was isolated from Ixodes uriae ticks (Nuttall et al., 1981 ). It is the only tick-borne orbivirus for which nucleotide sequence data are available (for genome segments 2, 5, 7 and 10: Moss et al., 1992
). Comparison of the corresponding protein sequences with those of insect-borne orbiviruses shows a considerable divergence (2333% aa identity).
Munderloh et al. (1994) established a number of tick cell lines, designated IDE and ISE, from eggs of Ixodes scapularis (the black-legged tick). The eggs were obtained from a tick collected from a hunter-killed white-tailed deer (Odocoileus virginianus) in western Wisconsin, near the St Croix River. We report here the isolation and molecular characterization of a virus, named St Croix River virus (SCRV), from the IDE2 cell line. In order to facilitate comparisons of individual proteins between virus species, we have added the abbreviations used by Grimes et al. (1998)
and Mertens et al. (2000)
to indicate protein function wherever possible.
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Methods |
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Isolation and purification of nucleic acids.
SCRV dsRNA was extracted from IDE2 cells by a commercially available guanidinium isothiocyanate-based procedure (RNA NOW reagent, Biogentex). Briefly, cells from a 75 cm2 culture flask were scraped off and pelleted by centrifugation at 800 g at 4 °C for 10 min. The supernatant was discarded and the pellet was dissolved in 500 µl of lysis reagent:6 M guanidinium isothiocyanate and then mixed with 500 µl of the extraction reagent. Two hundred µl chloroform was added and the mixture was shaken for 1 min, kept for 10 min on ice and then centrifuged at 12000 g for 10 min at 4 °C. The supernatant was recovered, mixed with 900 µl 100% isopropanol and incubated overnight at -20 °C. The RNA was pelleted by centrifugation at 18000 g for 10 min at 4 °C, washed with 75% ethanol, dried and dissolved in water. The dsRNA was purified further by precipitating high molecular mass ssRNA with 2 M LiCl as described elsewhere (Attoui et al., 2000 a ).
Cloning of the dsRNA segments.
The genome segments of SCRV were copied into cDNA, cloned and sequenced according to the single-primer amplification technique described previously (Lambden et al., 1992 ; Attoui et al., 2000a
, b
). Briefly, a defined 3'-amino-blocked oligodeoxyribonucleotide was ligated to both of the 3' ends of the dsRNA segments using T4 RNA ligase, followed by reverse transcription and PCR amplification with a complementary primer. PCR amplicons were analysed by agarose gel electrophoresis, ligated into the pGEM-T cloning vector (Promega) and transformed into competent E. coli XL-blue cells. Insert sequences were determined with M13 universal primers, the D-Rhodamine DNA sequencing kit and an ABI prism 377 sequence analyser (Perkin Elmer).
Sequence analysis.
All sequence alignments were performed with the CLUSTAL W software (Thompson et al., 1994 ) and the local BLAST program implemented in the DNATools package version 5.01.661 (written by S. W. Rasmussen; available at http://www.crc.dk/phys/dnatools.htm). Phylogenetic analyses were carried out with the program MEGA (Kumar et al., 1993
) using the p-distance determination algorithm. Sequence relatedness is reported as percentage identity. Tree drawing was performed with the help of the TreeView program (Page, 1996
). Comparisons of SCRV sequence data with those available from nucleic acid and protein databases were performed using the NCBI gapped BLAST program (http://www3.ncbi.nlm.gov/BLAST).
Additional orbivirus sequences were retrieved from databases, or have been published previously, including: (i) amino acid sequences of putative RNA-dependent RNA polymerases (RdRps) of representative strains from species within eight genera of the family Reoviridae (details of the sequences are listed in Table 1; the resulting tree is shown in Fig. 2
) and (ii) amino acid sequences (aa 393548 relative to BTV-10 sequence) of the T2 protein (subcore shell protein) of isolates from different orbivirus species (see Table 1
, the resulting tree is shown in Fig. 3
).
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Results |
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Analysis of VP2 of SCRV showed it to be homologous to the T2 protein, which forms the subcore shell of the orbivirus capsid [VP2(T2) of BRDV (Moss & Nuttall, 1994 ) and VP3(T2) of BTV (Grimes et al., 1998
)]. As a consequence of its important function in virus protein/RNA structure and assembly, the T2 protein is highly conserved (Grimes et al., 1998
; Gouet et al., 1999
), exhibiting very high levels of sequence identity (>91%) within a single orbivirus species (serogroup). Amino acid identities for this protein range from 95·5 to 98·7% for Wongorr virus, 94 to 98% for Corriparta virus, 99% for AHSV, 95·5 to 100% for BTV, 95·5% for EHDV, 92·3% for Wallal virus, 99% for Warrego virus and 98% for Palyam virus. However, the T2 protein shows lower levels of identity (3383%) between distinct orbivirus species. The amino acid sequence identity that was detected between the T2 proteins of SCRV and the other orbiviruses ranged from 26 to 33%. A phylogenetic comparison of the amino acid sequence of VP2 of SCRV aligned with all of the T2 protein sequences available for different orbiviruses is shown in Fig. 3
.
Genome hybridization analysis
Northern blot analysis was used in an attempt to find any significant nucleotide sequence relatedness between genome segment 2 of SCRV and the genomic RNA of other unsequenced and some unassigned orbiviruses (Mertens et al., 2000 ). cDNA probes were synthesized and labelled radioactively using an SCRV segment 2 clone as the template. These experiments consistently gave negative results and no significant hybridization was detected to the RNA of any of the orbiviruses listed in Table 2
. A directly comparable probe, made from cDNA of genome segment 3 of BTV-1 SA (encoding the T2 protein), was bound efficiently by RNA of both homologous and heterologous BTV serotypes. However, this probe also failed to bind RNA from any of the uncharacterized orbivirus species or the unassigned isolates used (data not shown).
Electron microscopy
Virus particles present in material pelleted from tissue culture supernatant showed an indistinct surface morphology (Fig. 4a), typical of intact orbivirus particles (Mertens et al., 1987
; Burroughs et al., 1994
). SCRV particles that were partially purified by sucrose gradient centrifugation showed a more defined surface structure, with ring-shaped capsomeres that are characteristic of orbivirus core particles (Mertens et al., 2000
). They also had a smaller mean diameter, suggesting that they had lost outer capsid components (Fig. 4b
). The more intact but unpurified SCRV particles had a mean estimated diameter of 59·4±3·1 nm, while the partially purified core particles had a mean diameter of 55·2±2·6 nm. Although these size estimates are smaller than those for BTV particles determined by X-ray crystallography (Grimes et al., 1998
), it is inevitable that staining and drying during sample preparation will affect the particle structure significantly. The particle dimensions observed are comparable to those of BTV prepared by similar techniques.
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Discussion |
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A cell line prepared by Munderloh et al. (1994) from eggs of Ixodes scapularis ticks, designated IDE2, was found to contain an endogenous virus that possesses a segmented dsRNA genome and could be visualized by agarose gel electrophoresis (Attoui et al., 2000a
). The virus was shown by electron microscopy to have typical orbivirus morphology and was formally identified as an orbivirus by the sequence analyses that are presented in this paper and discussed below. The replication characteristics and pathogenicity of this recently identified virus have never been studied before and are currently under investigation.
The genus Orbivirus contains insect-borne and tick-borne viruses and viruses with no known vectors. The insect-borne orbiviruses have been studied intensively and full-length genome sequences have been determined for the major veterinary pathogens BTV, AHSV and EHDV (Roy & Mertens, 1999 ). In contrast, relatively little is known about the molecular biology of the tick-borne orbiviruses, despite the fact that they are transmitted by a large number of tick species (including ticks of the genera Argas, Boophilus, Hyalomma, Ixodes, Ornithodorus and Rhipicephalus), and the species Great Island virus contains the largest number of distinct orbivirus serotypes. Only partial sequences are available for genome segments 2, 5, 7 and 10 of the tick-borne BRDV (Moss et al., 1992
). Comparison of the protein sequences of the insect-borne orbiviruses and BRDV revealed amino acid sequence identities ranging from 24 to 36% (Moss et al., 1990
, 1992
; Moss & Nuttall, 1995
).
The characterization of the full-length genome of SCRV has facilitated the analysis of its genetic relationship to previously reported orbiviruses. VP1 of SCRV was found to contain the signature motifs of RNA-dependent RNA polymerases (RdRps) from viruses of the family Reoviridae and to match with a number of other orbivirus polymerases (Mertens et al., 2000 ). Analysis of the protein sequences of the highly conserved RdRps showed amino acid sequence identity equal to or greater than 20% within a single genus of the family Reoviridae. The amino acid sequence identity of SCRV VP1 to RdRps of other orbiviruses is 3638%, confirming its classification within the genus Orbivirus. However, this value is lower than that detected for VP1(Pol) between the species AHSV, BTV and PALV, which ranged between 55 and 64%, demonstrating that SCRV is related more distantly to these previously characterized, insect-transmitted orbiviruses. Phylogenetic analysis of RdRp sequences from viruses belonging to eight different genera of the family Reoviridae also confirm the placement of SCRV VP1(Pol) within the orbivirus cluster (Table 3
and Fig. 2
).
The sequence variations of orbivirus T2 proteins correlate with virus serogroup (or species) (Gould, 1987 ; Gould & Pritchard, 1991
; Mertens, 1999
; Mertens et al., 2000
). The SCRV T2 protein is only 23% identical to BRDV VP2 and 2225% identical to the VP3(T2) proteins of other insect-transmitted orbiviruses. This identity is significantly lower than that detected between isolates from a single orbivirus species (>91% sequence identity). These analyses and comparisons confirm that SCRV belongs to the genus Orbivirus, but also that it does not belong to any of the previously recognized orbivirus species that have been sequenced (Fig. 3
).
Dot-hybridization assays were carried out under stringency conditions that permitted the detection of >85% nucleotide sequence identity. Using cDNA probes made from the genome segment 2 of SCRV or segment 3 of BTV, no cross-hybridization was detected to RNA from representative members of established orbivirus species or to some unassigned viruses. These findings support the classification of SCRV as a species that is distinct from any of the previously isolated orbiviruses.
Comparison of the proteins encoded by the genome segments of SCRV to those of insect-borne orbiviruses shows them to be highly divergent, with amino acid sequence identities ranging from 18 to 38%. The highest identity was found for VP1(Pol), reflecting its status as a highly conserved protein. At this stage, our knowledge of the genome sequences of tick-borne orbiviruses is limited by the data available. There is no evidence that SCRV is related more closely to BRDV than to insect-borne orbiviruses. Future sequence analyses of other tick-borne orbiviruses will reveal the genetic relationships between these viruses and will show if insect-borne and tick-borne orbiviruses represent different phylogenetic lineages (as observed in the case of flaviviruses; Billoir et al., 2000 ) or the same lineage.
The proteins encoded by SCRV genome segments 4, 5, 6, 8, 9 and 10 showed clear similarities to orbivirus proteins with known functional or structural roles (Fig. 1). Although lower identity scores were observed for the protein product of SCRV genome segment 7, this protein is considered likely to be homologous to the NS2(ViP) of other orbiviruses. Protein VP3 of SCRV shows a low but significant level of identity to the outer capsid protein, VP2, of the insect-borne orbiviruses and is therefore likely to be homologous. VP2 of BTV interacts with antibodies responsible for serum neutralization and is therefore subjected to antibody selective pressure. In consequence, the protein is highly variable, in a manner that correlates with virus serotype (serum neutralization type) (Mertens et al., 2000
). The low level of similarity of SCRV VP3 to any homologous orbivirus proteins is not therefore surprising.
Sequence characterization of SCRV has revealed its genetic relationship to other sequenced orbiviruses and has formed the basis of its identification as a new orbivirus species (now accepted by the ICTV). The availability of the complete genome sequence will facilitate the development of standardized serological and sequence-specific molecular assays (PCR or probe techniques) for the study of SCRV epidemiology in the field. Transovarial transmission is considered to be relatively common in ticks and may be essential for the survival of many tick-borne viruses (Turell, 1988 ). The ability of SCRV to remain infectious on the surface of the tick egg or under the conditions used for treatment and maintenance of tick eggs for almost a month is unknown. However, confirmation of transovarial transmission of SCRV in ticks will require further study, possibly involving surface sterilization of the eggs or infection of Ixodes scapularis ticks with crude or purified virus.
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Acknowledgments |
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Footnotes |
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Received 9 November 2000;
accepted 7 December 2000.