Complete sequence characterization of the genome of the St Croix River virus, a new orbivirus isolated from cells of Ixodes scapularis

Houssam Attoui1, Julie M. Stirling3, Ulrike G. Munderloh4, Frédérique Billoir2, Sharon M. Brookes3, J. Nicholas Burroughs3, Philippe de Micco1,2, Peter P. C. Mertens3 and Xavier de Lamballerie2

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


   Abstract
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Abstract
Introduction
Methods
Results
Discussion
References
 
An orbivirus identified as St Croix River virus (SCRV) was isolated from cells of Ixodes scapularis ticks. Electron microscopy showed particles with typical orbivirus morphology. The SCRV genome was sequenced completely and compared to previously characterized orbivirus genomes. Significant identity scores (21–38%) were detected between proteins encoded by segments S1, S2, S4, S5, S6, S8, S9 and S10 of SCRV and those encoded by segments S1, S3, S4, S5, S6, S7, S9 and S10, respectively, of Bluetongue virus (BTV), the prototype orbivirus species. The protein encoded by SCRV genome segment 3 (VP3) is thought to be the equivalent of VP2 of BTV. Segment 7 encodes a protein homologous to non-structural protein NS2(ViP) of BTV. Analysis of VP1(Pol) (segment 1) shows that SCRV is an orbivirus, distantly related to the other sequenced species. Blot hybridizations and sequence comparisons of the conserved protein encoded by genome segment 2 (the T2 subcore shell protein) with previously identified orbiviruses confirm that SCRV is a distinct orbivirus species, unrelated to another tick-borne species, Great Island virus. The presence of SCRV in cells prepared from tick eggs suggests that transovarial transmission of SCRV may occur in ticks.


   Introduction
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Abstract
Introduction
Methods
Results
Discussion
References
 
The genus Orbivirus includes 19 recognized species that, together with nine unassigned isolates, represent one of nine established and two newly proposed genera within the family Reoviridae (Orthoreovirus, Orbivirus, Rotavirus, Coltivirus, Aquareovirus, Cypovirus, Fijivirus, Phytoreovirus, Oryzavirus, Seadornavirus (proposed) and Entomoreovirus (proposed); Mertens et al., 2000 ). In a recent review of virus classification and taxonomy, parameters have been defined for the identification of virus species in each of the genera within the family Reoviridae (Mertens et al., 2000 ). An ability to ‘reassort’ genome segments is defined as the primary determinant of virus species (as discussed by Calisher & Mertens, 1998 ). However, species parameters also include the use of nucleotide or predicted amino acid sequence comparisons to examine the relationships that exist between virus isolates, and these methods can therefore be used to identify the members of the same or distinct virus species, or even genera.

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 (23–33% 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.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Virus propagation and electron microscopy.
The IDE2 cell line was found to contain an endogenous orbivirus (SCRV) that has established a persistent infection and can replicate with no visible cytopathic effect. IDE2 cells were grown at 31 °C in L-15B medium (Munderloh & Kurtti, 1989 ) as described elsewhere (Munderloh et al., 1994 ). The virus was pelleted from the cell culture supernatant, resuspended and partially purified by using a discontinuous 40/66% (w/w) sucrose gradient in 0·2 M Tris–HCl, pH 8·0. The virus was adsorbed to formvar/carbon-coated grids and stained with 2% methylamine tungstate for 20 s, rinsed in water and dried prior to being examined by electron microscopy on a JEOL 1200EX transmission electron microscope.

{blacksquare} 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 ).

{blacksquare} 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).

{blacksquare} 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 393–548 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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Table 1. Sequences used in phylogenetic analyses

 


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Fig. 2. Phylogenetic comparison of the viral polymerase [VP1(Pol)] proteins of SCRV, other orbivirus species and members of other genera within the family Reoviridae. The analysis (presented as a radial tree) was performed with the help of TreeView. Accession numbers and further details of the sequences and viruses used are included in Table 1(a).

 


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Fig. 3. Phylogenetic comparison of the T2 proteins (the major component of the subcore shell) of SCRV and other orbivirus species. This protein is equivalent to the VP3(T2) protein of BTV. Since many of the available sequences are incomplete, the analysis (presented as a radial tree) was based on partial sequences (aa 393–548 relative to BTV-10). Accession numbers and further details of the sequences and viruses used are included in Table 1(b).

 
{blacksquare} Genome hybridization analysis.
dsRNA was extracted from tissue culture cells infected with cell-adapted orbiviruses by using the protocol described above. The viruses included representative members of recognized Orbivirus species and some unassigned viruses. A list of these viruses is given in Table 2. Full-length cDNA of SCRV genome segment 2 (encoding the T2 subcore shell protein) was prepared and radiolabelled with [{alpha}-32P]dATP by nick translation with the Klenow enzyme as described by Sambrook et al. (1989) for use as a probe. Briefly, full-length segment 2 cDNA was denatured for 3 min at 95 °C then added to 32 µl water, 10 µl oligonucleotide ligation buffer, 2 µl 10 mg/ml BSA, 185 Bq [{alpha}-32P]dATP and 5 U Klenow enzyme. The mixture was incubated for 5 h at room temperature and then the probe was denatured at 65 °C. A cDNA probe of genome segment 3 of BTV-1 from South Africa (BTV-1 SA) was also produced and was used to detect BTV RNA (from BTV-1 SA, BTV-1 A and BTV-10) as a positive control.


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Table 2. Viruses used in dot-blot analysis

 
The preparations of viral dsRNA to be probed were denatured in 1 µl 10 mM methylmercuric hydroxide for 10 min at room temperature and spotted on Hybond-N transfer membrane at concentrations of 10, 100 and 1000 ng per spot (concentration determined by spectrophotometric measurement). The bound RNA was fixed to the filter by exposure to UV light for 5 min. The filters were incubated in pre-hybridization solution (6x SSC, 0·5% SDS, 10x Denhardt’s solution, 0·05 mg/ml yeast tRNA) at 60 °C for 2 h in a hybridization oven (Hybaid). The denatured probe was added followed by overnight incubation at 60 °C. The filters were washed twice in 50 ml 2x SSC, twice in 50 ml SSC+0·1% SDS and twice in 50 ml 0·1x SSC+0·1% SDS for 30 min each at 65 °C. These stringency conditions permit the detection of >85% nucleotide sequence identity (Mertens et al., 2000 ). The filters were wrapped in cellophane and exposed to X-ray film.


   Results
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Abstract
Introduction
Methods
Results
Discussion
References
 
Cloning SCRV cDNA
Segments 1–10 of the SCRV genome were cloned and sequenced. The corresponding sequences have been deposited in GenBank (see Table 3 for accession numbers). The lengths of the segments and their corresponding encoded proteins are given in Table 3. Analysis of the 5' and 3' non-coding regions (NCRs) showed that all of the segments except segment 6 share five conserved nucleotides at their 5' ends and three conserved nucleotides at their 3' ends (5'-GUAAU...UAC-3'; Table 3). Moreover, the first and last three nucleotides of all of the segments are inversely complementary.


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Table 3. Characteristics of dsRNA segments 1–10, putative encoded proteins and 5'- and 3'-NCRs of SCRV

 
Sequence analysis
According to results obtained by using the BLAST programs, the putative proteins encoded by SCRV segments 1, 4, 5, 6, 9 and 10 produced significant matches with those encoded by segments 1, 4, 5, 6, 9 and 10, respectively, of BTV (Fig. 1). The proteins encoded by SCRV segment 2 showed significant identity to those encoded by segment 3 of BTV-13 (25%) (insect-borne) and segment 2 of BRDV (23%) (tick-borne), while the proteins encoded by SCRV genome segment 8 and BTV-15 segment 7 showed 21% identity. SCRV segment 3 (VP3) encodes a protein that is comparable to the outer capsid protein, VP2, of the insect-borne orbiviruses according to CLUSTAL W, showing an identity of 25% to VP2 of BTV-10. The protein encoded by genome segment 7 of SCRV was compared to the NS2(ViP) protein (genome segment 8) of the insect-borne orbiviruses (using the same program) and an identity of 23% was demonstrated with NS2(ViP) of BTV-10.



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Fig. 1. Correspondence between the genomes of SCRV, BTV and BRDV. The putative functions of SCRV proteins were deduced by comparison to the already established functions of BTV proteins. The functions and the abbreviations (in parentheses) used to indicate these roles are taken from Mertens et al. (2000) . Percentage similarities and identities to SCRV sequences are shown. Where two percentages are shown, the values are amino acid identity and similarity obtained by using BLAST 2.0; single values represent amino acid identity calculated from alignments generated by CLUSTAL W in cases where BLAST did not produce significant matches. NS, Not sequenced. Continuous arrows indicate homologous segments for which significant amino acid identity scores were found using the BLAST analysis. Dotted arrows indicate homologous segments for which BLAST searches did not produce significant matches, while CLUSTAL W alignments showed significant patterns from which amino acid identity was calculated.

 
Fig. 2 shows a neighbour-joining tree of the amino acid sequence of VP1 of SCRV aligned with all of the VP1(Pol) sequences available for different orbiviruses as well as representative members from other genera within the family Reoviridae. Between 36 and 38% amino acid sequence identity was detected in VP1(Pol) between SCRV and the other orbiviruses. In a previous study, Attoui et al. (2000b ) found that the polymerase sequences of viruses belonging to a single genus within the family Reoviridae share identity values greater than 20%. The data presented here therefore confirm that SCRV is an orbivirus. However, these values are significantly lower than the amino acid sequence identity (55–64%) detected between the VP1(Pol) sequences from AHSV (isolate AHSV-9), BTV (isolates BTV-2, -10, -11 and -13) and PALV (isolate CHUV), demonstrating that SCRV is related only distantly to these insect-transmitted orbivirus species.

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 (33–83%) 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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Fig. 4. Electron micrographs of SCRV particles negatively stained with 2% methylamine tungstate. (a) Virus particles present in material pelleted from tissue culture supernatant. One of the particles shown was more permeable to stain than the others. (b) SCRV particles partially purified by sucrose gradient centrifugation. Bars, 50 nm.

 

   Discussion
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Introduction
Methods
Results
Discussion
References
 
Ticks of the family Ixodidae are known vectors for a number of aetiological agents, including the bacteria Borrelia burgdorferi, Ehrlichia chaffeensis and Rickettsia species, the protozoan Babesia microti (Munderloh et al., 1994 ) and a number of flaviviruses (McLean et al., 1985 ), orbiviruses and coltiviruses (Mertens et al., 2000 ).

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 36–38%, 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 22–25% 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.


   Acknowledgments
 
The authors wish to thank Allan Gould and Ian Pritchard for the electronic version of their sequences, Bob Shope and Nick Karabatsos for providing isolates of additional orbivirus species and unassigned viruses and Adam Meyer for adapting these viruses to replicate in cell culture. This study is partially supported by EU grant ‘Reo ID’ number QLRT-1999-30143. The work at IAH is also supported by the Ministry of Agriculture, Fisheries and Food, UK.


   Footnotes
 
The GenBank accession numbers of the sequences reported in this paper are AF133431, AF133432 and AF145400AF145407.


   References
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Abstract
Introduction
Methods
Results
Discussion
References
 
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Received 9 November 2000; accepted 7 December 2000.