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1 Unité des Virus Emergents EA3292, Etablissement Français du Sang Alpes-Méditerranée and Faculté de Médecine de Marseille, 27 Boulevard Jean Moulin, 13005 Marseille cedex 5, France
2 Chinese Centers for Disease Control and Prevention, 100 Ying Xin Jie, Xuan Wu Qu, Beijing 100052, China
3 Department of Pathology, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-0609, USA
4 Maladies Virales Emergentes et Systèmes d'Information UR 034, Institut de Recherche pour le Développement, Faculté de Médecine de Marseille, 13005 Marseille, France
Correspondence
Houssam Attoui
h-attoui-ets-ap{at}gulliver.fr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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A table showing the sequences used in RdRps phylogenetic analysis of seadornaviruses is available as supplementary material in JGV Online.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). It includes two species, Banna virus (BAV) and Kadipiro virus (KDV), which together with 15 unassigned isolates, represents one of 12 established genera within the family Reoviridae (Orthoreovirus, Orbivirus, Rotavirus, Coltivirus, Aquareovirus, Cypovirus, Fijivirus, Phytoreovirus, Oryzavirus, Mycoreovirus, Seadornavirus and Idnoreovirus; Mertens et al., 2005).
Seadornaviruses are arboviruses that are transmitted by anopheline and culicine mosquitoes and have genomes consisting of 12 segments of double-stranded RNA (dsRNA). The type species of the genus is BAV, and it has been isolated within Indonesia (from mosquitoes) and China (from humans with encephalitis and from mosquitoes). The isolates from humans in China were made from the sera and cerebrospinal fluids of infected patients. Isolates of BAV have been repeatedly obtained from Culex and Anopheles mosquitoes in China (Chen & Tao, 1996; Li, 1992; Liting et al., 1995).
In recent attempts to isolate arboviruses in China, a new virus strain obtained from Aedes dorsalis mosquitoes in the Liao ning province in the North-East of China was found to possess a 12 segmented dsRNA genome. This virus was designated Liao ning virus (LNV).
Here, we present the complete genomic characterization of this new virus, which was identified as a new seadornavirus species encompassing two different serotypes. Biological information regarding its ability to replicate within insect and mammalian cells is also presented, which shows that LNV possesses the typical characteristics of arboviruses. We also present a comparative study of mice infected with LNV or BAV, which shows distinct replication properties of the two viruses.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The lyophilized product was used to infect C6/36 cells (third passage) in L-15 medium at 27 °C as described previously (Attoui et al., 2000b), and a virus stock was prepared. The virus was subsequently plaque purified in C6/36 cells using SeaPlaque agarose (Cambrex) and Neutral red (ICN Biomedicals) to identify the plaques. One clone was further propagated, purified and used for analysis.
Virus purification and electron microscopy.
Infected cells and supernatant were centrifuged at 3000 g for 10 min and the supernatant was recovered. Virus was concentrated from the supernatant at 200 000 g for 1 h using an SW41 rotor on an Optiprep cushion (60 % Iodixanol in water).
Cells were lysed by treatment with deionized water (18 M
) and mixed with the virus from the supernatant. The suspension was mixed with 1 M Tris/HCl pH 7·5 to obtain a final concentration of 100 mM Tris/HCl. The mixture was treated with an equal volume of Vertrel XF (Dupont), centrifuged at 2000 g at 4 °C for 10 min and the supernatant was recovered, layered on top of a discontinuous CsCl gradient (40 and 55 % CsCl in 100 mM Tris/HCl pH 7·5) as described before (Burroughs et al., 1994), and centrifuged for 2 h at 210 000 g in an SW41 rotor. The light blue virus band was recovered from the interface of the 40 and 55 % layers.
The virus was adsorbed onto Formvar–carbon coated grids, stained with 2 % potassium phosphotungstate for 30 s and dried prior to being examined by electron microscopy on a Philips Morgagni 268 transmission electron microscope.
Replication of LNV in mosquito and mammalian cell lines.
The virus was inoculated into other cell lines. The mosquito cell lines included AA23 (Aedes albopictus), A20 (Aedes aegypti), AE (Aedes aegypti) and A w-albus (Aedes w-albus). The mammalian cell lines included L-929 (mouse fibroblast), BHK-21 (hamster kidney), BGM (monkey kidney), Hep-2 (human adenocarcinoma) and MRC5 (human embryo lung). For this purpose, 100 µl of a C6/36 LNV-infected culture supernatant was added to the cell monolayers and incubated at 27 °C (mosquito) or at 37 °C (mammalian) for 1 h. The cells were washed twice with PBS and the culture medium was added. At day 5 post-infection (p.i.), the RNA was extracted from some of the harvested cells and an aliquot of the mixed cells and supernatant was used in a second passage.
The extracted RNA was processed for agarose gel electrophoresis and RT-PCR using specific LNV primers as described below.
Isolation and purification of nucleic acids.
LNV dsRNA was extracted from C6/36 cells using a commercially available guanidinium isothiocyanate-based procedure (RNA Now reagent; Biogentex). Cells were solubilized in 1 ml RNA Now reagent and the RNA was extracted. The extracted dsRNA was further purified by precipitating high molecular mass single-stranded RNAs in 2 M LiCl as described earlier (Attoui et al., 2000a).
Viral RNA was extracted from infected mouse blood using the QIAmp RNA blood minikit (Qiagen), as described by the manufacturer.
Genome sequencing.
The genome segments of LNV were copied into cDNA, cloned and sequenced as described previously (Attoui et al., 2000a). 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 RT-PCR using a complementary primer. PCR amplicons were analysed by agarose gel electrophoresis, ligated into the pGEM-T cloning vector (Promega) and transfected into competent XL-blue Escherichia coli. Insert sequences were determined using M13 universal primers, the D-Rhodamine DNA sequencing kit and an ABI prism 377 sequence analyser (Perkin Elmer).
Sequence analysis.
Analysis of LNV sequence was performed by comparing the sequence of each segment with a database constructed with all available sequences from the family Reoviridae, using the local BLAST program implemented in DNATools package (version 5.2.018; S.W. Rasmussen, Valby Data Center, Denmark).
For phylogenetic analysis, the VP1 sequence of LNV, identified through sequence comparison as the putative virus RNA-dependent RNA polymerase (RdRp), was compared with the amino acid sequences of putative RdRps of representative strains of viruses representing the 12 genera of the family Reoviridae. GenBank accession numbers are provided in the Supplementary Table available in JGV Online. Sequence alignments were performed using the CLUSTAL W software program (Thompson et al., 1994). Phylogenetic analyses were carried out with the software program MEGA3 (Kumar et al., 2004) using the p-distance determination algorithm or the Poisson correction, and the neighbour-joining method for tree building.
RT-PCR of the RNA extract from the tested cell lines.
The dsRNA was copied into cDNA using random hexanucleotide primers as described previously (Attoui et al., 1998). Briefly, the dsRNA was denatured in 15 % DMSO by heating at 99 °C for 1 min and then immediately incubated on ice. Reverse transcription was carried out using the Superscript III reverse transcriptase (Invitrogen) at 42 °C. The resulting cDNA was PCR amplified using first round primers LNV12s1 (position 79–101, 5'-GGAAGAATCAATGCCGTAGCCAC-3') and LNV12r1 (position 584–561, 5'-GTGACGATCTTCTCTGAACCAGTG-3') to produce an amplicon of 506 bp, and second round primers LNV12s2 (position 105–128, 5'-CACTGGCTCCGGCTGTAGTAACAG-3') and LNV12r2 (position 539–516, 5'-CTGTTCGGATCATCTGGAATTTGA-3') to produce an amplicon of 435 bp.
Amplicons from the nested PCR were cloned into pGEM-T vector and 47 clones were sequenced.
Replication of BAV and LNV in mice.
Ten-week-old mice were inoculated intraperitoneally with plaque purified clonal LNV-NE9712 or BAV-Ch: 100 p.f.u. of live virus or 5000 p.f.u. of inactivated virus (using 0·1 % formaldehyde and heating to 70 °C). Blood (30–50 µl) was recovered from the caudal vein at days 0, 1, 3, 7 and 10, in tubes containing 20 µl 10 mM EDTA pH 8·0. The blood was extracted using the RNA Now reagent (Biogentex). RT-PCR detection of the viral genome was performed using the same protocol as described above for LNV. The primers for BAV were BAV11s1 (position 79–101, 5'-GACGATCCAAGTTCAGAATTTGA-3') and BAV11r1 (position 584–561, 5'-TATTAGGATCAACACTGTTTTGTT-3'), to produce an amplicon of 506 bp, and second round primers BAV11s2 (position 105–128, 5'-GCTGTCCGGGACGTTTTGTTTGTG-3') and BAV11r2 (position 539–516, 5'-CTGTCAACAAGTACGCTTGTTGTG-3'), to produce an amplicon of 435 bp.
For both viruses, amplicons from secondary PCR were cloned into pGEM-T vector and 47 clones were sequenced.
Serum was used in indirect immunofluoresence assays on infected C6/36 cells (BAV and LNV) and BHK-21 cells (LNV) for the detection of specific antibodies.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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LNV particles purified using CsCl gradient centrifugation showed a defined surface structure, with ring shaped capsomeres (Fig. 1) that are characteristic of core particles of member viruses of the family Reoviridae. This suggests that they had lost the outer capsid components (Fig. 1). These were comparable to BAV particles purified using CsCl ultracentrifugation (Mohd Jaafar et al., 2005a). These LNV core particles had a mean diameter of 55 nm.
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for GenBank accession numbers). The length of the segments and their corresponding encoded proteins are given in Table 1. A unique open reading frame spanned almost the whole length of each individual segment.
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). Moreover, the first and last 2 nt of all of the segments are inverted complements.
The deduced amino acid sequence comparison showed that the various proteins encoded by LNV genome segments matched proteins of viruses belonging to the family Reoviridae. The highest amino acid identities were with proteins of the seadornaviruses. All the proteins of LNV showed significant identities to their homologues encoded by genome segments of BAV and KDV (Fig. 2).
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shows a neighbour-joining tree of the amino acid sequences from RdRp of LNV aligned with all of the RdRp sequences available for different seadornaviruses as well as representative members from other genera within the family Reoviridae. Between 41 and 42 % amino acid sequence identity was detected in VP1(Pol) between LNV and the other seadornaviruses. A previous study (Attoui et al., 2002) described the polymerase sequences of viruses, belonging to a single genus within the family Reoviridae, as having identity values greater than 30 %. The data presented here (number of segments, terminal nucleotides and sequence relatedness to seadornavirus proteins) therefore confirm that LNV belongs to the genus Seadornavirus.
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). Amino acid identities were found to range between 80 and 96 % as shown in Fig. 2. It is noteworthy that the lowest amino acid identity was found in the VP10 protein, which is the homologue of the BAV VP9 outer coat protein. BAV VP9 has been shown to be involved in cell attachment and internalization of virus particles (Mohd Jaafar et al., 2005a, b). The homologue of BAV VP4 (outer coat protein) was also found to be VP4 in LNV. For this protein, amino acid identity between the two LNV isolates was 92 %.
Comparative sequence analysis with BAV and KDV
The sequence analysis showed that all segments of LNV correspond to segments in either BAV or KDV. As shown in Fig. 2, the correspondence between homologous genes is not a function of the electrophoretic mobility of the dsRNA segments. The total genome of LNV was found to be 20 739 nt long for LNV-NE9712 and 20 747 nt long for LNV-NE9731 (Table 1), a size similar to BAV (20 682) and KDV (20 985) (Attoui et al., 2005).
The first two and last three conserved terminal nucleotides of LNV are identical to those of BAV and KDV, while the remainder of the sequence of the conserved terminal regions is distinct (Table 2).
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The amino acid identity between homologous proteins of either BAV or KDV and LNV ranged from 18 to 42 %, as shown in Fig. 2. These values are comparable to those existing between homologous proteins of distinct virus species within a single genus of the family Reoviridae (Mertens et al., 2005). The highest identity was found within the polymerase genes.
Identification of putative viral enzymes
Analysis of the deduced amino acid sequences showed that VP1 of LNV contains the signature motifs of the family Reoviridae RdRps, including motif SGELTT (positions 712–717) and GDD (positions 753–755). As is the case for BAV and KDV, VP1 is most likely the viral RdRp and should be designated VP1(Pol).
The VP3 of LNV matched VP3 of both BAV and KDV (37–38 % amino acid identity). It contains the motif KALS, which conforms to the motif Kx(I/L/V)S that was described in the guanylyltransferases of other members of the family Reoviridae (Luongo, 2002; Qiu & Luongo, 2003; Mohd Jaafar et al., 2005c). An alignment of the amino acid sequences, in the vicinity of this motif, of guanylyltransferases of the family Reoviridae with that of seadornaviruses (including LNV) is shown in Fig. 4(a). Two histidine residues involved in the activity of the guanylyltransferase of orthoreoviruses and aquareoviruses have been described recently. The sequence of LNV VP3 contains two histidine residues that are conserved among VP3 of other seadornaviruses. The sequences neighbouring these two histidine residues exhibit similarities to sequences in the vicinity of the two histidines of orthoreoviruses and aquareoviruses (Fig. 4b).
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). Together, these findings suggest that the VP3 of LNV is the virus guanylyltransferase, which should be designated VP3(Cap).
Identification of similarities with proteins of known functions
The VP7 showed similarities to active motifs of serine/threonine protein kinases involved in signalling pathways, such as the Rho kinase of Xenopus laevis (GenBank accession no. AAC06351, 44 % amino acid identity over a stretch of 40 aa) and the Pirerulla species protein kinase (NP_863898, 26 % amino acid identity over a stretch of 122 aa).
VP9 showed similarity to the major core protein P3 of the rice ragged stunt virus (RRSV) (NP_620516, 34 % amino acid identity over a stretch of 43 aa).
VP11 showed similarities to various dsRNA-binding proteins including the Staufen protein (AAB70372, 38 % amino acid identity), ribonuclease III (AAS11516, 32 % amino acid identity), TAR protein of HIV which is an RNA-binding protein (AAH02028, 34 % amino acid identity) and the PKR protein (AAH16422, 36 % amino acid identity). BLAST analysis showed that VP11 matched dsRNA-binding domains (DRBDs) designated KOG3732, pfam00035 and cd00048 as shown in Fig. 4(c). In addition, VP11 matched the DRBDs of BAV (VP12, 35 %) and KDV (VP8, 26 %).
Growth of LNV in mammalian and mosquito cell lines
LNV grew in all mosquito cell lines except the Aedes aegypti AE cell line. A comparison of the electropherotypes of BAV, KDV and LNV is shown in Fig. 5(a). The presence of the LNV in the mosquito cells was identified by both RNA analysis on agarose gel and by RT-PCR (Fig. 5b and c).
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Replication of BAV and LNV in mice
For both viruses, blood samples recovered from day 0 to 12 were used in PCR assays. The samples recovered at day 1 were negative as shown by RT-PCR. Samples recovered at days 3 and 5 were found to be positive, suggesting virus replication and viraemia. Samples recovered at day 7 were positive for BAV and negative for LNV. Samples recovered at day 10 were negative for both viruses, suggesting a clearance of the viraemia. Antibodies to LNV and BAV were identified by Western blot at 12 days p.i. Earlier sera were repeatedly negative.
Mice were reinjected at day 12 with the same isolate (either BAV or LNV). Four days following the second injection, LNV-infected mice died with signs of haemorrhage at the nasal opening and under the skin. Virus genomes could be detected in the blood of these dead mice. This result was reproducible each time new mice were injected on two separate occasions with the live LNV (similar results were obtained regardless of the LNV isolate injected: LNV-NE9712 or LNV-NE9731). The virus genome was detected in the RNA from pieces of the tail and the limbs (3–5 mm long) of the dead mice that were homogenized. The virus genome was not detected in extracts of the brain.
By contrast, reinjection of BAV did not result in detectable virus replication or any form of disease.
Analysis of BAV and LNV populations from infected mice
The analysis of clones obtained from the PCR amplification of segment 11 of BAV showed that following the injection into mice of clonal BAV no significant virus divergence was observed. Only 14 nt changes out of 20 445 nt (47 DNA clones of 435 nt each) sequenced were observed, randomly distributed along the studied sequence (with an absence of hot spots), which is most probably due to errors of the reverse transcriptase and Taq polymerase.
In contrast, the sequence of the amplicon of LNV segment 12 in the injected mice differed significantly from the sequence of the injected strain. Sequence analysis showed clones with silent and non-silent mutations [26 positions mutated: three silent and 23 non-silent (corresponding to 120 nt changes in 47 clones)]. Some of these mutations were not randomly distributed (suggesting the presence of potential hot spots), and were repeatedly found in many clones. Mouse-specific nucleotide (and consequently amino acid) substitution profiles were observed (see alignment in Fig. 6) by DNA clone analysis. Amino acid sequence alignment is shown in Fig. 6. These results suggest that quasi-species exist for LNV.
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These results suggest that LNV-NE9712 and LNV-NE9731 represent two serotypes of LNV.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Li, 1992; Liting et al., 1995) and Indonesia (particularly in Java) (Brown et al., 1993), whilst KDV has not been reported to be isolated from humans or any other mammals. LNV isolation from Aedes dorsalis mosquitoes in the north-east of China constitutes evidence that the diversity of this genus is not only restricted to BAV and KDV. First, there are obvious arguments for the classification of LNV in the family Reoviridae. These include the size and morphology of the virion particles, the polysegmented nature of the dsRNA genome, the genetic relatedness of the putative LNV polymerase to those of member-viruses of the family Reoviridae and the identification in this protein of the signature motifs of RdRps of viruses belonging to this family. Second, the full-length genome characterization of LNV has established its genetic relationships to previously reported member-viruses of the genus Seadornavirus: (i) the viral genome is made up of 12 segments with conserved terminal sequences that are similar, mainly in the 3'NCR, to those of seadornaviruses; and (ii) all virus proteins have a significant identity with seadornavirus proteins. In particular, the amino acid identity observed in the polymerase confirms the status of LNV as a seadornavirus according to previously defined criteria (amino acid identity greater than 30 %). Within a given seadornavirus species, the amino acid identity within the polymerase is very high (for instance in BAV it is 93–99 %). Between LNV and either BAV or KDV the amino acid identity is compatible with viruses belonging to distinct species of the genus Seadornavirus. Accordingly, a proposal was made to the ICTV for the recognition of LNV as a new species and this has been formally accepted. Moreover, serum neutralization experiments demonstrate the existence of two serotypes. By analogy with BAV, the protein involved in serotype determination in LNV is suspected to be VP10 (80 % amino acid identity only between the two LNV serotypes). However, conclusive evidence about the role of VP10 as neutralizing epitope would still necessitate the use of specific antibodies to VP10.
The putative function of proteins encoded by the different genome segments of LNV was explored through sequence comparison to BAV and other proteins from databases. In particular, the polymerase function (VP1), the capping function (VP3) and the dsRNA-binding activity (VP11), and the cell attachment and neutralization epitope (VP10) were identified.
It is of special interest to note that the characteristics of LNV replication in mammalian cells are different from those of BAV and KDV. While BAV and KDV replicate only in mosquito cell lines, LNV was able to replicate not only in a large panel of mosquito cell lines but also in mammalian cell lines. This is comparable to the situation of many insect-borne arboviruses infecting humans, including within the family Reoviridae viruses such as Orungo virus and Lebombo virus (both orbiviruses; Tomori & Fabiyi, 1977).
BAV, KDV and LNV were able to replicate in mice and could be detected in blood a few days after intraperitoneal injection. BAV and KDV infections were not lethal, did not result in severe clinical presentations and produced protective immunity. Reinfection was not accompanied by detectable virus replication. The situation observed after LNV injection was different. Primary infection provoked a 5 days prostration after which all tested animals recovered. However, reinfection was followed by a new phase of virus replication and resulted in death at day 4 with signs of generalized haemorrhage. It is of interest to note that the first immunization with inactivated LNV followed by a second injection of live LNV was not followed by virus replication and did not cause death.
The exact mechanism by which LNV kills infected mice warrants further investigation, but the data presented here suggest that it is not due to an antibody facilitating effect since only primary infection by live LNV was followed by a secondary lethal infection.
The sequence variation in RNA viruses results from the error-prone nature of RdRps and the selective pressure, resulting in adaptation and evolution. The generally observed mean mutation rates of viral RNA genomes range between 10–4 and 10–5 mutations (although rates between 10–3 and 10–6 have been reported) per nucleotide per round of RNA replication (Pugachev et al., 2004). The high mutation frequencies of most RNA viruses might lead to the generation of genetically heterogeneous populations referred to as quasi-species. One of the most obvious examples is the case of Hepatitis C virus where the mutation rate was estimated to be as high as 8x10–2 within the hypervariable region of the genome (Herring et al., 2005). The sequence variation of BAV and LNV was investigated in infected mice. A clonal virus obtained by plaque purification (the genetic homogeneity was controlled by RT-PCR and cloning) was injected into mice and possible sequence variation was investigated after 5 days of infection. The sequence analysis of amplicons obtained from mouse blood showed no evidence for quasi-species in BAV-infected mice, but in amplicons from LNV-infected mice there were four times more nucleotide changes than expected from errors of Taq polymerase. This is compatible with the emergence of new variants that might lead to a quasi-species in LNV (which was not observed following LNV passage in cell cultures). One possible conclusion concerning the higher degree of sequence variation, which is detected in LNV, is either that the virus has a polymerase that shows a higher rate of error during the growth in mice than that of BAV or that the virus goes through significantly more rounds of replication in this host species. Further understanding of the determinants of dsRNA virus evolution is hampered by the paucity of similar reported information. New models are required to better document this question and attempt to explain the different replication properties of BAV and LNV.
LNV was isolated from the mosquito Aedes dorsalis. The mosquito is encountered in the North American subcontinent, in Europe and in Asia (Romanowski, 1989; Ciolpan et al., 1998). It is found in a variety of habitats including brackish and freshwater. This mosquito feeds on mammals (including humans and domestic animals) and birds. It has been considered as one of the possible vectors of West Nile virus (genus Flavivirus) in the North American continent (Medlock et al., 2005; Goddard et al., 2002). It is also considered to be one of the vectors of the Western equine encephalitis virus (genus Alphavirus) (Fulhorst et al., 1994; Kramer et al., 1998).
The availability of the complete genome sequence of LNV will facilitate the development of sequence-specific PCR assays for the study of LNV epidemiology in the field to establish the ecological cycle of this virus and its possible implication in mammalian diseases.
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ACKNOWLEDGEMENTS |
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Received 28 June 2005;
accepted 5 October 2005.
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