# Comparative sequence analysis of American, European and Asian isolates of viruses in the genus Coltivirus

Houssam Attoui, Rémi Charrel, Frédérique Billoir, Jean-François Cantaloube, Philippe de Micco, Xavier de Lamballerie

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Houssam Attoui, Rémi Charrel, Frédérique Billoir, Jean-François Cantaloube, Philippe de Micco, et al.. Comparative sequence analysis of American, European and Asian isolates of viruses in the genus Coltivirus. Journal of General Virology, 1998, 79, pp.2481-2489. 10.1099/0022-1317-79-10-2481. hal-05295874

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Distributed under a Creative Commons Attribution 4.0 International License
COMPARATIVE SEQUENCE ANALYSIS OF AMERICAN, EUROPEAN AND
ASIAN ISOLATES OF GENUS *COLTIVIRUS*

Houssam ATTOUI, Rémi N. CHARREL, Frédérique BILLOIR, Jean-François CANTALOUBE, Philippe DE MICCO and Xavier de LAMBALLERIE

Corresponding author

Address :

Laboratoire de Virologie Moléculaire,
Tropicale et Transfusionnelle
Faculté de Médecine de Marseille
Université de la Méditerranée
27 Boulevard Jean Moulin
13385 Marseille cedex 5
FRANCE

Phone : (33) 4 91 25 59 74

Fax : (33) 4 91 18 95 98

Running title : Sequence analysis of coltiviruses

Word count : Text (4178), summary (189), ref. (25)
Summary

In this study, the basis for the analysis and classification of viral isolates
grouped within genus *Coltivirus*, family *Reoviridae*, are discussed. Sequences
of double-stranded RNA segments from American (segments 9, 10, 11 and
12), European (segment 12) and Asian (segments 7 to 12) isolates were
characterised and polythetic criteria were defined for their taxonomic
classification. These criteria (including sequence analysis) permitted to
distinguish the different species and to classify them in two groups. In both
groups, sub-groups were defined according to the degree of homology
between the genomic sequences. American and European isolates are
classified within group A which includes sub-groups A1 {Colorado tick fever
virus (CTFV) species} and A2 {Eyach virus (EYAV) species}. Asian isolates
are classified in group B including sub-groups B1 {JKT-7075 virus species}
and B2 {JKT-6423 virus species}.

The proteins encoded by the sequenced genomic segments were analysed.
This permitted the identification of double-stranded RNA binding domains
in proteins encoded by segment 8 of sub-group B1 and segment 12 of sub-
group B2 isolates. A conserved pattern of amino acids in segment 7 of group
B isolates matched sequences in the catalytic domains of protein kinases.
Viruses having genomes of 12 segments of double-stranded RNA, that have been isolated from vertebrates, ticks and mosquitoes, are grouped within genus *Coltivirus*, family *Reoviridae*. The type species of this genus is the Colorado Tick Fever (CTF) virus (Murphy *et al.*, 1995). Other members are the Eyach virus isolated in Germany (Rehse-Küpper *et al.*, 1976) and the two Eyach antigenic variants (AR577 and AR578) isolated in France (Chastel *et al.*, 1984). In previous studies, an antigenic relationship between these American and European isolates was established (Chastel *et al.*, 1984; Karabatsos *et al.*, 1987) and the full-length nucleotide sequences of the 10th, 11th and 12th genomic segments of CTF virus were determined (Attoui *et al.*, 1997).

Other viruses were considered as tentative species of the genus : Indonesian isolates (Brown *et al.*, 1993) made from mosquitoes; and Chinese isolates (Xu *et al.*, 1990; Chen & Tao, 1996) made from mosquitoes, ticks and vertebrates.

The taxonomic relations between the viruses grouped within the genus *Coltivirus*
were only poorly explored, and did not permit the exact classification of the
different isolates. This is particularly due to the absence of precise information
describing the genetic relations between these viruses.

In this paper, we present a comparative sequence analysis of American, European and Asian isolates of genus *Coltivirus*. In order to define the taxonomic status of these isolates, species parameters have been established in agreement with those used for other genera of the *Reoviridae* family. Accordingly, members of a single *Coltivirus* species may be identified according to :

1. Genetic criteria:

(i). Analysis of electropherotypes by agarose gel electrophoresis.

(ii). RNA cross hybridisation assays (northern or dot blots, with probes made from viral RNA or cDNA).

(iii). RNA sequence analysis (including quantification of variation in genome segments and analysis of conserved RNA terminal sequences).
2. Serological comparisons by complement fixation and neutralisation assays.

3. Their ability to exchange genetic material by genome segment reassortment during dual infections, thereby producing viable progeny virus strains.

The application of these criteria to the analysis of American, European and Asian isolates permitted to gain insight about their taxonomic status within the genus. A classification was defined that is included in the forthcoming report of the International Committee on Taxonomy of Viruses. We also present here preliminary data concerning the putative proteins encoded by some of the genomic segments of these isolates as deduced from the comparison to protein sequences deposited in databases.
# MATERIALS AND METHODS

## Virus strains

American isolates : Florio strain (N-7180) of CTF virus was purchased at the American Type Culture Collection. Two other CTF virus isolates (R-1575 and 69V28) and a related virus (S6-14-03) were provided by Dr. N. Karabatsos.

European isolates (Eyach and AR578 viruses) were provided respectively by Dr. N. Karabatsos and Pr. C. Chastel.

Indonesian isolates (JKT-6423, JKT-6969, JKT-7043 and JKT-7075) and the Chinese isolate (Banna virus) were provided by Drs. R.B. Tesh and R.E. Shope.

## Cell lines

BHK-21 cells were cultured as monolayers or suspensions as previously described (Attoui *et al.*, 1997). C6/36 mosquito cells were cultured as monolayers in Leibovitz's L-15 medium supplemented with 5% FBS, 2% tryptose phosphate broth, penicillin G (100 IU/ml) and streptomycin (100 µg/ml).

## Virus propagation

The Florio strain of CTF virus was propagated in suspensions of BHK-21 cells as described elsewhere (Attoui *et al.*, 1997). The S6-14-03 virus was cultured in monolayers of BHK-21 cells. The two CTF strains (R-1575 and 69V28), the Eyach and the AR578 viruses were propagated by intracerebral inoculations of suckling mice and infected brains were recovered at the seventh day post-infection (pi). Indonesian isolates JKT-6423, JKT-6969, JKT-7043 and JKT-7075 as well as the Banna virus were propagated in C6/36 mosquito cells. Infected cells were recovered at the fourth day pi.

## Isolation and purification of nucleic acids

Viral dsRNA was extracted from infected cell cultures or suckling mouse brains using a guanidinium isothiocyanate derived procedure (RNA NOW, Biogentex). In the case of cell cultures, viral dsRNA was further purified by precipitation of single stranded RNAs in 2 M LiCl and electrophoresis on a 10% acrylamide gel as previously reported (Attoui *et al.*, 1997). RNA was extracted from the acrylamide
matrix by shaking over-night in TNE buffer, precipitated from the aqueous phase using 10 volumes of butanol and purified using the RNAide kit (Bio 101).

## Cloning of the genomic segments 7 to 12 of JKT isolates and Banna virus

The full-length genomic segments 7 to 12 of the JKT isolates and Banna virus were cloned and sequenced using a modification of the Lambden method (Lambden *et al.*, 1992; Attoui *et al.*, 1997). Briefly, a 3'-amino blocked oligodeoxyribonucleotide (PO4-5'-CCACGTGCCAGATGCTCTGGA-3'-amine, 2.5 mM) was ligated to both of the 3'-OH ends of the dsRNA (1 ng) using 10 U of T4-RNA ligase.

The tailed dsRNA product was purified using the RNAide kit and heat denatured (99°C, 1 min in 15% dimethyl sulfoxide). The cDNA was synthesised in presence of the complementary oligodeoxyribonucleotide primer (5'-GGTGCACGGTCTACGAGACCT-3', 1 mM) using 200 U of MMuLV superscript reverse transcriptase (Gibco BRL).

Full-length cDNA was PCR amplified using 2.5 U of Taq polymerase and 1 µM of the primer (5'-GGTGCACGGTCTACGAGACCT-3'). Thermal cycling parameters were as follows: one cycle of denaturation (90°C, 10 min) followed by 35 cycles of denaturation (94°C, 50 sec), annealing (65°C, 50 sec) and extension (72°C, 2 min. The cycling program ended by an extension step at 72°C for 10 min. PCR products were gel purified and ligated into PGEM-T vector (Promega).

Recombinant plasmids containing the full-length PCR products were transfected into competent *E. coli* XL-Blue cells. Both strands of cloned PCR product were sequenced using the M13 universal primers and the D.Rhodamine DNA sequencing kit and analysed on the ABI prism 377 sequence analyser (Perkin Elmer).

## RT-PCR amplification of genomic segments

The extracted viral dsRNAs from the CTF strains R-1575 and 69V28, the Eyach, the AR578 and the S6-14-03 viruses were resuspended in diethylpyrocarbonate
treated water.

The reverse transcription was realised as previously described, in the presence of random hexanucleotides and the MuMLV superscript reverse transcriptase (Attoui *et al.*, 1998). PCR primers were designed from the sequences of the 9th, 10th, 11th and 12th genomic segments of the Forio strain (N-7180) of CTF virus with the help of the Oligo software program (National Biosciences inc., Plymouth, MN, USA). Primer sequences are displayed in Table 1.

The PCR protocol using segment specific primers was described elsewhere (Attoui *et al.*, 1998). Briefly, the reaction was carried out in a one hundred microliters volume and the amplification mixture included : 10 mM Tris-HCl (pH8.8), 50 mM KCl, 1.5 mM MgCl₂, 0.1% Triton X-100, 0.2 mM each dNTP, 20 µl of the cDNA solution, 1 µM of each primer, 2.5 U of *Taq* DNA polymerase. The thermal cycling parameters were as described above except that the annealing was realized at 55°C. Amplification products were cloned and sequenced as described above.

## Sequence analysis

The open reading frames and their encoded putative proteins were determined using the DNA strider 1.1 program (Institut de Recherche Fondamentale, CEA, France). Sequence alignments were generated by the Clustal W software program (Metrowerks Inc.). Phylogenetic analyses were performed with the software program MEGA (Kumar *et al.*, 1993) using the p-distance determination algorithm, and the sequence relatedness were reported as percentage identity. Comparison of our sequences with those available from nucleic acid and protein databases were performed using the GENSTREAM's FASTA and BLAST software programs (EERIE, Nîmes, France) and the Pfam program (Sanger institute, UK).
# RESULTS

## Analysis of nucleic acid sequences from American and European isolates

A set of primers (S2259S/COLREV) was designed from the previously determined nucleotide sequence of segment 12 of the Florio strain. It permitted the amplification of the 12th genomic segments of all American and European strains. The generated amplicons were 437 nucleotide (nt.) for the American isolates and 440 nt. for the European isolates. The difference was due to an additional non-coding "GAG" trinucleotide at position 585 of the corresponding CTF sequence. Asian isolates could not be amplified using this set of primers.

The sequences of the corresponding amplicons were determined {GenBank accession numbers : CTF viruses (AF007173, AF007174 AF007175); Eyach viruses (AF007185 and AF0071886)}.

Primers issued from the sequences of the 9th, 10th and 11th segments of the Florio strain of CTF virus allowed the amplification of the viral RNAs from all American isolates, but not that of RNAs from European and Asian isolates. The generated PCR products were 584 nt. (segment 9), 221 nt. (segment 10) and 302 nt. long (segment 11). Their sequences were determined {accession numbers : segment 9 (AF007176-AF007178), segment 10 (AF007182-AF007184) and segment 11 (AF007179-AF007181)}.

The 12th segment of both American and European viruses could be amplified by the same set of primers; accordingly these isolates were placed in the same group (group A). A first subgroup (designated A1), encompassing all the American isolates, was identified. The 9th, 10th and 11th segments of all isolates from this subgroup could also be amplified by common sets of primers. The second subgroup (designated A2) included European isolates. The 9th, 10th and 11th segments of isolates from this subgroup could not be amplified by primers successfully used for the A1 subgroup.

When the nucleotide sequences of segment 12 of group A strains were compared, the percentage identity was found to be 94.37 to 96.78% within subgroup A1, 92.29% within subgroup A2, and 65.68% to 67.56% between subgroups A1 and A2. Detailed values are displayed in Table 2.

Within subgroup A1, similar degrees of nucleic acid identity were observed upon
analysis of the 9th,10th,11th, and 12th genomic segments. These ranged between
97.69 and 100% (segment 9), between 96.84 and 98.95% (segment 10) and from
90.6 to 94.74% (segment 11).

No significant homology was found when the sequences of group A isolates were
compared to those of Asian isolates.

The calculated G+C content for sequences of group A coltiviruses was found to
range between 48.4% and 51.6%.

Analysis of the putative amino acids sequences of American and European isolates

A sequence of 170 amino acids (A.A.) from the viral protein (VP) 9 of the Florio
strain was compared to homologous sequences of strains R-1575, 69v28 and S6-
14-03. Identity ranged from 99.22 to 100%. Database searching revealed, in the
VP9, the presence of the pattern {hhhhGx₄GKSxₙhhhhDD, where h indicates a
bulky hydrophobic residue} corresponding to the P-loop in the ATP binding site
of archeal ATPAses.

A 65 amino acid sequence from the VP10 of the Florio strain was compared to
homologous sequences of other American isolates. The amino acid identity ranged
from 96.84 to 98.95%. The comparison of the full-length VP10 to database
proteins revealed a partial matching (42%) between a 270 A.A. hypothetical
protein (MJ1287) of unknown function from the archeon *Methanococcus
jannaschii* (A.A 42 to 102) and the VP10 (A.A 40 to 100).

A 90 A.A sequence from the VP11 of the Florio strain was compared to the
corresponding VP11 sequences of other strains of subgroup A1. The amino acid
identity between these proteins ranged from 95.45 to 98.86%.

The percentage identity in VP12 ranged between 87.74 and 92.45% within
subgroup A1 isolates and was 87.74% within subgroup A2 isolates. It ranged from
51.89 to 55.66% between American and European isolates. Detailed records are
given in Table 2.

The VP11 and VP12 showed no significant matching with proteins from database.

Analysis of the nucleic acids sequences from Asian isolates
The full-length segments 7 to 12 from the JKT-6423, JKT-6969, JKT-7043, JKT-7075 isolates and the Banna virus were sequenced (GenBank accession numbers AF019908, AF019909 and AF052008-AF052035).

All Asian strains were assigned to a single group (B). The sequence analysis permitted the identification of two subgroups, B1 and B2, corresponding to different viral species. The electropherotype of subgroup B1 viruses (JKT-7075) corresponds to a 6-5-1 pattern and that of subgroup B2 viruses (isolates JKT-6423, JKT-6969, JKT-7043 and Banna virus) to a 6-6 pattern. The electrophoretic profiles are displayed in Figure 1 and the lengths of segments 7 to 12 are reported in Table 3.

Two genotypes could be distinguished in subgroup B2 according to the length of segments : genotype 2a including JKT-6423 and Banna virus and genotype 2b including JKT-6969 and JKT-7043 (Tables 3 and 4).

Within subgroup B2 the identity between homologous segments 8, 10, 11 and 12 ranged between 83 and 98%. By contrast, identity in segment 7 ranged between 72 and 98%, and in segment 9 between 54 and 99%. The comparison of the segments of subgroup B1 to corresponding segments of subgroup B2 showed that the highest degree of identity ranged between 46.67% (segment 9) and 52.16% (segment 12).

The G+C content calculated from the sequenced segments of group B isolates was 37-39%.

## Analysis of the 5' and 3' non coding regions of Asian isolates

The analysis of the non coding regions (NCR) of the cloned segments permitted the identification of conserved motifs located at the termini. In all positive strands of the genome segments from the subgroup B1 viruses, the motifs (5'-GUAU^A/U^A/UAA^A/U^A/UU-3') and (5'-A/G)^C/U/GAC3') were found in the 5' NCR and the 3' NCR respectively. In all positive strands of segments from subgroup B2 viruses, the motif (5'-GUAGAA^A/U^A/U^A/UU-3') was identified in the 5' NCR and the motif (5'-A^A/C^C/U/GAC-3') in the 3' NCR. Segments of all group B isolates have in common the first two nucleotides in the 5' NCRs (GU) and the last two
nucleotides in the 3' NCRs (AC). These dinucleotides are inverted complements.
Segment-specific inverted terminal repeats (ITRS), involving the conserved
motifs, were detected in all segments of the different isolates. These ITRS could
interact by base pairing thus forming secondary structures. Such possible
secondary structures were generated with the help of the MacStan software
program (Gast, 1994) and are displayed in Figure 2 for segments 7 to 12 of isolate
JKT-6423.

Analysis of the putative amino acids sequences of Asian isolates

The putative viral proteins encoded by the segments 7 to 12 of B group isolates
were determined. Like nucleic acid sequences, the analysis of the putative proteins
permitted to distinguish two different species : species JKT-7075 and species
JKT-6423. The lengths of the encoded proteins are reported in Table 3.

Amino acid sequence identity ranged between 85 and 100% for VP8, VP10, VP11 and VP12 of subgroup B2 isolates. The VP7 sequence identities ranged between 70.9 and 97.39%. The VP9 was the most variable, with 41.79 to 99.64% identity.

The comparison of the homologous proteins of subgroups B1 and B2 isolates
showed that the highest degrees of identity ranged between 17% (VP9) and 28%
(VP7). However, alignments of the different amino acid sequences from group B
isolates revealed partially conserved amino acid patterns in VP7 (35-93% identity)
and VP10 (39-100% identity).

Database searching showed that the partially conserved amino acids motif in the
VP7 proteins corresponded to the (ΔH-D--P-NΔ--D--G-ZKΔ-D, where Δ=I or L, Z
= I, L or V and - = any amino acid) pattern found in the catalytic domains of
different protein kinases, including the dsRNA dependent protein kinase (Figure
3).

Database searching using the BLASTP program showed that the sequences of
JKT-7075 VP8 (amino acids 5-63) partially matched a sequence of Ribonuclease
III (Figure 4). Similarly, a partial matching was found between the sequence of
VP12 (amino acids 3-63) from JKT-6423, JKT-6969, JKT-7043 and Banna virus,
and that of the maternal effect protein staufen (Figure 4). Theses sequences corresponded to dsRNA binding domains (DRBD) as deduced from the analysis by the protein family domain (Pfam) program.

The VP8 of other isolates than JKT-7075, the VP9 and VP11 proteins did not show a significant matching with proteins from databases. Despite the conserved pattern in the VP10 protein, similarity with database proteins was not significant.

Homologies between putative viral proteins of groups A and B isolates were insignificant.
DISCUSSION

The family *Reoviridae* includes members having polyphyletic origins (Koonin, 1992). Among viruses exhibiting a 12 dsRNA segmented genome, those that infect plants are grouped within genus *Phytoreovirus* and those isolated from vertebrates, ticks and mosquitoes are classified in the genus *Coltivirus*. The antigenic relationship between CTF (American) and Eyach (European) viruses isolates has been analysed (Chastel *et al.*, 1984; Karabatsos *et al.*, 1987; Rehse-Küpper *et al.*, 1976). The neutralisation of both American and European isolates by antibodies to the CTF virus (Karabatsos *et al.*, 1987) suggests that these viruses are antigenically related. In our experience, the fact that European strains can be detected in cell cultures by an indirect immunofluorescence assay using anti-CTF antibodies is consistent with these data.

Primers designed from the sequences of segments 9, 10, 11 and 12 of the Florio strain (Attoui *et al.*, 1997; 1998) of CTF virus permitted the PCR amplification of these segments from the R-1575, 69v28 and S6-14-03 viruses. The amplified sequences had the same lengths than those of the Florio strain and exhibited high degrees of nucleic acid and protein identities. The primers used for the amplification of segments 9, 10 and 11 did not amplify the homologous segments from the European viruses. However, primers S2259S/COLREV designed from the sequence of segment 12 of CTF virus amplified the 12th segment of the Eyach virus and one of its variants AR578. The identity to the homologous CTF virus sequences was 65.68 % to 67.56% for nucleic acids and 51.99 and 55.66% for amino acids.

These molecular findings are in agreement with the previously reported serological conclusions. They suggest the common phylogenetic origin of American and European strains and corroborate the geographical clusterisation suggested by serological data. Accordingly, American and European viruses were assigned to a single group designated *Coltivirus* group A. The nucleic and amino acid identities, the genome electropherotype of Eyach virus and the geographical origin of the isolates suggest that group A viruses belong to two different species : the CTF virus species (subgroup A1) that includes the American strains (virus S6-14-03 being a distinct serotype), and the Eyach virus species (subgroup A2)
including European viruses.

The molecular relationship between American coltiviruses and the Indonesian JKT-viruses had been previously analysed by Brown and colleagues (1993) using RNA-RNA hybridisation which did not produce any cross hybridisation signal. Furthermore, genomes of strains JKT-6423, JKT-6969, JKT-7043 on one hand and JKT-7075 on the other hand did not cross hybridise.

In this study, we cloned the segments 7 to 12 of JKT-7075, JKT-6423, JKT-6969, JKT-7043 and Banna viruses and determined their complete nucleotide sequences. Sequence analysis did not reveal any significant homology to group A coltiviruses in agreement with the findings of Brown and colleagues. Moreover, the G+C content calculated from the sequences of segments 7 to 12 of Asian isolates was 37-39%, which is clearly lower than that of group A coltiviruses (48.4-51.6%).

The analysis of the sequences of Asian isolates showed that nucleic acids exhibit 47.75-98.82% overall identity and revealed the presence of common amino acid patterns in segments 7 and 10. This permitted to assign Asian viruses to a second group (Coltivirus group B). Further investigations revealed the existence of two species represented by isolates of subgroups B1 and B2, the latter including two distinct genotypes.

The subgroup B1 is represented by the JKT-7075 virus. Its nucleic acid sequence identity to sequences of isolates belonging to subgroup B2 (JKT-6423, JKT-6969, JKT-7043 and Banna viruses) is about 49%. The comparison of the proteins of JKT-7075 to those of subgroup B2 viruses showed similarities only in VP7 and VP10.

For isolates of subgroup B2, the overall nucleic acid sequence identity in the conserved segments (8, 10, 11 and 12) is 83.3%-98.84% and ranges between 54.39 and 99.7% in the variable segments (7 and 9). The analysis of the nucleic and amino acids sequences of subgroup B2 viruses reveals the existence of two genotypes. The first one (2a) is represented by JKT-6423 and Banna viruses and the second one (2b) is represented by JKT-6969 and JKT-7043 viruses. In each genotype, the variable segments (7 and 9) are similar as regarding their nucleic and amino acids sequences.

The amino acid sequence analysis is in agreement with the nucleic acid findings. In each genotype, identity between homologous proteins is very high (over 85%).
However, in the case of VP10 of genotype 2b, a difference of 8 amino acids in the carboxyl terminal is noticed between isolates JKT-6969 and JKT-7043. The VP10 of JKT-7043 is 249 amino acids long (thus having the same length as VP10 from isolates of genotype 2a), while that of JKT-6969 is 241 amino acids long. However, the nucleic and amino acid sequence identity values are higher between segment 10 of JKT-6969 and JKT-7043 than between that of JKT-6969 and genotype 2a viruses.

The non coding regions of segments 10, 11 and 12 of CTF virus were previously analysed and conserved motifs were identified in their 5' and 3' NCRs. The conserved terminal motifs in the NCRs of the full-length segments of JKT isolates are different from those of CTF virus. Such conserved motifs can be detected in many viruses possessing multisegmented genomes and would probably play an important role in replication and sorting of RNA segments as well as in virus maturation (Anzola *et al.*, 1987; Xu *et al.*, 1989).

The first and last two nucleotides as well as the terminal repeats in the 5' NCRs and 3' NCRs of segments of group B isolates were found to be inverted complements. This suggests that the RNA transcript could be held in a circular form either by itself or via protein components (Anzola *et al.*, 1987; Theron and Nel, 1997). This finding has been previously reported for viruses such as CTF virus (Attoui *et al.*, 1997), infectious bursal disease virus (Mundt and Müller, 1995), wound tumor virus (Anzola *et al.*, 1987) and influenza virus (Hsu *et al.*, 1987), where a "panhandle" structure was described that would possibly function as a guiding site for the virus specific RNA-dependant RNA polymerase.

Altogether these data suggest a different phylogenetic origin for groups A and B isolates. Group A isolates share a number of characters that permitted their assignment to this group: (i) the degree of identity between nucleic acid sequences of segment 12 (65.68-96.78%); (ii) common amino acid patterns; (iii) common vectors (*Ixodidae ticks : Ixodes and Dermacentor species*) and hosts (vertebrates); (iv) similar G+C content (48.4% to 51.6%). All subgroup A1 isolates had the same geographical origin and similar genomic electrophoretic pattern.

Group B isolates also share a number of common characters : (i) the overall identity between nucleotide sequences is 47.75%-98,82% (51.42-98.96% for segment 12); (ii) common amino acids patterns; (iii) common vectors (*Culex* and
*Anopheles* mosquitoes ); (iii) similar G+C content (37% to 39%); (iv) a common geographical origin (south-eastern Asia). Within subgroup B2, all isolates have also similar conserved terminal sequences and genomic electrophoretic profiles. The conserved terminal sequences in subgroups B1 and B2 are similar and identical terminal dinucleotides are common to all group B isolates.

The genetic variability of the American isolates of CTF virus was analysed by Bodkin and Knudson (1987) using the RNA-RNA hybridisation methodology. These authors found that genes 4 and 6 are variable and reported that this finding is the consequence of a genetic reassortment phenomenon. Accordingly, as stated in the introduction section, the ability of viral strains to exchange genetic material by genome segment reassortment during dual infections may also be used as a parameter for the definition of species in the *Coltivirus* genus.

The possible functions of the putative proteins of coltiviruses were investigated. Regarding group A viruses, partial matching with proteins from databases was only found in VP9 and VP10 of CTF virus. An amino acid pattern described as being the ATP binding site in archeal ATPases (Koonin, 1997) was found in the VP9 while VP10 partially matched an amino acid sequence from a hypothetical protein of the archeon *Methanococcus jannaschii*.

In the group B isolates, DRBDs were detected in the VP8 (subgroup B1) and VP12 (subgroup B2). Within the *Reoviridae* family, proteins exhibiting a dsRNA binding activity have been identified such as the sigma-3 protein in reovirus type-3 (Schiff *et al.*, 1988) and the NSP3 protein in porcine rotavirus group C (Langland *et al.*, 1994). Despite the fact that the DRDB of sigma-3 protein is unrelated to that of NSP3 DRDB (which conforms to those of the group B VP8 and VP12), similar functions have been attributed to these two proteins. They were found to stimulate the translation of viral messenger RNAs by blocking the activation of dsRNA-dependant protein kinase (Langland *et al.*, 1994; Yue and Shatkin, 1997). It is therefore possible that the JKT proteins with DRBDs would have a similar function.

Finally, this study represents the first attempt for the determination of the taxonomic status of the existing viral isolates belonging to genus *Coltivirus*, as well as a mean for the assignment of viruses having genomes of 12 dsRNA that
might be isolated in the future.

The characterised sequences of isolates of genus *Coltivirus* would permit the
elaboration molecular procedures for detection these viruses.

However, in the absence of data on the structures and functions of viral proteins,
the actual mechanism of replication and the pathogenicity of these viruses further
investigations are required to gain more insight about the genus *Coltivirus*.

**Acknowledgements**

The authors wish to thank Dr. R.B. Tesh and Dr. R.E. Shope for kindly providing
the Indonesian and Chinese viral strains.
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<table><caption>**Table 1 :** Primers used in amplification of CTF viruses genomic segments.</caption><thead><tr><th>Primer name</th><th>Sequence (5'-->3')</th><th>Segment</th><th>Map position</th><th>Orientation</th></tr></thead><tbody><tr><td>M5720PS</td><td>CACTTGTGGCAGATCGGGGAGA</td><td>9 (M5)</td><td>*</td><td>sense</td></tr><tr><td>M5720PA</td><td>CACCGGCTTACTACCTCATTTTT</td><td>9 (M5)</td><td>*</td><td>antisense</td></tr><tr><td>M6TGAS</td><td>TGAAGATCTGGCGTGGCGAGAG</td><td>10 (M6)</td><td>970-991</td><td>sense</td></tr><tr><td>M6TGAA</td><td>GAACACACAGATGTATTCAGACAC</td><td>10 (M6)</td><td>1165-1188</td><td>antisense</td></tr><tr><td>S1S302</td><td>GGGAAGACGGACAATGTTCGTG</td><td>11 (S1)</td><td>367-388</td><td>sense</td></tr><tr><td>S1A302</td><td>CCATTCACCAACCTCGTCCCG</td><td>11 (S1)</td><td>646-666</td><td>antisense</td></tr><tr><td>S2259S</td><td>CTACTCGGATTTGAGTTAAGCCC</td><td>12 (S2)</td><td>224-246</td><td>sense</td></tr><tr><td>COLREV</td><td>CCGGGAGAATGATGCTAGG</td><td>12 (S2)</td><td>640-658</td><td>antisense</td></tr></tbody></table>

<footnote>* the primers were designed from a partial sequence of the ninth genomic segment.</footnote>
<caption>**Table 2.** Identity between sequences from *Coltivirus* isolates.</caption>

a)

<table><thead><tr><th>Total-sequence</th><th>BANNA</th><th>JKT-6969</th><th>JKT-7043</th><th>JKT-7075</th></tr></thead><tbody><tr><td>JKT-6423</td><td>87.25<br>(91.42)</td><td>84.39<br>(81.90)</td><td>84.22<br>(81.08)</td><td>47.95<br>(21.15)</td></tr><tr><td>BANNA</td><td></td><td>80.11<br>(77.61)</td><td>79.74<br>(76.74)</td><td>47.45<br>(20.81)</td></tr><tr><td>JKT-6969</td><td></td><td></td><td>98.82<br>(98.48)</td><td>49.16<br>(21.80)</td></tr><tr><td>JKT-7043</td><td></td><td></td><td></td><td>49.20<br>(21.34)</td></tr></tbody></table>

b)

<table><thead><tr><th>Segment-9</th><th>BANNA</th><th>JKT-6969</th><th>JKT-7043</th><th>JKT-7075</th></tr></thead><tbody><tr><td>JKT-6423</td><td>83.82<br>(89.75)</td><td>54.48<br>(41.79)</td><td>54.39<br>(41.79)</td><td>41.67<br>(17.09)</td></tr><tr><td>BANNA</td><td></td><td>55.12<br>(41.07)</td><td>55.21<br>(41.07)</td><td>40.60<br>(17.45)</td></tr><tr><td>JKT-6969</td><td></td><td></td><td>99.71<br>(99.64)</td><td>46.57<br>(18.68)</td></tr><tr><td>JKT-7043</td><td></td><td></td><td></td><td>46.67<br>(18.68)</td></tr></tbody></table>

c)

<table><thead><tr><th>Segment-7</th><th>BANNA</th><th>JKT-6969</th><th>JKT-7043</th><th>JKT-7075</th></tr></thead><tbody><tr><td>JKT-6423</td><td>85.64<br>(91.50)</td><td>73.22<br>(72.88)</td><td>72.95<br>(71.57)</td><td>49.47<br>(28.20)</td></tr><tr><td>BANNA</td><td></td><td>73.99<br>(72.22)</td><td>73.28<br>(70.92)</td><td>48.93<br>(27.54)</td></tr><tr><td>JKT-6969</td><td></td><td></td><td>98.10<br>(97.39)</td><td>51.22<br>(28.85)</td></tr><tr><td>JKT-7043</td><td></td><td></td><td></td><td>51.39<br>(27.87)</td></tr></tbody></table>

d)

<table><thead><tr><th>Segment-12</th><th>69V28</th><th>R-1575</th><th>S6-14-03</th><th>Eyach</th><th>AR578</th></tr></thead><tbody><tr><td>N-7180</td><td>95.98<br>(92.45)</td><td>96.78<br>(92.45)</td><td>94.37<br>(87.74)</td><td>66.22<br>(51.89)</td><td>66.76<br>(53.77)</td></tr><tr><td>69V28</td><td></td><td>95.44<br>(91.51)</td><td>95.17<br>(89.62)</td><td>66.76<br>(55.66)</td><td>67.56<br>(55.66)</td></tr><tr><td>R-1575</td><td></td><td></td><td>95.44<br>(92.45)</td><td>65.95<br>(54.72)</td><td>66.49<br>(55.66)</td></tr><tr><td>S6-14-03</td><td></td><td></td><td></td><td>65.68<br>(53.77)</td><td>65.95<br>(54.72)</td></tr><tr><td>Eyach</td><td></td><td></td><td></td><td></td><td>92.29<br>(87.74)</td></tr></tbody></table>
<table><caption>**Table 3 :** Segments 7 to 12 of group B coltiviruses : lengths of nucleotide and protein sequences.</caption><thead><tr><th rowspan="2">Gene</th><th colspan="5">Length of the sequence</th></tr><tr><th>JKT-6423</th><th>BANNA</th><th>JKT-6969</th><th>JKT-7043</th><th>JKT-7075</th></tr></thead><tbody><tr><td>7</td><td>1136 (307)</td><td>1137 (307)</td><td>1136 (307)</td><td>1136 (307)</td><td>1259 (347)</td></tr><tr><td>8</td><td>1119 (302)</td><td>1119 (302)</td><td>1119 (302)</td><td>1119 (302)</td><td>1140 (302)</td></tr><tr><td>9</td><td>1101 (283)</td><td>1100 (283)</td><td>1141 (280)</td><td>1141 (280)</td><td>1054 (303)</td></tr><tr><td>10</td><td>977 (249)</td><td>978 (249)</td><td>977 (241)</td><td>977 (249)</td><td>946 (260)</td></tr><tr><td>11</td><td>867 (180)</td><td>867 (180)</td><td>867 (178)</td><td>867 (178)</td><td>894 (263)</td></tr><tr><td>12</td><td>862 (207)</td><td>861 (207)</td><td>862 (207)</td><td>862 (207)</td><td>756 (190)</td></tr></tbody></table>

Values are given in base pairs for nucleotide sequences. Values between brackets indicate the number of amino acid residues in the protein encoded by the segment.
<table><caption>**Table 4.** List of species in the genus *Coltivirus*</caption><thead><tr><th>Species in the genus<br>{vector species : host}</th><th>sequence accession<br>numbers</th><th>assigned<br>abbreviations</th></tr></thead><tbody><tr><td colspan="3"><strong>Coltivirus group A</strong></td></tr><tr><td>1- Colorado tick fever virus species (subgroup A1)<br>{Ixodidae ticks : Rodents, Humans}<br>Over 22 isolates reported<br>2 serotypes : CTFV and S6-14-03</td><td>Segment 9 : AF007172<br>Segment 10 : AF000720<br>Segment 11 : U72694<br>Segment 12 : U5322</td><td>(CTFV)</td></tr><tr><td>2-Eyach virus species (subgroup A2)<br>{Ixodidae ticks : Possibly Humans}<br>(Eyach virus)<br>(AR577)<br>(AR578)</td><td>Segment 12 : AF007185</td><td>(EYAV)</td></tr><tr><td colspan="3"><strong>Coltivirus group B</strong></td></tr><tr><td>1-JKT-7075 species (Subgroup B1)<br>{Culex mosquitoes}<br>JKT-7075</td><td>Segment 7 : AF052023<br>segment 8 : AF052022<br>segment 9 : AF052021<br>segment 10 : AF052020<br>segment 11 : AF052019<br>segment 12 : AF019909</td><td>(JKT-7075)</td></tr><tr><td>2-JKT-6423 species (Subgroup B2)<br>{Culex and Anopheles mosquitoes : Humans, other vertebrates}<br>JKT-6423<br>JKT-6969<br>JKT-7043<br>BANNA virus.</td><td>Segment 7 : AF052018<br>Segment 8 : AF052017<br>Segment 9 : AF052016<br>Segment 10 : AF052015<br>Segment 11 : AF052014<br>Segment 12 : AF019908</td><td>(JKT-6423)</td></tr><tr><td colspan="3">Chinese isolates that belong most probably to JKT-6423 species based on serological and electrophoretic profiles analyses (Xu et al., 1990, Chen & Tao, 1996) : HN59, HN131, HN191, HN295.</td></tr></tbody></table>
<figure><img src="image_2.png" /><figcaption>Figure 2</figcaption></figure>
<table><caption>Figure 3.</caption><tr><td>7-7075</td><td>174-LAGLKALMLFHEHTNC</td><td>IHGDCNPSNIMCDKMGNVKIVD-211</td></tr><tr><td>MAPK</td><td>257-LATVKALNHLKENLKII</td><td>IHRDIKPSNILLDTNGNIKLCD-294</td></tr><tr><td></td><td>LA +KAL E+ IH D PSNI+ D GN+K+ D</td><td></td></tr><tr><td>7-Banna</td><td>156-HSSNPHLLHGDANPDNIMSDSNGYLKLVD-184</td></tr><tr><td>7-6423</td><td>156-HSSNQHLLHGDANPDNIMSDSEGYLKLVD-184</td></tr><tr><td>P34-PK</td><td>119-YSHGHRILHRDLKPQNLLIDRQGALKLAD-147</td></tr><tr><td></td><td>+S +LH D P N++ D +G LKL D</td><td></td></tr><tr><td>7-7043</td><td>162-YLHGDVNPENLMSDPNGYLKLVD-184</td></tr><tr><td>7-6969</td><td>162-YLHGDVNPENIMSDANGYLKLVD-184</td></tr><tr><td>Ser./Threo.PK</td><td>138-VAHRDIKPENILLDKNGNLKLAD-160</td></tr><tr><td></td><td>H D+ PENI+ D NG LKL D</td><td></td></tr><tr><td>P68-kinase</td><td colspan="2">IHRDLKPSNIFLVDTKQVKIGD</td></tr><tr><td>Consensus</td><td colspan="2">ΔH-D--P-NΔ--D--G-ZKΔ-D</td></tr></table>
<table><caption>Figure 4</caption><tr><td>8-7075</td><td>5-</td><td>KGMLQELCVKRGLELPVYEKLSKVGPDHAPTITVKLTANGIEVIEAASSRAQAEKLAAA-63</td></tr><tr><td>RNAseIII</td><td>159-</td><td>KTALQEWAQARGLPPPRYETLGRDGPDHAPQFRIAVVLASGETEEAQAGSKRNAEQAAA-217</td></tr><tr><td></td><td>K</td><td>LQE</td><td>RGL</td><td>P</td><td>YE</td><td>L</td><td>+</td><td>GPDHAP</td><td>+</td><td>+</td><td>E</td><td>EA</td><td>+</td><td>+</td><td>+</td><td>AAA</td></tr><tr><td>12-B2</td><td>3-</td><td>PVSVVHSFARSQGLPLNFETVGCEGPSHDPRFVIECKLLDFHQCT-D---SSKKRAIQKICVLI-63</td></tr><tr><td>MEPS</td><td>579-</td><td>PISQVHEIGIKRNMTVHFKVLREEGPAHMKNFITACIVGSIVTEGEGNGKKVSKKRAAEKMLVEL-643</td></tr><tr><td></td><td>P+S</td><td>VH</td><td>+</td><td>+</td><td>++</td><td>F+</td><td>+</td><td>EGP+H</td><td>F+</td><td>C</td><td>+</td><td>+</td><td>+</td><td>SKKRA</td><td>+K+</td><td>V</td><td>+</td></tr></table>
**Legend**

**Figure 1** : The electrophoretic profiles of the genomes of cultivable coltiviruses are displayed.

Lane M : Size marker labelled in base pairs.

Lanes 1 to 7 : genomes of CTF, S6-14-03, JKT-6423, Banna, JKT-6969, JKT-7043 and JKT-7075 viruses respectively.

**Figure 2** : Interaction between terminal sequences of the sense strands of segment 12 (a), 11 (b), 10 (c), 9 (d), 8 (e) and 7 (f) of JKT-6423 virus. The displayed structures were generated with the help of the MacStan software program. I denotes canonical base pairing while : denotes non-canonical base pairing.

**Figure 3** : Matching between protein kinases and putative proteins encoded by segment 7 of different group B *Coltivirus* isolates.

MAPK : human dual specificity mitogen activated protein kinase (accession number : P45985), P34-PK : cell division control protein-2 homologue (P34 protein kinase, accession number : P34112), Ser./Threo.PK : yeast probable Serine/Threonine-protein kinase YBR274w (accession number : P38147), P68-kinase : human double-stranded RNA dependent protein kinase (accession number : P19525). Shadowed sequences represent identical and similar (+) amino acids. Δ = I or L and Z = I, L or V and − in the consensus sequence represents any amino acid.

**Figure 4** : Matching of proteins of group B coltiviruses to proteins harbouring dsRNA
binding domains. RNAse III : *Rhodobacter capsulatus* Ribonuclease III (accession number : 52698), MEPS : *Drosophila melanogaster* Maternal Effect Protein Staufen (accession number : P25159). 8-7075 : VP8 from JKT-7075, 12-B2 : VP12 of subgroup B2 isolates. Shadowed sequences represent identical and similar (+) amino acids.

**Table 2.** a) : values calculated for segments 7+8+9+10+11+12 of group B viruses, b) and c) : values calculated for segments 9 and 7 respectively from group B viruses and d) : values calculated for segment 12 of group A viruses. The value between brackets represents amino acid identity while the other value represents the nucleic acid identity.