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Type Species |
(CTFV) |
The coltivirus genome consists of 12 segments of dsRNA. During replication, viruses are found in the cell cytoplasm, associated with granular matrices, arrays of filaments or “tubules” and fine kinky threads. Immunofluorescent staining reveals nucleolar fluorescence. Viruses are transmitted to vertebrate hosts by ticks and mosquito vectors.
Coltivirus particles are 60-80 nm in diameter having two concentric capsid shells with a core that is about 50 nm in diameter. Electron microscopic studies, using negative staining have shown that particles have a relatively smooth surface capsomeric structure and icosahedral symmetry (Fig. 11). Particles are found intimately associated with filamentous structures and granular matrices in the cytoplasm. The majority of the viral particles are non-enveloped, but a few acquire an envelope structure during the passage through the endoplasmic reticulum.
Physicochemical and Physical Properties
The buoyant density of the virus in CsCl is 1.38 g/cm3. Viruses are stable between pH 7 and 8, but lose infectivity at pH 3.0. At 4°C, the virus is stable for long periods when stored in presence of 50% fetal calf serum in 0.2 M Tris-HCl pH 7.8. Heating to 55°C considerably decreases the viral infectivity. Coltiviruses are fairly stable upon treatment with non-ionic detergents, sodium lauroyl sarcosine, or freon but the viral infectivity is abolished by treatment with sodium deoxycholate or sodium dodecyl sulfate. Moderate ultrasonic oscillation treatment does not destroy infectivity and can be used in virus purification. Viruses can be stored for long periods at -80°C, and infectivity is further protected by addition of 50% foetal calf serum.
The genome consists of 12 dsRNA segments that are named “genome segment 1” to “genome segment 12” in order of reducing molecular weight, or increasing electrophoretic mobility during agarose gel electrophoresis. The genome comprises approximately 21,000 bp and the segment length range between 3.75 kbp and 756 bp. In subgroup ‘A’ coltiviruses, the genomic RNA of CTFV migrates in three size classes during 1% agarose gel electrophoresis (AGE): the large (long) or L-segments (segments 1-4), the medium length or M-segments (segments 5-10) and the small (short) or S-segments (segments 11 and 12). The genome comprises approximately 28 kbp and the segment lengths range between 4.14 kbp and 675 bp (total Mr about 18 
106). Within Subgroup A, analysis of Eyach virus (EYAV) dsRNA by AGE showed a genome segments migration pattern that is significantly different from that of CTFV. The genomic RNAs from subgroup ‘B’ coltiviruses also exhibit two distinct electrophoretic profiles: a 6-6 electrophoretic profile for Banna virus (BAV-In6423) and a 6-5-1 electrophoretic profile for Kadipiro virus (KDV).
RNA cross hybridization analysis shows that CTFV isolates have remained relatively homogenous and distinct CTFV serotypes are difficult to define (although some variation does occur, for example in genome segments 4 and 6). The overall similarities between nucleotide sequences from the four smaller genome segments of different CTFV isolates ranged between 90% and 100%. The degree of similarity between nucleotide sequences of segment 12 from CTFV and EYAV isolates, ranged from 53% to 58%. Isolates of BAV show variation in their PAGE profiles, as a consequence of variations in primary sequence. The overall similarity in nucleotide sequences of these isolates ranges from 80% and 99%, with genome segments 7 and in particular 9 being most variable (similarities 54%-99%).
Based on those segments that have been analyzed, the length of the 5
-NTRs of the different coltiviruses is 17-40 nts, while that of the 3-NTRs is 35-249 nts. The G+C content from Subgroup A coltiviruses (48-52%) is different from Subgroup B coltiviruses (37-39%).
Viral proteins of CTFV were translated from dsRNA in a cell free system. Viral proteins of the other coltiviruses have not been characterized (Tables 8 and 9).
None reported.
None reported.
Genome Organization and Replication
In cells infected by CTFV, granular matrices are produced which contain virus-like particles. These structures appear similar to VIBs produced during orbivirus infections. In addition, bundles of filaments (tubules), characterized by cross-striations, are found in the cytoplasm and, in some cases, in the nucleus of infected cells. These may also be comparable to the ‘tubules’ found in orbivirus infected cells. There is no evidence for virus release prior to cell death and disruption, after which more than 90% of virus particles remain cell associated. Immunofluorescence, shows that viral proteins accumulate in the cytoplasm and could be detected from the 12th hour post-infection. Nucleolar fluorescence was also observed. Mosquito cells infected by EYAV show syncitial foci.
The Asian subgroup B coltivirus isolates replicate in mosquitoes cell lines, and considerable amounts of virus are liberated in the culture medium prior to cell death and gross CPE, which is attained by 40 hours post-infection with BAV, and 72 hours post-infection with KDV. Intracellular radio labeling of viral polypeptides has shown that unless a DNA analogue or actinomycin D is included at the time of infection with CTFV, label continues to be incorporated into cellular proteins. With subgroup B coltiviruses, label is incorporated predominantly into viral polypeptides, even in absence of inhibitors of DNA replication, demonstrating ‘shut off’ of host cell protein synthesis. It has been proposed that the subgroup B coltiviruses should be reclassified within a separate new genus on the basis of very low aa sequence homology with CTFV (<20%) and >35% difference in total genome size.
CTFV from North America and EYAV from Europe, which are classified as distinct species, show little cross-reaction in neutralization tests. An isolate, S6-14-03, obtained from a hare (Lepus californicus) in Northern California (CTFV-Ca), also regarded as a distinct species, is serologically related to Eyach virus. Assays using polyclonal or monoclonal antibodies failed to detect any cross-reactions between Subgroup B coltiviruses and CTFV. Four species, belonging to the two different subgroups of coltiviruses could therefore be distinguished. However, additional unassigned Chinese isolates require further analysis to confirm or rule out the presence of other species.
Coltiviruses have been isolated from several mammalian species (including humans) and from ticks and mosquitoes which serve as vectors. The tick species include Dermacentor andersoni, D. occidentales, D. albipictus, D. parumapertus, Haemaphysalis leporispalustris, Otobius lagophilus, Ixodes sculptus, I. spinipalpis, I. ricinus and I. ventalloi. The mosquito species include Culex vishnui, C. fuscocephalus, Anopeles vagus, A. aconitus and A. subpictus.
Ticks become infected with CTFV on ingestion of a blood meal from an infected vertebrate host. Both adult and nymphal ticks become persistently infected and provide an overwintering mechanism for the virus. CTFV is transmitted transstadially but not transovarially. Some rodent species have prolonged viraemias (more than 5 months) which may also facilitate overwintering and virus persistence. Humans usually become infected with CTFV when bitten by the adult wood tick D. andersoni but probably do not act as a source of reinfection for other ticks. Transmission from person to person has been recorded as the result of blood transfusion. The prolonged viraemia observed in humans and rodents is thought to be due to the intra-erythrocytic location of virions, protecting them from immune clearance.
CTFV is characterized in humans by an abrupt onset of fever, chills, headache, retro-orbital pains, photophobia, myalgia and generalised malaise. Abdominal pain occurs in about 20% of patients. Rashes are uncommon (less than 10%). A diphasic, or even triphasic, febrile pattern has been observed, usually lasting for 5-10 days. Severe forms of the disease, involving infection of the central nervous system, or haemorrhagic fever, or both, have been infrequently observed (nearly always in children under 12 years of age). A small number of such cases are fatal. Congenital infection with CTFV may occur, although the risk of abortion and congenital defects remains uncertain. Antibodies to Eyach virus have been found in patients with meningoencephalitis and polyneuritis but a causal relationship to the virus has not been established.
CTFV causes leukopaenia in adult hamsters and in about two-thirds of infected humans. Suckling mice, which usually die at 6-8 days post-infection, suffer myocardial necrosis, necrobiotic cerebellar changes, widespread focal necrosis and perivascular inflammation in the cerebral cortex, degeneration of skeletal myofibers, hepatic necrosis, acute involution of the thymus, focal necrosis in the retina and in brown fat. The pathologic changes in mice due to CTFV infection (in skeletal muscle, heart and brain), are consistent with the clinical features of human infection which may include meningitis, meningo-encephalitis, encephalitis, gastro-intestinal bleeding, pneumonia and myocarditis.
CTFV occurs in forest habitats at 4,000-10,000 ft. elevation in the Rocky Mountain region of North America. Antibodies to the virus have been detected in hares in Ontario and a virus isolate has been reported from Long Island, New York. Eyach virus appears to be widely distributed in Europe. The BAV isolates were made in 1987, from cerebrospinal fluids and sera of patients with febrile manifestations and encephalitis. The Indonesian isolates were made solely from mosquitoes. Although the disease clinical manifestations are not as well documented as for CTFV. BAV and KDV seems to be widespread in countries of the South-Eastern Asia.
List of Species Demarcation Criteria in the Genus
In common with the other genera within the family Reoviridae, the prime determinant for inclusion of virus isolates within a single coltivirus species is defined by their compatibility for reassortment of genome segments during co-infections, allowing the exchange of genetic information and generating viable progeny virus strains. However, data providing direct evidence of segment reassortment between isolates is very limited. Therefore, serological comparisons, comparisons of RNA sequence, cross-hybridization, analysis of electropherotypes and analysis of conserved RNA sequences in near terminal regions of the genome segments represent major factors used to examine the level of similarity that exists between isolates.
Members of a single Coltivirus species may be identified by:
1. |
An ability to exchange genetic material by genome segment reassortment during dual infections, thereby producing viable progeny virus strains. |
2. |
RNA cross hybridization assays (Northern or dot blots, with probes made from viral RNA or cDNA). Within a single species, the amount of RNA sequence similarity is higher than 74% under hybridization conditions of Tm (RNA) -36°C. |
3. |
Serological comparisons by complement fixation and neutralization assays. The CTFV species isolates show high levels of cross neutralization. Cross reactivity between CTFV and EYAV species (both subgroup A viruses) has only been detected by CF tests. Virus species do not cross-react significantly between subgroup A and B, by either neutralization or CF tests. |
4. |
RNA sequence analysis (e.g., segments 7 to 12). Within a single species, high levels of sequence similarities are observed in conserved segments, e.g., segment 12 shows less than 11% sequence variation within a single virus species. |
5. |
Comparisons of amino acid sequences (for example, those of the translation products of genome segment 12, Fig. 12) indicate that different species will contain less than 50% amino acid similarity. |
6. |
The analysis of electropherotypes by agarose gel electrophoresis. Within a single species the electropherotype is relatively uniform. However, deletions or additions can occur (as in segments 7 and 9 of the BAV species) resulting in variations in electropherotype. |
7. |
Analysis of conserved RNA terminal sequences. These sequences show conservation within species isolates. Sequences at the 3 |
8. |
Analysis of the G+C content of the viral RNA. Species belonging to the same subgroup have similar G+C content. RNA segments of CTFV and EYAV species, belonging to coltivirus subgroup A, have a G+C content ranging between 48% and 52%, while segments of BAV and KDV species, belonging to coltivirus subgroup B, have a G+C content ranging between 37% and 39%). |
CTFV-S6-14-03 from a hare collected in California in 1976 shows some one-way cross-reaction in serum neutralization tests with Eyach virus, but is clearly distinguishable and has been reported as a distinct serotype. Serological variants of Eyach virus (Eyach virus (France 577) and Eyach virus (France 578)) have also been reported. Recently, several Indonesian (BAV-In6423, BAV-In6969, BAV-In7043 and KDV-Ja7075) and Chinese virus isolates have been obtained (BAV-Ch, BAV-HN59, BAV-HN131, BAV-HN191 and BAV-HN295), which are serologically distinct from subgroup A coltiviruses. The analysis of these isolates permits the identification of four different species. The status of some Chinese isolates remains to be confirmed.
Official virus species names are in italics. Tentative virus species names, alternative names ( ), strains or serotypes are not italicized. Virus names, genome sequence accession numbers [ ], arthropod vectors and host species { } and assigned abbreviations ( ) are:
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Coltivirus subgroup A | ||
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Colorado tick fever virus (2 serotypes) |
(CTFV) | |
|
>22 isolates |
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Colorado tick fever virus |
Seg 9:[AF007172], Seg10: [AF000720] |
(CTFV) |
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California hare coltivirus S6-14-03 {Ixodidae ticks : Rodents, Humans } |
Seg 11: [U72694], Seg 12: [U5322] |
(CTFV-S6-14-03) |
|
Eyach virus |
(EYAV) | |
|
Eyach virus (Germany) |
Seg 12: [AF007185] |
(EYAV-Gr) |
|
Eyach virus (France-577) |
(EYAV-Fr577) | |
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Eyach virus (France-578) {Ixodidae ticks : Possibly Humans } |
(EYAV-Fr578) | |
|
Coltivirus subgroup B | ||
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Banna virus |
(BAV) | |
|
Banna virus (China) |
(BAV-Ch) | |
|
Banna virus (Indonesia-6423) |
Seg 7: [AF052018], Seg 8: [AF052017] |
(BAV-In6423) |
|
Banna virus (Indonesia-6969) |
Seg 9: [AF052016], Seg 10: [AF052015] |
(BAV-In6969) |
|
Banna virus (Indonesia-7043) {Culex and Anopheles mosquitoes : Humans, other vertebrates} |
Seg 11: [AF052014], Seg 12: [AF019908] |
(BAV-In7043) |
|
Kadipiro virus |
(KDV) | |
|
Kadipiro virus (Java-7075) {Culex mosquitoes} |
Seg 7: [AF052023], Seg 8: [AF052022] Seg 9: [AF052021], Seg 10: [AF052020] Seg 11: [AF052019], Seg 12: [AF019909] |
(KDV-Ja7075) |
Tentative Species in the Genus
Chinese isolates that belong most probably to Banna virus based on serological and electrophoretic profile analyses.
|
Banna virus (China-HN59) |
(BAV-HN59V) |
|
Banna virus (China-HN131) |
(BAV-HN131V) |
|
Banna virus (China-HN191) |
(BAV-HN191V) |
|
Banna virus (China-HN295) |
(BAV-HN295V) |
Phylogenetic Relationships Within the Genus
See Fig. 12.
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