Genus Thogotovirus

The morphology and morphogenesis of these viruses show similarities with the influenza viruses. Virions are spherical or pleomorphic, 80–120 nm in diameter, and filamentous forms occur. The virion envelope is derived from cell membrane lipids and bear surface glycoprotein projections, 10–14 nm in length and 4–6 nm in diameter. Virions contain six or seven segments of linear, negative sense single-stranded RNA. Total genomic size is about 10 kb. Like the influenza viruses, each viral RNA segment possesses conserved regions of semicomplementary nucleotides at the 3′ and 5′ termini and mRNA synthesis is primed by host-derived cap structures. Both the 3′ and 5′ sequences of virion RNA are required for viral RNA promoter activity and the cap-snatching mechanism appears unique (Leahy, Dessens & Nuttall, 1997).

The type species, Thogoto virus (THOV), contains six single-stranded RNA segments. Four of them encode gene products that correspond to the viral polymerase (PB1, PB2 and PA) and nucleocapsid protein (NP) of influenza viruses (Weber et al. 1998). However, the fourth largest segment encodes a surface glycoprotein that is unrelated to any influenza viral protein but instead shows striking sequence homology to a baculovirus surface glycoprotein (Morse, Marriott & Nuttall, 1992). The same is true for Dhori virus (DHOV) (Freedman-Faulstich & Fuller, 1990). This unique glycoprotein is probably the key to the ability of members of the Thogotovirus genus to infect ticks (Nuttall et al. 1995). Influenza viruses use sialic acid residues on the surface of vertebrate cell membranes as receptors for infecting cells. Ticks are devoid of sialic acid. Clearly Thogotovirus members have got round this problem by evolving a different mechanism of cell infection to that of influenza viruses. A recent comparison of the glycoprotein sequences of eight THOV isolates, two DHOV isolates and one Batken virus isolate with the glycoprotein sequence of 10 nucleopolyhedrosis viruses (insect baculoviruses) suggests that the sequence similarity may represent convergent evolution rather than a common ancestry for the encoding gene (S. Turner, personal communication).

THOV has been isolated from Boophilus and Rhipicephalus spp. ticks in Africa (Kenya) and Europe (Sicily) as well as from Amblyomma variegatum ticks in Nigeria and Hyalomma spp. ticks in Nigeria and Egypt. THOV has also been isolated in the Central African Republic, Cameroon, Uganda and Ethiopia, and in several countries of southern Europe. Infections in sheep have been associated with high levels of abortion. THOV also infects cattle, goats and mongoose, and there are two reported cases of infections in humans associated with clinical conditions (Woodall, 2001). DHOV has a somewhat different but overlapping geographical distribution to that of THOV, occurring in India, eastern Russia, as well as Egypt and southern Portugal. DHOV virus has been isolated from Hyalomma spp. ticks. There is no detectable serological reactivity between THOV and DHOV and the structural differences (THOV has six RNA segments and DHOV has seven) and sequence diversity of 37% and 31% in the nucleoprotein and the envelope protein, respectively, support their separate species status. Batken virus, isolated from mosquitoes and ticks from Russia, cross reacts serologically with DHOV and shares 98% identity in a portion of the nucleoprotein and 90% identity in a portion of the envelope protein. These and other data suggest that Batken virus is closely related to DHOV (Frese et al. 1997). None of the viral proteins of members of the Thogotovirus genus are related antigenically to those of influenza viruses.

THOV has been used extensively in experimental studies of saliva-activated transmission in which the virus has been shown to exploit the pharmacological properties of its vector's saliva as described by Nuttall & Labuda in this Supplement. It has also been used to investigate reassortment using temperature-sensitive mutants to follow the exchange of genomic segments between viruses. The ability of THOV to reassort has been demonstrated in both ticks and a vertebrate host (Davies et al. 1987; Jones et al. 1987). However, the significance of such genetic exchange in the epidemiology of this virus is unknown.

Genus Orbivirus

Virions are spherical in appearance but have icosahedral symmetry. Although no lipid envelope is present on mature virions, they can leave the host cell by budding through the cell plasma membrane. During this process they transiently acquire an unstable membrane envelope. Unpurified virus is often associated with cellular membranes. The outer capsid has an ordered structure with icosahedral symmetry. The surface layer of the core particle is composed of capsomeres arranged as hexameric rings. These rings give rise to the name of the genus. The core particle also contains a complete inner capsid shell surrounding the 10 segments of double-stranded RNA that comprise the viral genome. Minor core proteins are attached to the inner surface of the subcore at the five-fold symmetry axes. Replication is characterized by production of viral ‘tubules’ and viral inclusion bodies and may be accompanied by formation of flat hexagonal crystals of the major outer core protein in the cytoplasm of infected cells.

Orbiviruses are transmitted between vertebrate hosts by a variety of haematophagous arthropods. They do not appear to cause disease in their natural hosts. Most orbiviruses associated with birds have been isolated from arthropod vectors that feed on birds, but evidence of infection of birds has been obtained for only a few orbiviruses (Nuttall, 1993).

The genus Orbivirus currently comprises 20 serological groups (Roy, 2001). The type species, Bluetongue virus (BTV), causes an economically important disease of sheep and is transmitted by midges of the genus Culicoides. It is one of the best characterized arboviruses. Approximately 60 tick-borne orbiviruses have been identified (Table 2). Most of these are variants (serotypes or topotypes) of Great Island virus (formerly in the Kemerovo serogroup) transmitted by ixodid ticks that parasitize seabirds (guillemots or murres, puffins, penguins, gannets, gulls etc.); at least 40 viruses have been isolated from the common seabird tick, Ixodes uriae. The distribution of Great Island virus reflects the bipolar distribution of I. uriae (Main et al. 1973; Nuttall, 1984; Chastel, 1988). Although these viruses are confined to seabird colonies, the presence of antibodies in various mammals including humans living in close proximity to the colonies (Lvov et al. 1972), and the recovery of I. uriae from ‘land’ birds (Arthur, 1963), indicate the possibility of extension of their normal range.

The four viruses of the species Kemerovo virus are maintained in the Palearctic region among small mammals and birds by two related species of ixodid ticks: Kemerovo virus by Ixodes persulcatus in western Siberia (Libikova et al. 1964), Tribeč virus and Lipovnik virus by I. ricinus in central Europe (Gresíková et al. 1965) and Kharagysh virus by I. ricinus in Moldova (Skofertsa et al. 1972). This group of viruses is listed under Great Island virus by the International Committee on Taxonomy of Viruses (van Regenmortel et al. 2000). We have identified Kemerovo virus as a distinct species because it showed limited genome segment reassortment with three representatives of Great Island virus, and none with Chenuda virus, Essaouira virus or Mono Lake virus (Nuttall & Moss, 1989). Speciation of Kemerovo virus and Great Island virus may be at a transitional stage in which ancestral links can be detected under highly selective experimental conditions. We have assumed genetic exchange between these two species cannot occur in nature, particularly given their different ecologies.

The species Chenuda virus includes 7 different serotypes from soft ticks of the genera Argas and Ornithodoros parasitizing birds (Hoogstraal, 1973). Chenuda virus was originally isolated from Argas reflexus hermanni collected from a pigeon house in Egypt (Taylor et al. 1966). Related viruses have been isolated from O. coniceps from pigeon nests in the Chatkal mountains and O. maritimus ticks infesting gulls (Larus argentatus) on an island in the Caspian Sea (Baku virus), gull colonies in Morocco (Essaouira virus and Kala Iris virus), and gull and shag (Phalacrocorax aristotelis) colonies in Brittany (Moellez virus). Two isolates of Great Saltee Island virus (GS80-5, GS80-6) were from eggs and adult male O. maritimus feeding in a breeding colony of shags on a small island off the south-east coast of Ireland. This virus is classed as a possible member of the species based on its geographical location, and vector and host associations.

Huacho virus, Mono Lake virus and Sixgun City virus are classified by the International Committee on Taxonomy of Viruses as belonging to the species Chenuda virus (van Regenmortel et al. 2000). Members of an orbivirus species are distinguished by their ability to exchange genomic segments. Based on this definition, we have recognised a distinct species, Mono Lake virus. The basis for this speciation is the inability of Mono Lake virus to demonstrate genome segment reassortment when tested experimentally with representatives of Chenuda virus, Great Island virus, and Kemerovo virus (Nuttall & Moss, 1989). Mono Lake virus was originally isolated from a pool of 10 adult Argas cooleyi ticks collected in 1966 from the nest of the California gull (L. californicus) on an island in Mono Lake, California. Other species members are recognized by their antigenic cross-reactivity with Mono Lake virus. These are Huacho virus, isolated from O. amblus nymphs collected from rocks of a guano seabird colony at Punta Salinas, and Sixgun City virus from Argas cooleyi collected in the nests of cliff swallows (Petrochelidon pyrrhonota) in Texas.

Chobar Gorge virus has two serotypes, Chobar Gorge virus and Fomede virus. They are associated with bats. Wad Medani virus has been isolated from sheep and from ixodid ticks of the genera Boophilus, Hyalomma, Ambylomma and Rhipicephalus in the Sudan, West Pakistan, India, Russia, and Jamaica. The closely related Seletar virus was isolated from B. microplus in Singapore and Malaysia.

The Great Island orbiviruses associated with seabird colonies provide a fascinating model in the study of virus evolution. Main et al. (1973) found two distinct but closely related serotypes, Great Island virus and Bauline virus, among puffins and petrels on Great Island, with little or no indication of other serotypes. They suggested that the antigenic identity of serotypes may be maintained by the isolation of the primary hosts of the vector and that each serotype developed in separate demes on the island. Yunker (1975) suggested that each virus within defined complexes has been influenced by common patterns of geography, ecology and behaviour. These included a discontinuous distribution, the nidiculous activity of the ticks that maintained them and the homing-colonial instinct of the bird hosts. Isolation produces a large number of strong insular variants, which in some cases develop into separate serotypes. Experimental studies have shown that Great Island serogroup viruses from ticks collected in seabird colonies in Scotland, Ireland, Iceland, Newfoundland, and Macquarie Island in the sub-Antarctic, can swap RNA segments. Thus, despite their extensive geographical distribution, this group of viruses constitutes a single gene pool clearly representing one virus species (Nuttall & Moss, 1989).

Structural and genetic studies have revealed significant differences between the tick-borne orbiviruses and (BTV, the type species of the genus) that may provide clues to their different ecologies. The most notable is in the structure and function of the two outer capsid proteins. In BTV and other insect-borne orbiviruses, VP2 and VP5 are the protein components of the outer capsid. VP2, the major determinant of the virus serotype, carries neutralising epitopes and the binding site for the vertebrate cell receptor. In contrast, the major determinant of serotype in the tick-borne orbiviruses is VP5 while VP4 is the second component of the outer capsid. VP4 probably bears the binding site for the vertebrate cell receptor as it determines neurovirulence. Interaction of VP4 and VP5 of Great Island virus (GIV) modulates viral pathogenicity. Thus, by reassortment, new viruses can be produced experimentally that are more or less virulent than either of their parental viruses (Nuttall et al. 1992). The significance of these studies for the natural ecology of GIV is unknown, particularly as the virus does not cause overt disease in its seabird hosts. However, a 4 year epidemiological study of guillemots (Uria aalge), the main amplifying hosts for GIV on the Isle of May, Scotland, revealed that GIV infections show a positive association with breeding success and might even influence colony structure (M. Nunn, personal communication). More needs to be done to determine the impact of GIV infections on seabirds and the role genetic reassortment plays in virus survival.

Comparison of three-dimensional models of BTV and Broadhaven virus (Great Island virus) indicate remarkable similarity except for differences in accessibility of the outer shell proteins (Schoehn et al. 1997). This may reflect the need to access and cleave VP2 of BTV within the midgut of its Culicoides vector. Cleavage of VP2 exposes the core protein, VP7, that bears the Arg–Gly–Asp (RGD) motif involved in insect cell infection. In contrast to insects, bloodmeal digestion in ticks occurs intracellularly. Furthermore, none of the viral proteins of tick-borne orbiviruses have been reported to carry a RGD motif. Hence tick-borne viruses most likely have evolved a mechanism of tick cell infection that does not rely on proteolysis in the midgut, unlike thier insect-borne relatives.

Genus Coltivirus

The most important feature distinguishing coltiviruses from the other members of the Reoviridae family is a genome comprising 12 segments of double-stranded RNA. During replication, viruses are found in the cell cytoplasm but immunofluorescent staining also reveals nucleolar fluorescence. Coltiviruses are transmitted to vertebrate hosts by ticks as well as mosquitoes (Brown et al. 1993). Tick-borne members of the genus are included in the Coltivirus subgroup A and mosquito-borne coltiviruses in subgroup B although it has been proposed that the latter group constitute a separate genus.

Coltivirus particles are 60–80 nm in diameter having two concentric capsid shells with a core that is about 50 nm in diameter. Electron microscope studies using negative staining reveal particles that have a relatively smooth surface capsomeric structure and icosahedral symmetry. 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 passage through the endoplasmic reticulum.

Coltiviruses have been isolated from several mammalian species (including humans) and from ticks and also mosquitoes. Colorado tick fever virus (CTFV), the type species of the genus, is transmitted by ticks and causes an acute febrile illness in humans. There appears to be a single serotype involved in human infections. Because of the ease of isolation of the virus from patients and ticks, the geographical distribution has been determined as essentially that of the major vector, Dermacentor andersoni, in mountainous northwestern USA and Canada. Other tick species from which CTFV has been isolated include Dermacentor occidentalis, D. albipictus, D. parumapertus, Haemaphysalis leporispalustris and a species of Otobius. Vertebrate reservoirs include rodents, ground squirrels, pine squirrels, chipmunks, meadow voles and porcupines (Burgdorfer, 1977).

CTFV persists within erythrocytes in human patients and in experimentally infected animals (Emmons et al. 1972; Hughes, Casper & Clifford, 1974). 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 trans-stadially and also trans-ovarially as demonstrated in Dermacentor andersoni ticks experimentally, and by the finding of related viruses from field collected Ixodes larvae in France and Germany (Calisher, 2001). Some rodent species have prolonged viraemias (more than 5 months), which may also facilitate overwintering, and virus persistence. Humans usually become infected when bitten by adult D. andersoni ticks 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 (Emmons et al. 1972).

In 1972, a second serotype of CTFV was isolated from Ixodes ricinus ticks collected near Tubingen, West Germany (Rehse-Kupper et al. 1976). This virus is now recognised as a distinct species, Eyach virus. The virus has been associated with meningo-encephalitis in humans based on detection of specific IgM and IgG in patients with the disease. California hare coltivirus, isolated from Lepus californicus, is antigenically related to Eyach virus and may represent a distinct species. Several unassigned Chinese coltivirus isolates require further characterisation.

Genus Bunyavirus

Classification of viruses belonging to the family Bunyaviridae was originally based on their serological relationships. These data have now been supplemented and largely supported by biochemical and genetic analyses. Viruses of the genus Bunyavirus are serologically unrelated to members of other genera.

At the genome level, the most important distinguishing feature is the consensus terminal nucleotide sequence of the L, M, and S genome segments. The N and NS_s proteins are encoded in overlapping reading frames by the S RNA and are translated from the same complementary mRNA by alternative AUG initiation codon usage.

The genus Bunyavirus comprises over 150 viruses representing nearly 50 viral species. Most bunyaviruses are mosquito-borne and some have been isolated from Culicoides midges; only a few tick-borne viruses have been recorded (Table 3). Isolates of Bahig virus and Matruh virus (representatives of Tete virus) originate from Africa (Egypt, South Africa, Nigeria), Europe (Italy), Cyprus and Japan. Most isolates have been made from migratory birds whereas a few isolates are from ticks, yet it is believed ticks are the main vectors. Estero Real virus is the only other bunyavirus believed to be tick-borne.

Genus Nairovirus

The genus Nairovirus is named after Nairobi sheep disease virus (NSDV) and contains some of the most important tick-borne pathogens. It was divided into seven serogroups: Crimean-Congo haemorrhagic fever, Dera Ghazi Khan, Hughes, Nairobi sheep disease (NSD), Qalyub, Sakhalin and Thiafora group (Clerx, Casals & Bishop, 1981; Zeller et al. 1989a). These have now been replaced by 7 viral species, the NSD serogroup comprising two species: Dugbe virus and NSDV. All but one species (Thiafora virus) are tick-borne viruses (Table 3).

Virions are morphologically similar to other members of the family with very small surface units, which appear as a peripheral fringe. The L RNA segment is considerably larger than the L segments of other members of the family (Marriott & Nuttall, 1996), and the S segment does not encode a non-structural protein. The M segment encodes a single gene product, which is processed in a complex and poorly defined manner to yield the structural glycoproteins and at least three non-structural proteins. Processing of Gn (G2) is dependent on subtilase SKI-1, a cellular protease, whereas Gc (G1) processing is not. Determination of glycoprotein processing in ticks might provide important insights into nairovirus replication in the vector. Recent progress in applying reverse genetic technology to CCHFV offers the real prospect of understanding the biology of this important virus genus.

The most distinctive biological feature of nairoviruses is that they are all tick-borne, with relatively few isolates known also from mosquitoes (Dugbe virus and Ganjam virus) or Culicoides midges, although insect-borne transmission is unlikely. CCHFV is the most medically important member of the genus and the best-studied representative. This virus was originally isolated independently in two distant parts of the world (Democratic Republic of Congo and the Crimea) and subsequent laboratory comparison demonstrated the two viruses to be identical (Hoogstraal, 1979). It has one of the widest geographical distributions of the medically important arboviruses (Nuttall, 2001). Infections in humans result in haemorrhagic fever with severe typhoid-like symptoms and mortality rates up to 50% (Watts et al. 1989). Livestock movements and migratory birds play an important role in the transport of infected ticks. Although CCHFV has been isolated from at least 31 different tick species and subspecies (including two argasid species), the principal vectors are ixodid ticks of the genus Hyalomma (such as H. marginatum, H. rufipes, H. anatolicum and H. asiaticum). In contrast to the observation in ixodid ticks, CCHFV failed to replicate in argasid ticks indicating that CCHFV isolated from argasid ticks in nature merely represents virus survival in the recently ingested blood-meal (Shepherd et al. 1989). The distribution of CCHFV in scattered enzootic foci in sub-Saharan Africa, the Middle East and southern Eurasia falls within the limits of the distribution of the genus Hyalomma and human infections have been associated principally with bites by ticks of this genus (Swanepoel et al. 1987). Direct transmission from human to human is a frequent cause of nosocomial infections. Hares (Lepus spp.), hedgehogs (Erinaceus and Hemiechinus spp.) and cattle are probably important amplifying hosts in nature, although virus screening of wild and domestic animals has failed to identify viraemic host species. Transmission from infected to uninfected ticks co-feeding on non-viraemic hosts (e.g. sheep and ground-feeding birds) may be an effective transmission route (Nuttall, 2001).

The International Committee on Taxonomy of Viruses lists Kodzha virus as a variant of CCHFV and cites under this virus two strains, C68031 and AP92. Strain AP92 is from Greece where evidence of the disease in humans has not been recorded. The Greek strain is phylogenetically distinct from all other CCHFV strains examined to date and warrants a distinct name, as indicated in Table 3. Other viruses related to CCHFV, Hazara virus and Khasan virus, likewise have not been associated with disease.

Dera Ghazi Khan virus (DGKV) was isolated originally from Hyalomma dromedarii collected from a camel in western Pakistan (reference strain JD 254). At least 5 other related viruses are known. Unlike DGKV from ixodid species, the relatives have been isolated from argasid ticks, except for Abu Mina virus for which the tick vector is unknown.

Until recently, Dugbe virus was classified in the NSD serogroup but is now recognized as a distinct species. It is antigenically and genetically related to CCHFV but of comparatively low pathogenicity, which makes it a good model for experimental studies. Dugbe virus replicates and persists trans-stadially in orally infected Amblyomma variegatum ticks and can subsequently be transmitted by tick feeding to a vertebrate host (Steele & Nuttall, 1989). The virus has been isolated from numerous other tick species (e.g. Hyalomma truncatum, Boophilus decoloratus and Rhipicephalus appendiculatus) but has a much more restricted geographical distribution compared with CCHFV, possibly because it is not associated with birds.

Nairobi sheep disease virus (NSDV) is highly pathogenic for sheep and goats, and causes disease in humans. Some of the earliest work on tick-borne virus transmission is recorded for NSDV (Daubney & Hudson, 1931). More recent studies have been limited by the high degree of containment needed to handle this virus. Genetic and serological data indicate that Ganjam virus is an Asian variant of NSDV.

The largest group of nairoviruses representing a distinct species is that of Hughes virus. Hughes group viruses are transmitted by Ornithodoros ticks of the ‘capensis’ complex associated with seabirds, such as O. maritimus and O. denmarki, and the ixodid seabird tick, I. uriae. Hughes virus was first isolated from O. denmarki ticks collected in 1962 on Bush Key, Dry Tortugas, Florida and subsequently from the same tick species from Soldado Rock, off the south-west corner of Trinidad. The virus was also isolated from the blood of nestling terns (Sterna fuscata) captured on Soldado Rock. The number and geographical range of Hughes group viruses is comparable to that of orbiviruses of the Great Island group, which also circulate in seabird colonies (Table 2). The abundance of these two virus groups may simply reflect the level of activity in collecting ticks and isolating viruses from the attractive habitats of seabirds. Alternatively, evolutionary pressures resulting from unique features of their ecology may have given rise to a large number of distinguishable virus variants.

Qalyub virus was originally isolated from Ornithodoros erraticus in Egypt and many subsequent isolates were obtained from the same tick species (Darwish & Hoogstraal, 1981). The related Bandia virus was isolated from O. sonrai, a member of the O. erraticus group, and from rodents in Senegal. The capacity of viruses in this group to serve as human pathogens is unknown.

Genus Phlebovirus

The surface morphology of phleboviruses is distinct in having small round subunits with a central hole. The S RNA exhibits an ambisense coding strategy. It is transcribed by the virion RNA polymerase to a subgenomic virus-complementary sense mRNA that encodes the N protein and, from full-length antigenome S RNA, to a subgenomic virus sense mRNA that encodes a non-structural protein, NSs. The M segment gene order and coding strategy varies. Tick-borne phleboviruses (e.g. Uukuniemi virus) encode only G1 and G2 glycoproteins whereas the mosquito- and sandfly-vectored phleboviruses also have coding information for a non-structural protein, NSm (Schmaljohn & Hooper, 2001).

The Phlebovirus genus comprises some 30 viruses transmitted by phlebotomine sand-flies, mosquitoes, or ceratopogonids of the genus Culicoides. The type species is Rift Valley fever virus transmitted by mosquitoes. At least 10 viruses transmitted by ticks were formerly assigned to an independent Uukuvirus genus but are now included within the Phlebovirus genus based on similarities in their coding strategies (Simons, Hellman & Pettersson, 1990). Uukuniemi virus was originally isolated in southeast Finland from I. ricinus ticks (reference strain S 23). Subsequently, it was isolated in many other European countries predominantly from I. ricinus ticks. This virus appears to straddle terrestrial and marine biotopes, having been isolated from I. uriae collected in seabird colonies on the island of Runde in Norway (strain Ru E82). The other representatives of the species have been isolated repeatedly from passerines and seabirds or from their ticks, particularly in Scandinavia and in other parts of Palaearctic and Nearctic regions. They include Grand Arbaud virus (from Argas reflexus ticks in the Rhone delta of France), Manawa virus (from Argas abdussalami in West Pakistan), Oceanside virus (from Ixodes uriae collected at Three Arch Rocks and Yaquina Head, Oregon and Flat Iron Rock, California), Ponteves virus (from Argas reflexus, France), Precarious Point virus (from Ixodes uriae collected in penguin rookeries on Macquarie Island in the sub-Antarctic) and the closely related Murre virus and RML 105355 virus, St. Abbs Head virus (from I. uriae, east Scotland), and Zaliv Terpeniya virus (from I. uriae collected in Sakhalin and Kamchatka regions, Far-eastern Russia and the Murmansk region in the European part of Russia) (Table 3).

In addition to the bunya-, nairo-, and phlebovirus tick-borne members of the Bunyaviridae, there are at least 15 other tick-borne viruses that have yet to be assigned to a particular genus. The best known of these is Bhanja virus isolated from ticks of the genera Haemaphysalis, Boophilus, Amblyomma and Hyalomma in India, Africa (Nigeria, Cameroon, Senegal) and Europe (Italy, Balkan states). Palma virus is an isolate from Portugal closely related to Bhanja virus. Others include Silverwater virus from Haemaphysalis leporispalustris collected from snowshoe hare in Ontario and Upolu virus from Ornithodoros ticks collected in a tern colony (Sterna sp.) on a coral atoll in the Great Barrier Reef. Issyk-Kul virus appears to be transmissible by both mosquitoes and ticks, and has been associated with febrile illness in humans in Tajikistan.

Genus Flavivirus

Most flaviviruses (at least 50 species) are transmitted to vertebrate hosts by mosquitoes. They include such medically important pathogens as Yellow fever virus, Japanese encephalitis virus, West Nile virus (WNV), the four serotypes of Dengue virus and several others. Tick-borne viruses currently comprise 12 species and there are at least 14 species that are zoonotic viruses transmitted between rodents or bats with no known arthropod vector.

Flavivirus virions are enveloped, roughly spherical, with a diameter of 40–60 nm. The virion contains a nucleocapsid core of 20–30 nm composed of a single capsid protein. The envelope contains two virus-encoded proteins (envelope and membrane proteins). Immature, intracellular virions contain a precursor membrane protein, which is proteolytically cleaved during virus maturation. The viral genome is a single molecule of single-stranded RNA of approximately 11 kb which is positive-sense and infectious. The virion RNA appears to be identical to the mRNA. Three structural proteins and seven non-structural proteins are encoded from one long open reading frame flanked by terminal noncoding regions that form specific secondary structures required for genome replication, translation or packaging. Viral proteins are synthesized as part of a polyprotein of more than 3000 amino acids, which is co- and post-translationally cleaved by viral and cellular proteases (Lindenbach & Rice, 2001).

The envelope glycoprotein (E protein) is the major structural protein and plays an important role in membrane binding and inducing a protective immune response following virus infection. It carries epitopes detected by neutralization and haemagglutination-inhibition tests that have been used to identify different flaviviral subgroups and species. Within the E gene three genetic markers have been identified. These are a type specific tripeptide hypervariable region which provides a unique genetic signature for individual flaviviruses (Shiu, Ayres & Gould, 1991); a subgroup-specific pentapeptide motif (Gao et al. 1993); and a hexapeptide insertion (EHLPTA) typical of TBEV complex viruses (Marin et al. 1995).

Determination of the crystallographic structure of a soluble form of the E protein of a TBEV revealed that, unlike the spikes seen on many viruses, the flavivirus envelope protein is situated parallel to the virion surface in the form of head-to-tail homodimeric rods. Residues that influence binding of monoclonal antibodies occur on the outward facing surface of the protein (Rey et al. 1995). Interestingly, the putative receptor binding site of some mosquito-borne flaviviruses contains a putative integrin-binding motif Arg–Gly–Asp which is not found in the E protein of TBEV. There is an obvious analogy with the orbiviruses (see above).

All flaviviruses are serologically related, as demonstrated in binding assays such as ELISA and by haemagglutination-inhibition using polyclonal and monoclonal antibodies. Neutralization assays are more discriminating and have been used to define several serocomplexes within the genus Flavivirus. Comparative analyses of the nucleotide and amino acid sequence of the E gene have been used to investigate the phylogeny of flaviviruses (Marin et al. 1995). Cell fusing agent virus (CFAV) (tentatively placed in the genus Flavivirus) E gene was used as the outgroup based on the distant relationship of CFAV to tick- and mosquito-borne flaviviruses. The analyses showed that tick- and mosquito-borne flaviviruses diverged as two distinct and major genetic lineages. Interestingly, analyses of the E and non-structural NS5 gene sequences to compare mutational regimes indicated that mosquito-borne flaviviruses are evolving almost twice as fast as tick-borne flaviviruses (Marin et al. 1995; Gould, Zanotto & Holmes, 1997).

The tick-borne flaviviruses are currently classified into two groups: the mammalian tick-borne virus group and the seabird tick-borne virus group (Table 4). Three seabird tick-borne flaviviruses are recognized. These are Meaban virus (from France), Saumarez Reef virus (from Australia) and Tyuleniy virus (from Russian Far East). We have also placed Gadgets Gully virus (GGYV) in the seabird tick-borne virus group because of its known ecology. However, the International Committee on Taxonomy of Viruses lists it together with the mammalian group based on phylogenetic studies. Analysis of the E gene of GGYV places this virus at the edge of the mammalian tick-borne flavivirus cluster, possibly indicating that this virus is at the cusp between the two groups (Gaunt et al. 2001).

Medically, by far the most important tick-borne flaviviruses are those classically grouped into the TBEV serogroup, the most important being TBEV. TBE was recognised clinically in the Far East of the former USSR (now Russia) in the early 1930s. Severe cases of encephalitis were observed in humans residing in formerly unhabited areas. A special expedition was organised in 1937 by L. A. Zilber to determine the cause of the disease. Virus isolates were obtained from the blood of patients and from Ixodes persulcatus ticks. The disease has several names, including Russian spring-summer encephalitis (RSSE), Far Eastern encephalitis, and forest spring encephalitis (Zilber & Soloviev, 1946). In 1948, a similar though less severe form of encephalitis affected humans residing in central Bohemia, Czech Republic. The virus recovered from the blood of a patient and from I. ricinus ticks was related to the isolates from RSSE cases (Rampas & Gallia, 1949). Similar or milder forms of the disease, called biphasic meningoencephalitis, were observed in other central and eastern European countries. TBEV is endemic over a wide area – covering Europe, northern Asia and China. Several thousand human cases are recorded annually, with considerable variation from year to year. In general, the Far Eastern subtype of TBEV cause severe disease in humans with a mortality that can be 50% in some outbreaks; disease associated with the European subtype is less severe and mortality is usually under 5% (Gresíková & Calisher, 1988). Based on genome analyses, recently a third Siberian subtype has been established. Members of this subtype are known to cause chronic infections in humans.

Louping ill virus (LIV) derives its name from the disease of sheep (louping ill), which has been recognized in southern Scotland for at least two centuries (Smith & Varma, 1981). ‘Louping’ refers to the characteristic behaviour of ‘leaping’ shown by sheep infected with LIV. The disease is found throughout much of the upland sheep farming areas of Scotland, northern and southwestern England, Ireland and Wales affecting sheep and red grouse (Lagopus scoticus), with other species of domestic animals and humans affected less frequently. The main vector is I. ricinus (Reid, 1984). Now two subtypes of LIV are recognized in the British Isles (Irish and British subtypes), as well as in Norway. In Turkey and Spain, the disease has been attributed to infections with Spanish and Turkish subtypes of LIV. The virus can cause severe encephalitis in humans; however, there are only about 35 recorded cases and they have been confined mostly to laboratory workers, veterinary surgeons, farmers and abattoir workers (Smith & Varma, 1981).

Powassan virus (POWV) is the North American member of the TBE subgroup although there are several records of POWV from ticks and mosquitoes collected also in Russia. The virus was originally isolated from a pool of Dermacentor andersoni ticks collected in 1952 in Colorado (Thomas, Kennedy & Eklund, 1960) and derives its name from the town in Northern Ontario where the first fatal case of the encephalitic disease was recognized. About 20 cases of disease associated with POWV infection have been reported in North America and there was one fatal case recorded in Russia (Artsob, 1988). Deer tick virus, is a newly recognized relative of POWV isolated from Ixodes and Dermacentor spp., and from human brain (Telford et al. 1997).

Omsk haemorrhagic fever (OHF) was first reported in 1941 when physicians recorded sporadic cases in the forest steppes of Omsk Region-Siberia among muskrat hunters. Though closely related to TBEV, Omsk haemorrhagic fever virus (OHFV) is unique with respect to both the clinical features of the disease (haemorrhagic symptoms contrasting with the encephalitis) and the ecology of the virus. The primary tick vector of OHFV is Dermacentor reticulatus and the hosts are voles, particularly the narrow-skulled vole, Microtus gregalis. Another tick species, Ixodes apronophorus, plays a role together with its main host, the water vole. D. marginatus and I. persulcatus ticks play secondary roles in the transmission cycles of OHFV (Lvov, 1988).

Kyasanur Forest disease virus (KFDV) is another highly pathogenic member of the tick-borne flaviviruses producing haemorrhagic disease in infected humans. Since the first record of the disease in 1957 in Karnataka State, India, epidemics have occurred every year in the affected region. The highest recorded incidence was in 1983 with 1555 cases and 150 deaths. Preceding the first human epidemic, a large number of sick and dead monkeys had been noticed in the nearby forest area. KFDV was isolated from dead monkeys, sick patients and from Haemaphysalis and other tick species. Clinically, the disease is particularly interesting because, in contrast to most of the other tick-borne flaviviruses that cause encephalitis, KFDV causes a haemorrhagic disease even more severe than that associated with OHFV (Banerjee, 1988). Alkhurma virus, a close relative of KFDV, was first isolated in 1995 from patients with haemorrhagic fever in Saudi Arabia (Zaki, 1997; Charrel et al. 2001). Little is known of the ecology and epidemiology of this virus.

Langat virus, which is transmitted by Ixodes granulatus, is the least pathogenic representative of the mammalian tick-borne flavivirus group. This virus was isolated in Malaysia (Karabatsos, 1985). Because of its naturally low virulence and serological cross-reactivity with TBEV it was assessed as a candidate vaccine against TBE in Russia with disastrous consequences.

The remaining members of the group, namely Kadam virus, Karshi virus, Royal Farm virus and Gadgets Gully virus, are not considered human pathogens and consequently knowledge of their ecology and epidemiology is limited (Karabatsos, 1985).

Ticks are potential reservoirs of at least two mosquito-borne flaviviruses. The evidence is based on the isolation of YFV from the eggs and emergent larvae of an ixodid tick species collected in the field (Germain et al. 1979). West Nile virus (WNV) is included in Table 4 because, although clearly a mosquito-borne virus, it has been isolated repeatedly from bird-infesting argasid and ixodid ticks in arid regions of Russia and from Hyalomma detritum in a desert region of Turkmenistan (Lvov et al. 1975). The five isolates of WNV from O. maritimus ticks collected in the nest site of gulls (Larus argentatus) were from Glinyanyi Island in the Caspian Sea, where mosquitoes are reported to be absent. For WNV, long-term survival and co-feeding transmission of the virus has been demonstrated in laboratory studies with Ornithodoros ticks (Vermeil, Lavillaureix & Reeb, 1959; Lawrie et al. in press).

The surprisingly broad spectrum of ecological conditions occupied by tick-borne flaviviruses is worthy of further comment. As we have seen, they range from the seabird colonies occupied by Ixodes uriae as the dominant tick species, to the typically forested habitats of the Northern Hemisphere with I. ricinus and I. persulcatus as the dominant tick species. In addition, representatives of the TBEV serogroup have dispersed across Asia and Europe in a cline (continuous or gradual evolution across a geographic area). The most divergent lineages (often incorrectly referred to as the most ancient viruses) appeared in the east and the more recent lineages emerged in the west, with LIV at the extreme west of this geographical distribution (Zanotto et al. 1995).