32 Mosquito-borne arboviruses

The arboviruses are all single-stranded RNA viruses, although they belong to five different viral families. Several important human pathogens belong to the mosquito-borne arboviruses including yellow fever, Japanese encephalitis and Rift Valley Fever. They cause a wide range of illnesses from unrecognized infection to severe systemic disease with hemorrhagic complications and/or encephalitis with a high mortality. A similar range of illnesses is seen in infected animals.

Arboviruses have several unique characteristics, these include; an ability to infect and be transmitted by mosquitoes, ticks, midges, sandflies, bugs, fleas, blackflies and horseflies. They infect vertebrate hosts which may amplify the virus for invertebrate vectors that feed on infected vertebrates. They have the ability to replicate in arthropods, with little pathology and in vertebrates often with significant pathology. Many arboviruses are zoonotic.

Control methods depend on the epidemiology of particular viruses, but epidemic vector control through control of insect breeding sites and the use of insecticide spraying have been successfully used in the past. Effective vaccines are available for yellow fever and Japanese encephalitis.

In 1881, before viruses were recognized, Carlos Finlay, a Cuban physician, proposed that yellow fever might be transmitted by mosquitoes. Despite the fact that previous alternative theories had not proven to be correct, Dr Finlay’s idea was followed up (Reed 1901) and the concept that some diseases could be spread by arthropods heralded the beginning of arbovirology as we know it today. By 1930 the laboratory mouse was beginning to be used as a test animal in virology and Max Theiler reported that newborn mice inoculated intraperitoneally with Yellow Fever virus (YFV) died of encephalitis (Theiler 1930). At about the same time, Louping ill virus (LIV), which is antigenically related to YFV, was shown to be transmitted by ticks, rather than mosquitoes. This virus was also isolated using mice (Greig et al. 1931). For many years afterwards, arboviruses were preferentially isolated using newborn mice. The first classification of these viruses was based on their ability to replicate in and be transmitted by arthropods, but it was subsequently shown that some related viruses were not transmitted by arthropods. As more viruses were isolated, new tests were developed to distinguish them. Infected suckling mice were used as hosts for detecting neutralizing antibodies to the New World equine encephalitis arboviruses (Casals and Webster 1944; Lennette and Koprowski 1944) and this was soon followed by the adaptation of complement fixation tests to demonstrate that antibodies specific for rabies or poliovirus or newly isolated arboviruses did not necessarily cross-react with all the isolated viruses (Casals 1944; Casals and Webster 1944). As even more viruses were isolated and antigenic tests performed, it became clear that some were distantly related whereas others were more closely related to each other (Casals 1957). The haemagglutination test, originally developed to study influenza virus (Hirst 1941), was then modified (Casals and Brown 1954; Sabin and Buescher 1950) and adapted to the study of arboviruses. The haemagglutination-inhibition test, complement fixation test and neutralization test have proved to be exquisitely sensitive and practical tools for diagnosis and studies of virus antigenic interrelationships. Indeed, these serological procedures have stood the test of time and even though many of the taxonomic decisions made today are primarily based on sequence data, relatively few major changes have been required when compared with those based on serological studies.

RNA viruses that are transmitted to vertebrate species by haematophagous (blood-feeding) arthropod vectors (including mosquitoes, ticks, sandflies, midges/gnats, bugs etc.) are known as arboviruses because they are arthropod-borne viruses. The term arbovirus reflects their ecological characteristics and is not taxonomic. Replication of the virus in the arthropod is a crucial element of the virus life cycle. However, as far as is known no significant pathological changes result from these infections, at least in the arthropods that subsequently replicate and transmit the virus to a vertebrate host. When an arthropod takes an infectious blood meal, the virus enters the midgut and infects the epithelial cells. If the virus survives transfer across the barrier of the midgut wall (Paulson et al. 1989), it enters a variety of tissues and replicates. The salivary glands of vectors competent to transmit the virus become infected, and the virus can then be transmitted to vertebrate hosts during subsequent feeding periods (Takahashi and Suzuki 1979). Transmission of the virus to vertebrate hosts may or may not result in pathology and clinical signs of infection. The time that the virus takes to infect the arthropod and to replicate and then be transmitted to a vertebrate host is known as the period of extrinsic incubation (World Health Organization (WHO) 1985). Infected female arthropods may also transmit the virus vertically (transovarially) through eggs, and infected males may transmit the virus to females during mating. Conventionally, the virus replicates in the infected vertebrate host to produce a viraemic infection that provides a source of virus for feeding haematophagous arthropods to continue the biological cycle. However, there is also evidence of virus transmission between co-feeding arthropods, in the absence of viraemia (Gould et al. 2003; Higgs et al. 2005; Jones et al. 1997; Labuda et al. 1997; Mead et al. 2000). The significance of non-viraemic arbovirus transmission is not yet fully understood although it clearly plays an important role in arbovirus pathogenesis, survival, dispersal and evolution. Alternatively, under some circumstances, viruses may adhere physically to the proboscis of a blood-sucking arthropod, without infecting the arthropod, and be transmitted mechanically to vertebrate hosts. Whilst this very rare form of mechanical transmission may occur, it would not be unique to arboviruses and is not a recognized method of arbovirus transmission.

More than five hundred viruses were registered in the International Catalogue of Arboviruses (Karabatsos 1985) by July 1985. This was the last issue of the Catalogue, but the number of identified arboviruses continues to increase. All recognized arboviruses are now listed within their families and genera in the VIIIth Report of the International Committee for the Taxonomy of Viruses (Fauquet et al. 2005). In general, individual arboviruses are primarily associated with a single family of arthropods, such as mosquitoes, ticks, midges, flies, bugs, etc. However, this is not an absolute rule, for example, whilst West Nile virus and several other closely related viruses are primarily associated with mosquito transmission, they may also infect and be transmitted by ticks (Theiler and Downs 1973). Nevertheless, the mosquito is clearly the predominant species for evolution and transmission of these viruses because phylogenetic analyses always group them in the clades occupied by mosquito-borne flaviviruses rather than tick-borne flaviviruses (Gould et al. 2003). The same rules apply to other arboviruses that are primarily transmitted by ticks or sandflies, i.e, some of them may also be transmissible by mosquitoes. Another complicating issue, in defining arboviruses, is the fact that some do not have recognized arthropod vectors and may be described as having ‘no known vector’ (NKV) even though they are antigenically and phylogenetically clearly members of arbovirus genera (Porterfield, 1980). Whilst this could reflect the lack of evidence from environmental samples, it seems more likely that such viruses do not have arthropod vectors and thus represent either archival or descendant lineages within the genera, that have evolved this characteristic. One more complicating factor is that some genera included in the Arbovirus Catalogue clearly are not arboviruses, for example, hantaviruses and arenaviruses. Nevertheless, the history of their discovery, their genome strategies, epidemiological and/or ecological characteristics together with the specialized facilities required for their study are traditionally associated with laboratories having expertise in arbovirology, hence their inclusion in the Arbovirus Catalogue.

By the mid-twentieth century sufficient serological data had accumulated to enable the categorization of arboviruses into Groups A, B and C, corresponding respectively to the viruses known today as the families Togaviridae (genus Alphavirus), Flaviviridae (genus Flavivirus) and Bunyaviridae (genera, Bunyavirus and Phlebovirus) (Fauquet et al. 2005). As more viruses were isolated and defined on the basis of antigenicity, together with other biological characteristics, and sequence data, arboviruses were also recognized as being present in the additional families, Reoviridae (genus Orbivirus) and Rhabdoviridae (genus Vesiculovirus). It should be emphasized that each of these five virus families consists of genera that contain arboviruses and other genera that do not contain arboviruses. Moreover, as stated earlier, a substantial proportion of arboviruses are primarily associated with arthropods other than mosquitoes. However, this chapter focuses only on mosquito-borne arboviruses (Table 32.1) and primarily on those that cause significant disease in vertebrate species, including humans. New arboviruses are constantly being isolated and characterized. Since it takes considerable time before they are listed in the latest catalogues of viruses it is impossible to provide an absolutely up to date precise taxonomic and biological description of them. Nevertheless, the most robust current listings can be found in the VIIIth Report of the ICTV (Fauquet et al. 2005).

Genus Alphavirus (Mukhopadhyay et al. 2006; Strauss and Strauss 1994; Weaver et al. 2005). Images of alphaviruses illustrating their structural details can be obtained at the following website; http://www.bio.indiana.edu/facultyresearch/faculty/Mukhopadhyay.html.

New species continue to be isolated and characterized but currently there are 29 recognized species in the genus Alphavirus. Virions, ∼70 nm in diameter, are spherical with a lipid bilayer containing heterodimeric protein spikes composed of two glycoproteins E1 and E2. Some flaviviruses also contain a third envelope protein E3. The heterodimers are organized in a T=4 icosahedral lattice consisting of 80 trimers. The enclosed nucleocapsid core consists of 240 copies of capsid protein and a single copy of the genomic RNA. The one to one relationship between glycoprotein heterodimers and nucleocapsid proteins is important in virus assembly. The structure of the E1 glycoprotein has been resolved by crystallography. E1 is the fusion protein for virus entry into the acidic cytoplasmic endosomes. The E2 glycoprotein extends outwards from the envelope and forms the petals of the spike that cover the underlying E1 protein fusion peptide at neutral pH. The four non-structural proteins are defined as nsP1, nsP2, nsP3 and nsP4. The genomic RNA is positive stranded and serves as the mRNA for translation of the four non-structural viral proteins which are cleaved from a polyprotein precursor by the viral-encoded protease in nsP2. The non-structural proteins replicate viral RNA. The nsP1 protein is probably involved in capping of viral RNAs and initiation of negative-strand RNA synthesis. In addition to its protease function the nsP2 also provides the helicase for RNA replication. The nsP3 protein is also required for RNA replication and the nsP4 protein is believed to be the viral RNA polymerase. During RNA replication, a negative-stranded copy is produced and used as a template for the synthesis of genome-sized positive strand RNA and subgenomic 26S mRNA corresponding to the 3’ third of the viral genome and encoding the viral structural proteins (Fig. 32.1).

The non-structural proteins function in the cytoplasm of infected cells in association with membrane surfaces and attachment appears to be mediated by nsP1 palmitoylation. The capsid protein assembles with the viral RNA to form the viral nucleocapsids in the cytosol. Glycoproteins are translocated via the Golgi apparatus to the plasma membrane and assembled nucleocapsids bud through these membranes, thus acquiring a lipid envelope containing the integral membrane glycoproteins, E1 and E2.

The VIIIth Edition of the International Committee for the Taxonomy of Viruses (ICTV) currently lists 29 species in the genus Alphavirus and family Togaviridae (Weaver et al. 2005). All arthropod-borne alphaviruses are antigenically related but most can be distinguished in cross-reactivity tests (Chanas et al. 1976; Clarke and Casals 1958; Karabatsos 1975; Porterfield 1961) with which they have been divided into 8 antigenic complexes: Eastern, Western, and Venezuelan equine encephalitis, Trocara, Middelburg, Ndumu, Semliki Forest and Barmah Forest. In addition, Southern elephant seal virus (SESV) has been isolated from the Seal Louse in the southern Oceans, indicating that alphaviruses can circulate in Antarctica and the non-arthropod-borne species, Salmon pancreatic disease virus (SPDV) has also been identified. These two viruses are antigenically unrelated to the other recognized alphaviruses. Based on sequence data, and with the exception of these two widely disparate viruses, the arthropod-borne alphaviruses share a minimum of about 40% amino acid identity in the more divergent structural proteins and 60% in the non-structural proteins. A phylogenetic tree based on the sequence of the envelope gene of all known alphavirus is presented in Fig. 32.2. The tree identifies the region of the world (Old World or New World) where the viruses are known to be indigenous. Western equine encephalitis virus (WEEV) and direct descendants of this virus differ from the other alphaviruses in being recombinant viruses. It is believed that an Old World virus related to Sindbis virus (SINV) recombined with a New World virus, probably an ancestor of Eastern equine encephalitis virus (EEEV) to produce WEEV (Weaver et al. 1997).

Ten alphaviruses are considered to be of significant importance in terms of public health. Indeed with the recent emergence of Chikungunya fever as a major human pathogen in Asia and potentially globally (de Lamballerie et al. 2008), the alphavirus profile has been significantly raised (Chevillon et al. 2008). Alphaviruses that circulate in the Old World most commonly cause febrile illness and painful arthralgias or polyarthralgias, particularly in the small joints. A characteristic macular-papular rash often appears three to five days after illness onset. In severe cases the joints are swollen and tender, and rheumatic signs and symptoms may persist for weeks or months following the acute illness. In general, these infections are rarely fatal and only infrequently result in encephalitic disease (Lewthwaite et al. 2009) following infection with Old World alphaviruses such as CHIKV, or o’nyong nyong virus (ONNV) in Africa/Asia, SINV and closely related viruses (Ockelbo, Whataroa) which are widespread throughout the Old World, or Ross River virus (RRV), and Barmah Forest virus (BFV) which are confined to Australia. In contrast with these Old World diseases, the New World alphaviruses VEEV, EEEV, and WEEV present a different epidemiological and clinical picture. VEEV is divided into six distinct antigenic subtypes (Walton and Grayson 1988; Young 1972; Young and Johnson 1969). Subtypes IAB and IC are associated with major epidemics and equine epizootics during which equine mortality due to encephalitis can reach 83%. In 1995, a major outbreak in Venezuela and Colombia, was associated with the VEEV subtype IC. This epidemic resulted in roughly 100, 000 human cases, with more than 300 fatal encephalitis cases (Diaz et al. 1997). Other recent epidemics indicate that VEEV still represents a serious public health problem (Weaver et al. 1996). In humans, while the overall mortality rate is low (<1%), neurological disease, including disorientation, ataxia, mental depression, and convulsions, can be detected in up to 14% of infected individuals, especially children (Johnson and Martin 1974). Neurological sequelae in humans are also common (Leon 1975). However, most human infections are either asymptomatic or present as a nonspecific febrile illness or aseptic meningitis. In rare cases, the fever and headache may progress through nausea and vomiting to somnolence or delirium and coma with seizures, impaired sensorium, and paralysis being commonly observed. The severity of neurological involvement and sequelae is greater with decreasing age. Horses are more susceptible than humans to these viruses but are considered to be dead-end hosts. Moreover, veterinary vaccines are available to reduce the risk of clinical disease. EEEV and WEEV, are widespread throughout the eastern and western regions of North America, including Canada, and also South America and Cuba. They are transmitted to horses by infected ornithophilic (bird-biting) mosquitoes that thrive in wetland habitats. Highlands J virus (HJV), a close relative of WEEV, is not known to be pathogenic for humans but appears to be an important pathogen of several wild bird species. VEEV also causes encephalitic disease in horses and, occasionally, humans bitten by mosquitoes normally associated with the horses (Weaver 2005; Weaver and Barrett 2004; Weaver et al. 1997, 2005;). The natural life cycle of VEEV involves small mammals, particularly rodents in forest environments more frequently found in South America. Several other related alphaviruses are recognized in the Americas but in most cases they are not known to cause disease in humans or animals.

Currently approximately 60 species are recognized in the genus Flavivirus although during the past decade several new unclassified flaviviruses have been isolated from mosquitoes in different regions of the world (Cook et al. 2006; Crabtree et al. 2003; Farfan-Ale et al. 2009; Hoshino et al. 2007; Kihara et al. 2007; Kim et al. 2009; Morales-Betoulle et al. 2008; Sang et al. 2003). Recent reviews of the Genus Flavivirus include (Gould et al. 2003; Heinz 2000; Thiel et al. 2005). Many of these new viruses appear to be insect viruses, i.e. they do not infect vertebrates. It is possible that they will ultimately be recognized as a new genus within the family Flaviviridae. Other recently isolated but not yet classified flaviviruses appear to be conventional arboviruses but their natural vertebrate hosts are not necessarily recognized as yet.

Infectious (mature) flaviviruses are spherical particles (50 nm) with a relatively smooth surface and no distinct projections. They have an electron-dense core (30 nm) surrounded by a lipid membrane. The core consists of positive-polarity genomic RNA (11 kbp) and capsid (C) protein (12K). The lipid membrane incorporates an envelope glycoprotein (E, 53K) and a membrane glycoprotein (M, 8K). The immature (intracellular) virions contain a precursor membrane protein (prM, 18K), the proteolytic cleavage of which occurs in the secretory pathway, during egress of virions from infected cells.

The E glycoprotein mediates virus binding to cellular receptors and thereby directly affects virus host range, virulence, and immunological properties by inducing protective antibodies. The E-protein ectodomain (N-terminal 395 amino acids) consists of 90 homodimers folded in a ‘head-to-tail’ manner and orientated parallel to the membrane surface. The E protein contains three structural domains, each based on β-sheets: the central domain I, the dimerization (fusion) domain II, and receptor domain III (dI, dII, and dIII). The C-terminal 101 residues of the E protein form a stem anchor region consisting of two stem a′-helices and two transmembrane a′ -helices that anchor the E protein into the lipid bilayer. Domain II contains a hydrophobic fusion peptide consisting of 13 residues that are highly conserved between all flaviviruses. The fusion peptide is located on the tip of domain II and plays a central role in fusion of the virion membrane to cellular endosomal membranes resulting in release of virion RNA into the cytoplasm. The E-dimers form a ‘herringbone’ configuration; three quasiparallel E-dimer molecules making up the main structural asymmetric unit of the shell. The fivefold symmetry axes are generated by appropriate positioning of five domain IIIs and their lateral surface is accessible to cellular receptors and neutralizing antibodies. The M protein protrudes through holes formed between dimerization domains of E molecules. Following acid pH-dependent fusion of the E protein with cytoplasmic endosomes, the 5’ and 3’ untranslated regions (UTRs) of the released genomic RNA provide signals to initiate translation of the open reading frame (ORF) which is then processed by cellular signalases and viral serine protease to produce three structural (C, prM, and E) and seven nonstructural (NS1 through NS5) proteins. The N-terminal region of the NS3 protein provides the viral protease function. NS2B probably acts as a co-factor by anchoring the NS3 protein. Replication of new viral RNA requires direct interaction between the 5’ and 3’ UTRs resulting in the formation of double-stranded RNA replicative forms and genome circularization. Specific RNA secondary structures within the UTRs provide essential promoter and replication enhancer functions. Flavivirus RNA replication is semiconservative and asymmetric. The replicative intermediate presents a partially double-stranded RNA with nascent, and displaced, plus-sense ssRNA molecules undergoing elongation. Capping of the 5’UTR occurs on the displaced plus-strand RNA; the NS3 and NS5 proteins provide nucleoside triphosphatase and guanylyl-methyltransferase activities, respectively. The C-terminal domain of NS5 protein acts as the viral RNA polymerase and the C-terminal domain of NS3 functions as the helicase. The functions of other nonstructural proteins are less precisely identified. The NS1 glycoprotein is translocated in the endoplasmic reticulum and secreted together with virions in mammalian but not in mosquito cells. The NS2A protein associates with the NS3 helicase domain, the NS5 protein, and the 3’UTR. It may be involved in viral RNA trafficking. The NS2B protein is a membrane-anchored cofactor of the serine proteinase, NS3 and also has membrane permeability modulating activity. The hydrophobic NS4A protein in conjunction with the NS1 protein probably anchors the polymerase complex to cell membranes (Gritsun and Gould 2008; Thiel et al. 2005). For colour images representing the above description of the flavivirus life cycle see Gritsun and Gould (2008).

About 60% of the currently classified flaviviruses are primarily transmitted between vertebrates by mosquitoes and these viruses have been roughly divided into those associated primarily with Aedes spp., typified by YFV and Dengue virus (DENV) and those associated primarily with Culex spp., mosquitoes, typified by West Nile virus (WNV) and Japanese encephalitis virus (JEV). Indeed the entire genus shows striking relationships between epidemiology, disease association and biogeography (Gaunt et al. 2001). Although the flaviviruses have a wide vertebrate host range their global distribution tends to be specific for individual viruses and is largely dependent on the dispersal and/or accumulation of arthropods via associated activities such as transportation of humans, animals and commercial goods, bird migration, irrigation, deforestation or remediation projects, human demographic changes (Gould et al. 2003, 2006; Gould and Higgs 2009; Gould and Solomon 2008; Reiter 2008). YFV provides an excellent example of how humans have been responsible for the dispersal of viruses. In the wild, YFV circulates in the central and West African jungles and the surrounding savannah regions, and also in some of the Caribbean Islands and the South American rain forests. The virus circulates between mosquitoes and monkeys living in the canopy of the African jungles, with little evidence of disease in the simian species suggesting that this cycle has existed for a long time. In contrast, in the South American jungles, YFV causes fatal infections in non-immune howler monkeys with which the virus is most often associated, suggesting that YFV was introduced relatively recently. Indeed, this is almost certainly the case. It is the widely held opinion that YFV was introduced many times, from Africa to the New World during the trading of African slaves between these countries. These trading activities occurred for more than 400 years during which YFV became established in the New World countries of Latin America (Bloom 1993; Gould et al. 2003; Tabachnick, 1991). Dengue virus was also almost certainly introduced from Africa to the New World during this period of slave trading. However, after this type of trading ceased and trading routes from Asia were increasing, the introduced Dengue virus species were of Asian origin rather than African origin. By the late 1970s many Asian strains of Dengue virus had been introduced into the New World and this led to the first cases of dengue haemorrhagic fever been observed in Cuba (Guzman et al. 1984; Rico-Hesse et al. 1997). Viruses in the Japanese encephalitis virus (JEV) complex are most frequently associated with ornithophilic (bird biting) mosquitoes, in particular Culex spp., Their geographic distribution is therefore largely dependent upon the migratory patterns of birds. Based on phylogenetic relationships and the known geographic distribution of these viruses, it is believed that the ancestral lineage originated in Africa and evolutionary descendants of this African virus were gradually dispersed by migratory birds into Europe, Asia and Australia (Gould et al. 2004). Whilst WNV appears to be dispersed efficiently via migratory birds in both the Old World and the New World (Gould and Higgs 2009; Malkinson et al. 2002; Owen et al. 2006), its transition across the Oceans to the New World was almost certainly the result of accidental introduction via an aeroplane or ship. The former mode of transportation is favoured because the first outbreaks of encephalitis in birds, horses and humans appear to have occurred not too distant from the major international airport in New York (Gould et al. 2003; Roehrig et al. 2002). Another member of the JEV complex, Usutu virus (USUV), is the most recently recognized emergent virus of this group. Prior to 2001, USUV had only ever been identified in Africa. However, a sudden outbreak of encephalitis in birds in Vienna (Austria) led to the discovery of its emergence out of Africa, presumably for the first time (Weissenbock et al. 2002). This virus has apparently continued to disperse in Europe (Buckley et al. 2003, 2006; Rizzoli et al. 2007) and was recently identified in mosquitoes in Spain (Busquets et al. 2008). Other antigenically related viruses in the JEV complex, such as Murray Valley encephalitis virus, Alfuy virus and Kunjin virus are found in Australia and are assumed to be direct descendants of the African lineage that has evolved as JEV, WNV, USUV etc. In the Americas, St Louis encephalitis virus, and many related viruses such as Ilheus virus, Rocio virus and others are presumed to have been introduced from Africa during the intensive period of slave trading (Gould et al. 2003).

There have been several recent reviews of the Genus Orthobunyavirus (Elliott 2008; Fauquet et al. 2005; Nichol et al. 2005; Putkuri et al. 2007). In general, the virions of bunyaviruses are spherical or pleomorphic, 80–120 nm in diameter and display surface glycoprotein projections of 5–10 nm which are embedded in a lipid bilayered envelope approximately 5 nm thick. The viral genomes are segmented consisting of 3 unique negative or ambisense ssRNA molecules, designated L (large), M (medium) and S (small) totalling 11–19 kb. The terminal nucleotides of each segment are base-paired forming non-covalently closed, circular RNAs (and nucleocapsids). These terminal sequences are conserved amongst viruses in any one genus but are different from those in any other genus within the family. The L segment encodes the viral RNA polymerase enzyme. The M segment encodes the Gc protein which is the major target of neutralizing antibodies and acts as a ligand for cellular receptors on erythrocytes and is therefore involved in haemagglutination inhibition (Gonzalez-Scarano et al. 1982; Grady et al. 1983; Kingsford and Hill 1983) and also in viral entry, playing a critical role in fusion between the viral envelope and the cellular membrane (Pekosz et al. 1995; Plassmeyer et al. 2007). Gc undergoes a pH-dependent conformational change associated with cell-to-cell and virus-to-cell fusion (Gonzalez-Scarano 1984, 1985; Jacoby et al. 1993; Pekosz and Gonzalez-Scarano 1996). The M segment also encodes a Gn glycoprotein (Gonzalez-Scarano et al. 1982; Kingsford et al. 1983), which is required for targeting the Gc protein to the Golgi apparatus from the endoplasmic reticulum (Bupp et al. 1996). Gn probably also functions in cell-to-cell fusion (Jacoby et al. 1993; Plassmeyer et al. 2005) and may be the protein responsible for viral attachment in the mosquito midgut (Ludwig et al. 1991). The S segment encodes the N protein which is possibly involved in early and late RNA synthesis, and the NSs which is an interferon antagonist and acts at the level of transcription by inhibiting RNA polymerase II-mediated transcription. For detailed colour images of the general characteristics and life cycle of bunyaviruses see (Elliott 2008).

There are estimated to be more than 300 different virus species in the genus Orthobunyavirus, only a few of which are transmitted by mosquitoes and associated with significant human disease outbreaks. The mosquito-transmitted bunyaviruses are widespread globally but in common with other arboviruses, individual species occupy geographic niches largely dependent on their dispersal patterns and the particular mosquito vectors and vertebrate hosts with which they have established their life cycles. For example, in the California encephalitis virus serogroup which contains 12 species of antigenically related viruses, La Crosse virus (LACV) is an important cause of encephalitis in children (LeDuc 1987) in the USA. Its principal vector Aedes triseriatus prefers woodland habitats such as tree holes where squirrels and chipmunks serve as the vertebrate hosts, but these mosquitoes also frequently lay their eggs in small pools of water in car tyres, buckets, cans, etc in urban and rural settings and the emerging mosquitoes may then feed on humans, causing localized outbreaks. In order for this to be possible it is necessary for LACV to be passed vertically in the female mosquito to the eggs that retain the infectious virus through the next generation of mosquitoes. This form of long-term survival and transmission has been recognized for many years (Tesh and Gubler 1975). Tahyna virus (TAHV) circulates widely in central and southern Europe and also in China (Lu et al. 2009). TAHV usually causes a febrile flu-like illness but neurological complications are sometimes observed. Its closest relative, Snowshoe hare virus (SSHV) circulates widely in Canada and northern regions of the USA amongst a wide variety of mosquito and wild/domestic animal species. A similar virus is also found in Russia. In addition to febrile illness SSHV may cause encephalitis. Another related virus, Lumbo virus (LUMV) is found in Africa (Elliott 2008; Putkuri et al. 2007) and may therefore represent an early lineage of the antigenic complex. Inkoo virus (INKV) is widespread in most countries in northern Europe. It was first isolated from its principle mosquito vector Aedes communis in Finland in 1964 (Brummer Korvenkontio et al. 1973). Phylogenetic studies indicate that the closest relative to INKV is Jamestown Canyon virus, found in the US (Campbell and Huang 1999; Vapalahti et al. 1996). Whilst there are very few studies on the origin of these bunyaviruses, the phylogentic trees imply that they have dispersed widely across the oceans and northern land masses. Since many related bunyaviruses are found in Africa, and are not associated with significant disease in humans, Africa would appear to be a reasonable candidate for their evolutionary origins.

The Genus Phlebovirus in the Bunyaviridae family (Gould and Higgs 2009; Zuckerman et al. 2004) contains one virus, Rift Valley fever virus (RVFV) that is a major human and animal pathogen. RVFV is primarily transmitted to animals and humans by a wide range of Aedes spp. mosquitoes, (Hoch et al. 1985; Turell et al. 1996) although it is also transmitted by phlebotomine species, hence its inclusion in the genus Phlebovirus. RVFV causes severe disease with high rates of abortion and fatal infections in livestock but appears to be relatively harmless in wild species that roam the plains in Africa. The virus was first identified in 1931 during an investigation into an epidemic on a farm in the Rift Valley of Kenya (Daubney et al. 1931) but it had almost certainly been responsible for outbreaks in different regions of Africa for many years before 1931. Humans that come into close contact with the blood, excreta and infected mosquitoes associated with clinically infected animals may also become infected. Most human cases are relatively mild, but some individuals develop much more severe symptoms that may present as ocular disease (0.5–2% of patients), meningoencephalitis (less than 1%), with residual neurological deficit and occasional fatalities, or haemorrhagic fever (less than 1%) with a case:fatality rate as high as 50%. RVFV is widespread throughout Africa and recently caused epidemics in the Arabian Peninsula, presumably being introduced either by infected mosquitoes or animals supplied for commercial purposes (Gould and Higgs 2009). There has been significant speculation as to whether or not this virus will continue to disperse out of Africa beyond the Arabian Peninsula. The opinion of some experts is that this is unlikely. However, others believe it would be most surprising if RVFV did not spread further.

Vesiculoviruses are members of the virus family Rhabdoviridae (genus Vesiculovirus). They are primarily associated with Phlebotomine sandflies and blackflies but several vesiculoviruses (tentatively classified in the genus) have been isolated from mosquito species. Whilst in the western hemisphere the vesiculoviruses are predominantly associated with disease in ungulates, particularly cows and horses, human infections usually presenting as flu-illness are recognized in people most frequently associated with infected animals. However, Chandipura virus (CHAV), principally associated with phlebotomine sandflies, was recently shown to be the cause of a human outbreak in India that involved 329 children of which 183 died following the development of acute encephalitis (Rao et al. 2004). If CHAV continues to produce epidemics with high fatality rates in India (and/or elsewhere), this could raise the profile of the vesiculoviruses as human pathogens.

In general, members of the genus Orbivirus do not infect humans and for many orbiviruses the invertebrate vector of importance in terms of disease in animals, is the midge (Culicoides). Bluetongue virus of sheep and cattle, African Horse sickness virus and Epizootic haemorrhagic disease of some wild ruminants, are the most well known viruses in this genus but in general they are not associated with transmission by mosquitoes. However, some less notorious orbiviruses are vectored by mosquitoes and may infect a large variety of wild animal species but these mosquito-borne arboviruses are not generally considered to be of significant importance to animal health.

Clinical diagnosis of the important mosquito virus infections can be reliable where infection is common, but definitive diagnosis requires laboratory confirmation either by virus isolation or the detection of antibody. The most widely used approaches used for diagnosis are RT-PCR tests for virus detection and detection of virus specific IgM in serum or CSF samples. A number of RT-PCR assays have been described for the clinically important viruses: JEV (Huang et al. 2004) Dengue (Lanciotti 2003).

Commercial assays for serological diagnosis are available (Guzman et al. 2004). However, serological diagnosis of DENV can be difficult to interpret because of the extensive antigenic cross reactions between Dengue scrotypes and other flaviviruses.

The mosquito-borne arboviruses cause a considerable amount of human morbidity and mortality worldwide. Dengue virus (DENV) in particular is an increasingly important human pathogen, comprising four serotypes (DENV-1, DENV-2, DENV-3, DENV-4) which cause epidemics in all tropical and many sub-tropical regions of the world. The number of dengue fever cases per year is estimated to exceed 50 million with approximately 2.5% of the 500, 000 cases of dengue haemorrhagic fever resulting in fatal infections, and in some poorly developed areas it may reach 20% (WHO, Fact sheet N°117, March 2009). In general, other arboviruses are not as widespread, but one or two, e.g. CHIKV and WNV have become much more widely dispersed and are causing larger epidemics than appears to have been the case 50 or more years ago. This is largely due to, increasing mosquito abundance, human mobility, agricultural and commercial activities, altered utilization of land, water and forests, impacts resulting from military activities and possibly early effects of climate change (Gould and Higgs 2009). There is therefore an increasing need for more extensive arbovirus disease control strategies.

Undoubtedly, the most effective method for controlling disease due to mosquito-borne arboviruses would be to eradicate the mosquitoes. Such methods have been shown to be effective, the most well known being the control of YFV at the beginning of the twentieth century in Cuba and in specific areas of the Panama Canal where the construction engineers were working. This success is largely attributed to William Gorgas and his colleagues; see website http://www.archives.state.al.us/famous/w_gorgas.html</url|>. Whilst mosquito eradication campaigns proved to be highly successful in controlling YFV, more than a century ago, attempts to control dengue fever in Singapore and in Cuba during the past twenty years, using a similar strategy have been only partly successful in reducing DENV incidence but DENV is constantly being re-introduced into these countries from neighbouring countries where the mosquito control measures are less successfully applied, making the total eradication of the virus a seemingly impossible task.

The live attenuated yellow fever virus vaccine known as 17D-204 (and also 17DD) is considered to be one of the most successful virus vaccines every produced. It was originally derived as an attenuated variant of wild type YFV by extensive serial passage in mice and subsequently in chick embryo eggs. It has been in use for over 70 years and millions of doses have been used almost certainly saving many lives in Africa and Latin America. However, as described earlier, YFV has a sylvatic existence in the African and Latin American jungles and this feature of the virus provides it with a safe reservoir from where it can re-emerge amongst human populations that have failed to maintain immunity levels by vigilant implementation of immunization programmes. The vaccine is described in detail in chapter 38. Inactivated vaccines against JEV and TBEV are also available for human use and they are often recommended to westerners travelling to endemic areas such as Asia, in the case of JEV. However, these vaccines are not approved for use in all countries. China has successfully developed a series of JEV vaccines, both inactivated and live attenuated, that have been used throughout large areas of China and other regions of Asia, where JEV is known to circulate. More recently widespread campaigns have also been implemented to immunize children in India in the hope of reducing the high levels of morbidity and mortality recorded annually. Currently, there are no other widely available vaccines for human protection against the known pathogenic arboviruses. However, vaccines have been produced against a variety of arboviruses for limited use in individuals exposed to the viruses through the nature of their occupations. For example, limited quantities of inactivated human vaccines against VEEV, KFDV, and RVFV, have been produced and used in military and laboratory personnel. However, none of these vaccines has been approved for large-scale use and with the possible exception of KFDV in India, the use of these vaccines has gradually being reduced to virtually zero. Currently, several projects have been initiated to develop vaccines against DENV, WNV, JEV, VEEV and CHIKV using a variety of methods including the development of chimaeric flaviviruses that use:

Several of these new potential human vaccines are currently undergoing clinical trials.

Several veterinary vaccines have already been developed to protect horses against a variety of arboviruses, in particular, VEEV, EEEV, and WNV. In general these vaccines are proving to be efficacious with few serious side effects in horses.

In addition to the use of virus vaccines to control infections due to mosquito-borne arboviruses, a large number of antiviral compounds are being tested for their ability to control infections that have already been initiated. At the moment no effective single antiviral therapeutic agent has been identified although several show promise and are currently undergoing clinical trials.

In conclusion, prior to the introduction of WNV into North America through New York, the global research effort on arboviruses was in relative decline when compared with malaria, hepatitis B and C, or HIV. However, the widespread dispersal of WNV throughout North America that commenced in 1999, together with the increasing epidemicity of DENV, and the recent emergence, in 2005, of CHIKV as a rapidly spreading threat to human populations throughout Asia, and potentially globally, has stimulated renewed interest in arboviruses, particularly in their rapid diagnosis, pathogenicity, modes of transmission and dispersal, their evolution and their control. Taking an optimistic viewpoint, the application of modern molecular technologies, and bioinformatics tools, combined with improved epidemiological modelling, more extensive sampling methods, a greater understanding of the disease process, and better medical support should increase our capacity to reduce the levels of human and animal suffering, due to arboviruses during the foreseeable future.