Bluetongue Virus

Description

Bluetongue is a non-contagious, arthropod-borne disease of ruminants, caused by bluetongue virus (BTV). The clinical severity of disease is variable, but is characterised by inflammation of mucous membranes, haemorrhages and oedema¹. Although cattle are the main reservoir of infection, sheep are more severely affected and often suffer a cyanotic tongue, lending the disease its name. The virus has been isolated from hosts and vectors on all continents(excluding Antartica)², despite being initially recognised in Africa in the late 19th and early 20th centuries³. Originally thought to be a disease of tropical and sub-tropical regions, bluetongue has shown a propensity to become established in temperate areas, and in recent years has spread North, through the Mediterranean Basin, to become endemic in many European countries including the UK. Although BTV's transmission and epidemiology is dependent on insect vectors, bluetongue greatly influences the global trade of ruminants as it is included on the Office International des Epizooties List A of animal diseases⁴.

Aetiology

Bluetongue virus particle. Source: Wikimedia Commons; Author: CDC (2007)

Bluetongue virus is a species of the genus Orbivirus, within the Reoviridae family. The Reoviridae are non-enveloped and possess a double-stranded RNA genome contained in an outer-shelled icosohedral capsid. The BTV genome is arranged into 10 segments and encodes 7 structural and 4 non-structural viral proteins². The BTV receptor is currently unknown, but is proposed to included sialic acid and junctional adhesion molecules. After interaction with this receptor, the virus enters an endolysosome where the capsid is partially digested to allow the genome into the cell. Replication begins at this partially uncoated stage since the virus particles contain all the necessary enzymes⁵. First, the dsRNA is transcribed to form positive sense RNA, of which some is delivered to cytoplasm for ribosomal translation and the remainder is packaged into partially assembled virions. Complementary negative sense RNA is then formed in the virions, to give a dsRNA genome. Complete virus particles are released from the cell.

All BTV’s share group antigens, which can be demonstrated by agar gel diffusion tests, fluorescent antibody tests and the group reactive ELISA¹. There are 24 distinct serotypes, which are are distinguished by epitopes on the outer capsid protein VP2⁴, encoded by L2, the only serotype-specific BTV gene. Serotypes are differentiated using serum neutralisation tests, although there is some degree of cross-reactivity between serotypes¹. Numerous strains of bluetongue virus also exist, and these are characterised by molecular analysis.

Hosts

All ruminants are susceptible to bluetongue virus infection, including sheep, goats, cattle, deer, buffaloes, camels and antelopes. Sheep are most severely affected, and disease is occasionally seen in goats. Although cattle BTV infection is significant in the epidemiology of disease, the condition is generally subclinical in this host. Mortalities in white-tailed deer due to bluetongue¹.

Pigs and horses do not become infected with BTV, but may act as a food source for the Culicoides midges that transmit bluetongue virus to ruminants. Their habitats may also provide areas suitable for vector breeding.

Transmission and Epidemiology

BTV is transmitted by biting insects. Although vertical and venereal transmission between ruminant hosts can occur, it is insignificant in the overall epidemiology of bluetongue.

Vectors

The arthropod vector for bluetongue virus is the Culicoides biting midge. These insects take blood meals from vertebrate hosts and breed in damp, dung-enriched soil enriched, and so are abdunant in the vicinity of domestic livestock. Once eggs are laid in soil, Culicoides progresses through four larval stages and pupates before becoming an adult midge. The lifecycle is greatly influenced by temperature: in temperate regions such as Britain, the adult midge population declines in October and is absent by December. The fourth larval stage overwinters, and adults re-appear the following April. The environment affects the activity of adult midges in a variety of ways. Culicoides survive around 10 days in warm weather but up to one month when conditions are cooler and are most active at night, from an hour before sunset to an hour after sunrise. Activity is decreased by windy conditions, and increased during the day when the weather is dull. Bluetongue virus replicates more rapidly in vector species when temperatures are warmer.

Classically, the major vector for BTV is Culicoides imicola. This midge is found throughout Africa, the Middle East, southern Asia, Portugal, Greece, Corsica, Sardinia, Sicily and areas of Italy¹, and its distribution appears to be extending northwards. However, C. imicola has not yet been demonstrated in the United Kingdom. The Culicoides species found in the British Isles are C. pulicais and C. obsoletus, which are also common across central and northern Europe. Knowledge of the distribution of these species in the UK is incomplete but the insects tend to gather where breeding sites and hosts occur in tandem, with the highest midge concentrations in areas containing cattle, horses and pigs. Removal of livestock decreases populations of Culicoides by a factor of 10 to 20¹, but some persist by feeding on wild animals and man. Hill sites have fewer midges as climatic conditions are less favourable, and sheep have a less positive influence on distribution.

In Britain, studies are ongoing to determine midge distribution, seasonal incidence and the competency of the various Culicoides species to act as BTV vectors.

Vector Competence

Certain information can help inferences be made regarding BTV vectors in Britain. Both C. obsoletus and C. pulicaris have been implicated in transmission before. Previously, BTV has been isolated from C. obsoletus in Cyprus, and African horse sickness virus (another Orbivirus) in Spain. C. obsoletus and C. pulicaris were also the most abundant Culicoides species detrected in the 1999 BTV epizootic in Greece and Bulgaria, and so are strongly suspected of acting as vectors in this case. They may also have mediated outbreaks in Serbia, FYR Macedonia, Croatia and Bosnia in 2001-2002, where C. imicola has not been recorded. Both species are therefore contenders to transmit bluetongue virus in the UK.

A British population of C. obsoletus has been shown to have and oral susceptibility rate of less than 2%¹, suggesting that C. obsoletus is likely to be an inefficient or minor vector of BTV in the UK. However, it is possible that a high abundance or survival rate may compensate for this low vector competence. Indeed, C. brevitarsis, the major Australian vector of BTV, has an extremely low experimental competency yet is an effective vector in the field.

Two other Culicoides species, C. nuberculous and C. impunctatus, exist in mainland Europe and the UK, and have been experimentally infected with bluetongue virus.

Epidemiology

When Culicoides feed on a bluetongue-infected host, they become persistently infected with the virus for the duration of their lives⁴. The global distribution of BTV is related only to these competent insect vectors²: although vertical and venereal transmission of bluetongue is possible, these routes do not influence the epidemiology of BTV. The vector species varies with location and is poorly characterised in some areas, including northern Europe. However, as described above, it has been demonstratated that ambient temperature has profound effects on vector survival and feeding activity, and the replication of BTV in the insect⁶. Therefore, the relationship between temperature and effective transmission of BTV is likely to be a limiting factor to the spread of BTV, which may potentially be overcome by global warming.

It is appears that in different regions of the world, specific serotypes of bluetongue virus have co-evolved with vector species of insect vector⁷. For example, different serotypes of BTV are prevalent in the USA to those in those the Caribbean and Central America although there is no real geographic barrier separating these areas, and animals move between the regions. This is thought to be due to the variation in the species of vector insects: Culicoides sonorensis is the primary vector in the USA and is most competent for serotypes 10, 11, 13 and 17, but in the Caribbean and Central America C. insignis carries serotypes 1, 2, 3, 4, 6, 8, 11, 12, 13, 14 and 17⁴.

A variety of other hosts have been implicated in the lifecycle of BTV infection. Serological evidence indicates that large African carnivores are infected with BTV, whereas smaller predators that co-habit with them are not, suggesting that large carnivores are infected through feeding on BTV-infected ruminants [2]. Inadvertent contamination of a canine vaccine with BTV confirmed that dogs are susceptible to BTV infection, indeed pregnant bitches that received this contaminated vaccine typically aborted and died [1]. There is no evidence, however, that dogs or other carnivores are important to the natural cycle of BTV infection.

Impact of Global Warming

Although BTV has been the subject of intense molecular and structural studies, the epidemiology and geographic dispersal of BTV have also been a major subject of interest to virologists and entomologists, because this virus is pathogenic for a range of domestic and wild ruminants. Seasonal incursions of the virus from Africa into more temperate latitudes, sometimes accompanied by disease, have occurred under favourable climatic conditions, but the recent introduction of serotype BTV-8, and the establishment of a transmission cycle that has resulted in its spread into northern Europe including the UK (see below), is of significant economic importance. BTV is a member of the genus Orbivirus in the family Reoviridae but, unlike many other arboviruses, does not infect humans and therefore is not zoonotic. There are 24 recognised serotypes of the virus, which contain between 10 and 12 segments of double-stranded RNA. Until recently BTV was considered to be almost exclusively a disease of some European breeds of sheep that, for commercial purposes, have been distributed widely in Africa, Asia and Australasia. In cattle and goats, clinical disease has been considered rare, and much milder than in sheep.76 However, recent observations suggest that cattle frequently show disease symptoms resulting from infection by the BTV-8 serotype that is currently circulating in northern Europe (see below). There is evidence that infected midges are carried on the wind for long distances,77,78 and it has been postulated that the major epidemics of bluetongue, in regions where disease occurs only sporadically, result from windborne carriage of infected Culicoides from distant endemic areas.79 Competent midges may be infected when biting viraemic vertebrates. The probability of infection depends in part on the genotype of the midge, the strain of virus, the level of viraemia and environmental factors.80 The extrinsic incubation period (the period between feeding on infected blood and the appearance of virus in the saliva of the arthropod vector) is 1—2 weeks. Contrary to the BTV strains referred to above, the recent appearance of BTV-8 in northern Europe, including the UK, has unexpectedly been accompanied by the appearance of overt disease and mortality in cattle. Moreover, as the result of currently unpublished evidence reported by Dr Oura on 20 March 2008,81 it is now recognised that healthy infected animals may remain ELISA- and RT-PCR-positive for at least 4 months.82 This observation helps to explain how BTVpositive animals may be detected in mid-winter in the UK when midge transmission activity is presumed to be minimal. Symptoms of BTV infection in sheep are variable but typically include fever. Facial oedema results in swelling and soreness of the lips and nose with mucopurulent discharge, which is exacerbated by champing to produce frothy saliva. The term ‘bluetongue’ is derived from the cyanosis of the tongue that is observed in some cases. Erosion of the coronal band above the hooves and musculoskeletal damage cause pain and lameness, inducing the sheep to adopt a posture similar to that shown in Figure 5. BTV circulates widely throughout tropical and subtropical regions, but until relatively recently the disease had been observed only infrequently in some areas of southern Europe. However, during the past decade, six strains of BTV are known to have spread across 12 European countries, and significantly the virus has gradually dispersed further north in central and western Europe. This dispersal has probably been driven by the northward expansion of the range of Cu. imicola, the main BTV vector, and by climate change, which has probably contributed to increased persistence Figure 5 Posture often observed in cases of bluetongue infection in sheep (source: http://129.186.78.52/DiseaseInfo/ ppt/bluetongue.ppt#17). during winter, consequently increasing the subsequent risk of transmission over larger geographical regions83 and an extended period of time. To the north of the Cu. imicola range, other species (Cu. obsoletus, Cu. pulicaris, Cu. chiopterus and Cu. dewulfi) with distributions extending across central and northwestern Europe84 were probably involved in the appearance of BTV-8 in Belgium, France, Luxembourg, Germany and the Netherlands in August 2006, and subsequently in the UK in September 2007.85 This presence of multiple vectors of BTV-8 appears to apply to large parts of northern Europe and has almost certainly contributed to the dramatic spread of this arbovirus across this area. In addition to the impact of climate change on vector range expansion and the northerly establishment of BTV-8, the commercial transportation of asymptomatic infectious ruminants and the wind-borne dispersal of infected midges are believed to be highly significant contributory factors to the rapid dispersal of the virus. Understanding this sequence of events may aid predictions of the emergence of other vector-borne pathogens, such as the more devastating African horse sickness virus, another animal pathogen in the genus Orbivirus that may be transmitted by several of the same vectors as BTV. Another important observation has appeared as the result of the incursion of BTV into northern Europe. Conventional opinion has previously considered it extremely unlikely that BTV could be transmitted vertically to newborn offspring. New evidence suggests that this virus may be transmitted across the bovine placenta to infect the fetus, causing an unusually high rate of malformed, stillborn and weak calves born on holdings with a known history of BTV infection.86 At the time of writing, this observation has not been confirmed through systematic investigation. Nevertheless, whether or not this represents an acquired new characteristic of BTV-8 clearly needs close attention. Transplacental infection has only previously been associated with attenuated BTV vaccine viruses. In further support of these reports, the recent unpublished finding of imported heifers in Northern Ireland, leading to the suspicion that newborn calves infected in utero can act as virus reservoirs for the Culicoides vector, is another worrying development that needs immediate investigation Methods for controlling BTV include reducing exposure of the animals to the competent midges, the use of insecticides to dissuade the insects from biting the animals, and the use of vaccines. While the strategies of reducing exposure and using insect repellents might reduce the levels of BTV transmission, clearly these measures cannot be expected to eradicate BTV from northern Europe. Vaccination is associated with several practical difficulties. Firstly, there are 24 serotypes of BTV, and while there is some antigenic cross-reactivity between different serotypes, the preparation of a single live attenuated virus multivalent vaccine to protect against all 24 is impractical, partly because different serotypes may outcompete each other in the vaccine, partly because at the moment only BTV-8 is circulating in northwestern Europe and partly because of the costs and time involved in producing a multivalent vaccine. Moreover, the use of live attenuated vaccines presents a low but potential risk of reversion to virulence, or in some circumstances the possibility of reassortment of the RNA gene segments between different serotypes of BTV. However, for reasons beyond the control of the manufacturers, the production of a vaccine in time to prevent the reemergence of BTV-8 in northern Europe during 2008 is proving to be seriously problematic. It will be interesting to see whether or not BTV-8 is brought under control in the UK and northern Europe during 2008. Non-infectious vaccines based on engineered recombinant proteins are also under development, but in addition to the requirement for multiple dosing, these vaccines are likely to be expensive and therefore not favoured by farmers.

Pathogenesis

• Transfer occurs through blood from viremic animals via biting midges (Culicoides spp.)
• Replication in haematopoietic and endothelial cells of blood vessels
• Clinical signs vary between species, with sheep most severely affected
  • Pyrexia
  • Ocular and nasal discharge
  • Drooling from mouth uclers
  • Swelling of the mouth, head and neck
  • Lameness
  • Subdural hemorrhages
  • Inflammation of the coronary band
• Cattle as the main reservoir
• A blue tongue is rarely seen as as a clinical sign of infection
• Resulting loss of condition, reduction in wool an meat production, which can be followed by death

Diagnosis

The typical clinical signs of bluetongue enable a presumptive diagnosis, especially in areas where the disease is endemic. Suspicion is confirmed by the presence of petechiae, ecchymoses, or hemorrhages in the wall of the base of the pulmonary artery and focal necrosis of the papillary muscle of the left ventricle. These highly characteristic lesions are usually obvious in severe clinical infections but may be barely visible in mild or convalescent cases. These lesions are often described as pathognomonic for bluetongue, but they have also been observed occasionally in other ovine diseases such as heartwater, pulpy kidney disease, and Rift Valley fever. Hemorrhages and necrosis are usually found where mechanical abrasion damages fragile capillaries, such as on the buccal surface of the cheek opposite the molar teeth and the mucosa of the esophageal groove and omasal folds. Other autopsy findings include subcutaneous and intermuscular edema, skeletal myonecrosis, myocardial and intestinal hemorrhages, hydrothorax, hydropericardium, pericarditis, and pneumonia. In many areas of the world, bluetongue in sheep, and especially in other ruminants, is subclinical and, therefore, laboratory confirmation based on virus isolation in embryonated chicken eggs, susceptible sheep, or cell cultures, or the identification of viral RNA by PCR is necessary. The identity of isolates may be confirmed by the group-specific antigen-capture ELISA, immunofluorescence, immunoperoxidase, serotype-specific virus neutralization tests, or hybridization with complementary gene sequences of group- or serotype-specific genes. For virus isolation, blood (10-20 mL) is collected as early as possible from febrile animals into an anticoagulant such as heparin, sodium citrate, or EDTA and transported at 4°C to the laboratory. For longterm storage where refrigeration is not possible, blood is collected in oxalate-phenol-glycerin (OPG). Blood to be frozen should be collected in buffered lactose peptone and stored at or below -70°C. Blood collected at later times during the viremic period should not be frozen, as lysing of the RBC or thawing releases the cell-associated virus, which may then be neutralized by early humoral antibody. The virus does not remain stable for long at -20°C. In fatal cases, specimens of spleen, lymph nodes, or red bone marrow are collected and transported to the laboratory at 4°C as soon as possible after death. A serologic response in ruminants can be detected 7-14 days after infection and is generally lifelong. Current recommended serologic techniques for the detection of bluetongue virus antibody include agar gel immunodiffusion and competitive ELISA. The latter is the test of choice and does not detect cross-reacting antibody to other orbiviruses, especially anti-EHDV (epizootic hemorrhagic disease virus) antibody. Various forms of virus neutralization test, including plaque reduction, plaque inhibition, and microtiter neutralization can be used to detect type-specific antibody.

Clinical Signs

The course of the disease in sheep can vary from peracute to chronic, with a mortality rate of 2-30%. Peracute cases die within 7-9 days of infection, mostly as a result of severe pulmonary edema leading to dyspnea, frothing from the nostrils, and death by asphyxiation. In chronic cases, sheep may die 3-5 wk after infection, mainly as a result of bacterial complications, especially pasteurellosis, and exhaustion. Mild cases usually recover rapidly and completely. The major production losses include deaths, unthriftiness during prolonged convalescence, wool breaks, and possibly reproductive loss. In sheep, bluetongue virus causes vascular endothelial damage, resulting in changes to capillary permeability and subsequent intravascular coagulation. This results in edema, congestion, hemorrhage, inflammation, and necrosis. The clinical signs in sheep are typical. After an incubation period of 4-6 days, a fever of 105-107.5°F (40.5-42°C) develops. The animals are listless and reluctant to move. Clinical signs in young lambs are more apparent, and the mortality rate is higher (up to 30%). About 2 days after onset of fever, additional clinical signs such as edema of lips, nose, face, submandibular area, eyelids, and sometimes ears; congestion of mouth, nose, nasal cavity, conjunctiva, and coronary bands; and lameness and depression may be seen. A serous nasal discharge is common, later becoming mucopurulent. The congestion of nose and nasal cavity produces a “sore muzzle” effect, the term used to describe the disease in sheep in the USA. Sheep eat less because of oral soreness and will hold food in their mouths to soften before chewing. They may champ to produce a frothy oral discharge at the corners of the lips. On close examination, small hemorrhages can be seen on the mucous membranes of the nose and mouth. Ulceration develops where the teeth come in contact with lips and tongue, especially in areas of most friction. Some affected sheep have severe swelling of the tongue, which may become cyanotic (‘blue tongue”) and even protrude from the mouth. Animals walk with difficulty as a result of inflammation of the hoof coronets. A purple-red color is easily seen as a band at the junction of the skin and the hoof. Later in the course of disease, lameness or torticollis is due to skeletal muscle damage. In most affected animals, abnormal wool growth resulting from dermatitis may be observed. The pathogenesis of bluetongue in cattle seems to differ from that in sheep and is based on immediate IgE hypersensitivity reactions. Clinical signs in cattle are rare but may be similar to those seen in sheep. They are usually limited to fever, increased respiratory rate, lacrimation, salivation, stiffness, oral vesicles and ulcers, hyperesthesia, and a vesicular and ulcerative dermatitis. Susceptible cattle and sheep infected during pregnancy may abort or deliver malformed calves or lambs. The malformations include hydranencephaly or porencephaly, which results in ataxia and blindness at birth. White-tailed deer and pronghorn antelope develop severe hemorrhagic disease leading to sudden death. Pregnant dogs abort or give birth to stillborn pups and then die in 3-7 days.

Pathology

Complete loss of integrity of epithelium. Uncommon.

• Characteristic of Bluetongue Virus,
• Epithelium lost and haemorrhage produces blue / black discoloration of the tongue, hence the name.

• Grossly:
  • Infarctions -> necrosis
  • Haemorrhage
• Histologically:
  • Necrosis -> calcification or regeneration (depends on age of lesion)

Treatment

• BTV is NOTIFIABLE
• Vigilance in recognizing clinical signs
• Restriction of movement:
  • Protection Zone: 100km radius around infected premises, movement within zone allowed but not in or out
    • Vaccination within PZ using appropriate serotype is encouraged but still voluntary
  • Surveillance Zone: 50km radius beyond PZ
• Vector control: ectoparasiticides, etc.

Prophylactic immunization of sheep remains the most effective and practical control measure against bluetongue in endemic regions. Three polyvalent vaccines, each comprising 5 different bluetongue virus serotypes attenuated by serial passage in embryonated hens’ eggs followed by growth and plaque selection in cell culture, are widely used in southern Africa and elsewhere, should epizootics of bluetongue occur. A monovalent modified live virus vaccine propagated in cell culture is available for use in sheep in the USA. Live-attenuated vaccines should not be used during Culicoides vector seasons because they may transmit the vaccine virus(es) from vaccinated to nonvaccinated animals, eg, other ruminant species. This may result in reassortment of genetic material and give rise to new viral strains. Abortion or malformation, particularly of the CNS, of fetuses may follow vaccination of ewes and cows with attenuated live vaccines during the first half and the first trimester of pregnancy, respectively. Passive immunity in lambs usually lasts 4-6 mo. The control of bluetongue is different in areas where the disease is not endemic. During an outbreak, when one or a limited number of serotypes may be involved, vaccination strategy depends on the serotype(s) that are causing infection. Use of vaccine strains other than the one(s) causing infection affords little or no protection. The vector status, potential risk from vaccine virus reassortment with wild-type viral strains, virus spread by the vectors to other susceptible ruminants, and reversion to virulence of vaccine virus strains or even the production of new serotypes also should be considered. Although a number of noninfectious vaccines are in development, they are not yet commercially available. Control of vectors by using insecticides or protection from vectors by moving animals into barns during the evening hours lowers the number of Culicoides bites and subsequently the risk of exposure to bluetongue virus infection.

Links

• Defra - Bluetongue
• BTV Control in Cattle and Sheep (Intervet)
• Institute for Animal Health - Bluetongue
• Bluetonguevirus.org - BTV information and resource portal

References

1. Defra (2002) Technical Review - Bluetongue : The Virus, Hosts and Vectors.
2. Gibbs, E P J and Geiner, E C (1994) The Epidemiology of Bluetongue. Comparative Immunology, Microbiology and Infectious Diseases, 17(3-4), 207-220.
3. Spreull, J (1905) Malarial catarrhal fever (bluetongue) of sheep in South Africa. Journal of Comparative Pathology and Therapeutics, 18, 321-337.
4. MacLachlan, N J (2004) Bluetongue: A Review and Global Overview of the Only OIE List a Disease that is Endemic in North America. Proceedings of the 55th Annual Meeting of the American College of Veterinary Pathologists (ACVP) and 39th Annual Meeting of the American Society of Clinical Pathology (ASVCP), p1237.
5. Carter, G R and Wise, D J (2005) A Concise Review of Veterinary Virology, IVIS.
6. Mullens, B A et al (1995) Effects of temperature on virogenesis of bluetongue virus serotype 11 in Culicoides variipennis sonorensis. Medical and Veterinary Entomology, 9, 71-76.
7. Gould, A R and Hyatt, A D (1994) The orbivirus genus: diversity, structure, replication and phylogenetic relationships. Comparative Immunology, Microbiology and Infectious Diseases, 1, 163-188.
8. Merck & Co (2008) The Merck Veterinary Manual (Eighth Edition), Merial.
9. Dal Pozzo, F et al (2009) Bovine infection with bluetongue virus with special emphasis on European serotype 8. The Veterinary Journal, 182(2), 142-151.
10. MacLachlan, N J et al (2009) The Pathology and Pathogenesis of Bluetongue. Journal of Comparative Pathology, 141(1), 1-16.
11. Barratt-Boyes, S M and MacLachlan, N J (1995) Pathogenesis of bluetongue virus infection of cattle. Journal of the American Veterinary Medical Association, 206(9), 1322-1329.
12. Afshar, A (2004) Bluetongue: Laboratory Diagnosis. Comparative Immunology, Microbiology and Infectious Diseases, 17(3-4), 221-242.
13. Gould, E A and Higgs, S (2009) Impact of climate change and other factors on emerging arbovirus diseases. Transactions of the Royal Society of Tropical Medicine and Hygiene, 103(2), 109-121.
14. MacLachlan, N J (1994) The pathogenesis and immunology of bluetongue virus infection of ruminants. Comparative Immunology, Microbiology and Infectious Diseases, 17(3-4), 197-206.