Bluetongue disease

Bluetongue disease (also called catarrhal fever) is a non-contagious, insect-borne viral disease of ruminants, mainly sheep and less frequently of cattle, goats, buffalo, deer, dromedaries and antelope. There are no reports of human transmission. It is caused by the Bluetongue virus.

Pathogen
The pathogenic virus, Bluetongue virus of the genus Orbivirus, is a member of the Reoviridae family. There are 24 serotypes. It is transmitted by a midge, Culicoides imicola and other culicoid species.

Bluetongue Virus
Bluetongue virus (BTV), a member of Orbivirus genus within the Reoviridae family causes serious disease in livestock (sheep, goat, cattle). Partly due to this BTV has been in the forefront of molecular studies for last three decades and now represents one of the best understood viruses at the molecular and structural levels. BTV, like the other members of the family is a complex non-enveloped virus with seven structural proteins and a RNA genome consisting of 10 double-stranded (ds) RNA segments of different sizes. Data obtained from studies over a number of years have defined the key players in BTV entry, replication, assembly and exit and have increasingly found roles for host proteins at each stage. Specifically, it has been possible to determine the complex nature of the virion through 3D structure reconstructions (diameter ~ 800 &Aring;); the atomic structure of proteins and the internal capsid (~ 700 &Aring;, the first large highly complex structure ever solved); the definition of the virus encoded enzymes required for RNA replication; the ordered assembly of the capsid shell and the protein sequestration required for it; and the role of host proteins in virus entry and virus release. These areas are important for BTV replication but they also indicate the pathways that may be used by related viruses, which include viruses that are pathogenic to man and animals, thus providing the basis for developing strategies for intervention or prevention.

BTV is the type species of the genus Orbivirus within the family Reoviridae. The Reoviridae family is one of the largest families of viruses and includes major human pathogens (e.g., rotavirus) as well as other vertebrate, plant and insect pathogens. Like the other members of the family, Orbiviruses which encompass, besides BTV, the agents causing African horse sickness (AHSV) and epizootic hemorrhagic disease of deer (EHDV), have the characteristic double-stranded and segmented features of their RNA genomes. However, unlike the mammalian reoviruses, Orbiviruses comprising 14 serogroups, are vectored to a variety of vertebrates by arthropod species (e.g., gnats, mosquitoes and ticks) and replicate in both hosts. BTV, the etiological agent of Bluetongue disease of animals, is transmitted by Culicoides species. In sheep BTV causes an acute disease with high morbidity and mortality. BTV also infects goats, cattle and other domestic animals as well as wild ruminants (e.g., blesbuck, white-tailed deer, elk, pronghorn antelope, etc.). The disease was first described in the late 18th century and was believed for many decades to be confined to Africa. However, to date BTV has been isolated in many tropical, subtropical and temperate zones and 24 serotypes have been identified from different parts of the world. Due to its economic significance BTV has been the subject of extensive molecular, genetic and structural studies. As a consequence it now represents one of the best characterised viruses.

Unlike the reovirus and rotavirus particles, the mature BTV particle is relatively fragile and the infectivity of BTV is lost easily in mildly acidic conditions. BTV virions (550S) are architecturally complex structures composed of 7 discrete proteins that are organised into two concentric shells, the outer and inner capsids, and a genome of 10 dsRNA segments. The outer capsid, which is composed of two major structural proteins (VP2 and VP5), is involved in cell attachment and virus penetration during the initial stages of infection. Shortly after infection, BTV is uncoated, i.e. VP2 and VP5 are removed, to yield a transcriptionally active 470S core particle which is composed of two major proteins VP7 and VP3, and the three minor proteins VP1, VP4 and VP6 in addition to the dsRNA genome. There is no evidence that any trace of the outer capsid remains associated with these cores, as has been described for reovirus. The cores may be further uncoated to form 390S subcore particles that lack VP7, also in contrast to reovirus. Subviral particles are probably akin to cores derived in vitro from virions by physical or proteolytic treatments that remove the outer capsid and causes activation of the BTV transcriptase. In addition to the seven structural proteins, three non-structural (NS) proteins, NS1, NS2, NS3 (and a related NS3A) are synthesised in BTV-infected cells. Of these, NS3/NS3A is involved in the egress of the progeny virus. The two remaining non-structural proteins, NS1 and NS2, are produced at high levels in the cytoplasm and are believed to be involved in virus replication, assembly and morphogenesis.

Current Research
Bluetongue virus (BTV) is well characterized both genetically (the sequence was completed in 1989) and structurally. Understanding of the molecular biology of the virus and mapping the role of each protein in virus life cycle has benefited significantly through the availability of recombinant BTV proteins and sub-viral particles. In addition the structure of BTV proteins, core and virionparticles have contributed greatly to understanding the mechanism of protein–protein interaction in the virus assembly pathway of BTV and other orbiviruses. Most importantly, information gained from these studies has laid sound foundation for the generation of safe BTV vaccines with the possibility of use in animals in the near future. Latterly, studies have concentrated on the fundamental mechanisms that are used by the virus to invade, replicate in and escape from susceptible host cells. Progress has been made in understanding the structure and entry of intact virus particles, the role of each enzymatic protein in the transcription complex, the critical interactions that occur between the viral non-structural proteins and viral RNA and the role of cellular proteins in non-enveloped virus egress.

Despite these advances, some critical questions remain unanswered for the BTV life cycle and a more complete understanding of the interactions between the virus and the host cell is required for these to be addressed. For example, although progress has been made in the identification of signals for the recruitment of BTV RNA segments into the virion assembly site in the host cell cytoplasm, it has not been possible yet to determine how exactly each genome segment is packaged into the progeny virus. It is also not apparent when and how these genome segments wrap around the polymerase complex once the RNA has been recruited. One of the major drawbacks of research with BTV and other members of Reoviridae has been the lack of availability of a suitable system for genetic manipulation of the virus. This has been a major obstacle in understanding the replication processes of these viruses. However, one of the recent developments in the field of BTV research has been to rescue live virus from transfection of BTV transcripts. There is no doubt that this will be soon extended to establish in vitro manipulative genetic system and will be utilized to address some of these remaining questions.

Very little is known of the intracellular trafficking of newly generated virions although there are some indications of involvement of the cytoskeleton, intermediate filaments and vimentin during BTV morphogenesis. Host–virus interactions during virus trafficking will be one of the future areas needing intense attention. Recent work has revealed unexpected and striking parallels between the entry and release pathways of BTV and pathways involved in entry and release of enveloped viruses. These parallels may be the result of an enveloped ancestor virus or because there are a limited number of cellular pathways that can be useful for the egress of large protein complexes from cells. It is notable that the NS3 glycoprotein of BTV is an integral membrane protein that is functionally involved in virus egress by bridging between the outer capsid protein VP2 and the cellular export machinery. Although no cell-free enveloped form of BTV has been isolated, budding of BTV particles from infected cells at the plasma membrane are quite apparent. The exact role of NS3 in this process and the role of host proteins (Annexin II and p11, Tsg101 and MVB) and their contribution in the release of non-enveloped viruses, such as BTV, remains to be clarified.

Epidemiology
Bluetongue has been observed in Australia, the USA, Africa, the Middle East, Asia and Europe. Its occurrence is seasonal in the affected Mediterranean countries, subsiding when temperatures drop. It has been spreading northward since October 1998, perhaps as a result of global warming. In August 2006, cases of bluetongue were found in the Netherlands, then Belgium, Germany, and Luxembourg. In September 2007, the UK reported its first ever suspected case of the disease, in a Highland cow on a rare breeds farm near Ipswich, Suffolk.

Although the disease is not a threat to humans the most vulnerable common breeds in the UK are cattle, goats and sheep.

Symptoms
Major signs are high fever, excessive salivation, swelling of the face and tongue and cyanosis of the tongue. Swelling of the lips and tongue gives the tongue its typical blue appearance, though this sign is confined to a minority of the animals. Recovery is very slow.

The incubation period is 5–20 days, and all signs usually develop within one month. The mortality rate is normally low, but is high in susceptible breeds of sheep. In cattle and wild ruminants infection is usually asymptomatic despite high virus levels in blood.

Treatment
There is no efficient treatment. Prevention is effected via quarantine, inoculation with live modified virus vaccine and control of the midge vector, including inspection of aircraft.

Although the tongues of human patients with some types of heart disease may be blue, this sign is not related to bluetongue disease.

Vaccine
Outbreaks in southern Europe have been caused by serotypes 2 and 4, and vaccines are available against these serotypes. However, the disease found in northern Europe (including the UK) in 2006 and 2007 has been caused by serotype 8: a vaccine is not yet available. Vaccine companies Merial and Intervet are developing vaccines against serotype 8 and the associated production facilities.

Since it is a vector-borne disease and transmitted to healthy livestock from infected animals by blood feeding Culicoides spp., attenuated, live virus vaccines are not desirable. The flexibility of baculovirus expression vectors and the capacity of the baculovirus genome to accommodate large amounts of foreign DNA allowed us to exploit the system for the simultaneous expression of multiple BTV genes by a single recombinant virus. Based on the 3D structural data, recombinant viruses were prepared to express either VP3 and VP7, the two major core proteins, or VP3 and VP7 together with VP2 and VP5, i.e. all four major structural proteins. Expression of VP3 and VP7 resulted in CLPs that were similar in size and appearance to cores prepared from BT virions. Similarly, the simultaneous expression of four proteins resulted in the assembly of virtually homogeneous doublecapsid particles. When the 3D structure of CLPs and VLPs were analysed by Cryo-EM both types of particles were clearly comparable to authentic cores and virions, and exhibited essentially the same basic features and full complement of the two or four proteins. VLPs synthesized by recombinant baculoviruses were also characterized further at the biological and immunological levels and compared to those of the native virion. VLPs exhibited high levels of haemagglutination activity similar to those of authentic BTV. Further antibodies raised to the expressed particles contained high titres of neutralizing activity against the homologous BTV serotype emphasizing their authenticity at functional level. Since VLPs elicited strong neutralizing antibodies in guinea pigs, it can be anticipated that VLPs should also elicit protective responses in sheep against BTV infection. Consequently, a number of experiments were performed to examine the protective efficacy of VLPs (10-200 µg per sheep) in 1-year old BTV free sheep divided in groups (4-8 sheep/group). All sheep were challenged by subcutaneous inoculation of 1 ml of infective sheep blood containing virulent virus at day 117 or at 14 months. The challenged sheep developed neither clinical signs nor viraemia, indicating a suppression of BTV replication. The post-challenge blood samples of the sheep that did not receive vaccine and only received saline, contained infectious BTV and these sheep developed high neutralizing antibody titres indicative of a primary infection. Protective immunity to BTV disease was obtained by vaccinating sheep with doses of 10 µg or more of BTV VLPs which resulted in long-lasting protection against homologous BTV challenge. Some preliminary evidence was obtained for crossprotection, depending on the challenge virus and the amounts of antigen used for vaccination.

Related diseases
African horse sickness is related to Bluetongue and is spread by the same midges (Culicoides species). It can kill up to 90% of the horses it infects.