RNA segment 9 exists as a duplex concatemer in an Australian strain of epizootic haemorrhagic disease virus (EHDV): Genetic analysis and evidence for the presence of concatemers as a normal feature of orbivirus replication.

This paper reports a concatemeric RNA in a strain of epizootic haemorrhagic disease virus (EHDV) serotype 5. Sequencing showed that the concatemeric RNA contains two identical full-length copies of genome segment 9, arranged in series, which has apparently replaced the monomeric form of the segment. In vitro translation demonstrated that the concatemeric RNA can act as a viable template for VP6 translation, but that no double-sized protein is produced. Studies were also performed to assess whether mutations might be easily introduced into the second copy (which might indicate some potential evolutionary significance of a concatemeric RNA segment), however multiple (n=40) passages generated no changes in the sequence of either the upstream or downstream segments. Further, we present results that demonstrate the presence of concatemers or partial gene duplications in multiple segments of different orbiviruses (in tissue culture and purified virus), suggesting their generation is likely to be a normal feature of orbivirus replication.

The evolution of two homologues of the core protein VP6 of epizootic haemorrhagic disease virus (EHDV), which correspond to the geographical origin of the virus.

Epizootic haemorrhagic disease virus is a 10-segmented, double-stranded RNA virus. When these ten segments of dsRNA are run on 1% agarose, eastern (Australia, Japan) and western (North America, Africa, Middle-East) strains of the virus can be separated phenotypically based on the migration of genome segments 7-9. In western strains, segments 7-9 are roughly the same size and co-migrate as a single RNA band. In eastern strains, segment 9 is smaller, so while segments 7 and 8 co-migrate, the segment 9 RNA runs faster than its western homologue. Translation experiments demonstrated that these two segment 9 homologues are both functional and produce proteins (VP6) of different sizes-something that has not been reported in any other orbivirus species to date. Sequence analysis suggests that eastern and western versions of segment 9 (VP6) have likely evolved as a response to adaptive selection in different geographical regions via gene duplication and subsequent mutation. These significant findings are considered unusual given the conserved nature of VP6 and its presumed role as the viral helicase. It is not currently known what the biological relevance of each homologue is to the virus.

Genetic and phylogenetic analysis of the outer-coat proteins VP2 and VP5 of epizootic haemorrhagic disease virus (EHDV): comparison of genetic and serological data to characterise the EHDV serogroup.

The outer-coat proteins, VP2 and VP5, of epizootic haemorrhagic disease virus (EHDV) are important for host cell binding during the initiation of infection. They are also known to determine virus serotype. This study presents a complete genetic and phylogenetic analysis of these proteins (and the genes that code for them) to allow comparison of the selective pressures acting on each and the correlation of genetic sequence data with serotype. Accession numbers, gene and protein sizes, ORF positions, G+C contents, terminal hexanucleotides, start and stop codons and phylogenetic relationships are all presented. The results show that VP2 is highly variable, is under great pressure to adapt and can be correlated with serotype. While also variable, VP5 appears to be under less adaptive pressure than VP2 but still shows some correlation with serotype. Seven serotypes of EHDV have been defined in this study, although the results do show that some serotypes are extremely closely related--and highlight the benefit of using both molecular and serologic analyses. Analysis of the terminal hexanucleotides showed that the 5' terminus is under greater purifying selection than the 3'. Evidence is also presented that both segments 2 and 6 (coding for VP2 and VP5 respectively) have grown via gene duplication and subsequent mutation.

The pathology and pathogenesis of bluetongue.

Bluetongue (BT) is an insect-transmitted viral disease of wild and domestic ruminants and, occasionally, other species. Amongst domestic livestock, BT is most common in certain breeds of sheep whereas asymptomatic BT virus (BTV) infection of cattle is typical in enzootic regions. BT in cattle can be a feature of specific outbreaks, notably the current epizootic in Europe caused by BTV serotype 8. BTV replicates within mononuclear phagocytic and endothelial cells, lymphocytes and possibly other cell types in lymphoid tissues, the lungs, skin and other tissues. Infected ruminants may exhibit a prolonged but not persistent viraemia and BTV is associated with erythrocytes during the late stages of this prolonged viraemia. The pathogenesis of BT involves injury to small blood vessels in target tissues, but the relative contributions of direct virus-induced cytolysis and virus-induced vasoactive mediators in causing endothelial injury and dysfunction are presently unclear. The lesions of BT are characteristic and include: haemorrhage and ulcers in the oral cavity and upper gastrointestinal tract; necrosis of skeletal and cardiac muscle; coronitis; subintimal haemorrhage in the pulmonary artery; oedema of the lungs, ventral subcutis, and fascia of the muscles of the neck and abdominal wall; and pericardial, pleural and abdominal effusions. Transplacental transmission of BTV in ruminants, with subsequent fetal infection, is a property of specific virus strains, especially those propagated in embryonated eggs or cell culture. The outcome of BTV infection of fetal ruminants is age-dependent, with distinctive cavitating lesions of the central nervous system in animals that survive infection in early gestation. Immune competence to BTV arises by mid-gestation, and animals infected in late gestation can be born viraemic and without significant brain malformations.

Clinical signs and pathology shown by British sheep and cattle infected with bluetongue virus serotype 8 derived from the 2006 outbreak in northern Europe.

Four poll Dorset sheep and four Holstein-Friesian cattle were infected with the northern European strain of bluetongue virus (BTV), BTV-8, to assess its pathogenicity in UK breeds. The time course of infection was monitored in both species by using real-time reverse transcriptase-PCR (RT-PCR), conventional RT-PCR and serology. Two of the sheep developed severe clinical signs that would have been fatal in the field; the other two were moderately and mildly ill, respectively. The cattle were clinically unaffected, but had high levels of viral RNA in their bloodstream. Real-time RT-PCR detected viral RNA as early as one day after infection in the cattle and three days after infection in the sheep. Antibodies against BTV were detected by six days after infection in the sheep and eight days after infection in the cattle. Postmortem examinations revealed pathology in the cattle that was more severe than suggested by the mild clinical signs, but the pathological and clinical findings in the sheep were more consistent.

Development and initial evaluation of a real-time RT-PCR assay to detect bluetongue virus genome segment 1.

Since 1998, multiple strains of bluetongue virus (BTV), belonging to six different serotypes (types 1, 2, 4, 8, 9 and 16) have caused outbreaks of disease in Europe, causing one of the largest epizootics of bluetongue ever recorded, with the deaths of >1.8 million animals (mainly sheep). The persistence and continuing spread of BTV in Europe and elsewhere highlights the importance of sensitive and reliable diagnostic assay systems that can be used to rapidly identify infected animals, helping to combat spread of the virus and disease. BTV has a genome composed of 10 linear segments of dsRNA. We describe a real-time RT-PCR assay that targets the highly conserved genome segment 1 (encoding the viral polymerase--VP1) that can be used to detect all of the 24 serotypes, as well as geographic variants (different topotypes) within individual serotypes of BTV. After an initial evaluation using 132 BTV samples including representatives of all 24 BTV serotypes, this assay was used by the European Community Reference Laboratory (CRL) at IAH Pirbright to confirm the negative status of 2,255 animals imported to the UK from regions that were considered to be at risk during the 2006 outbreak of BTV-8 in Northern Europe. All of these animals were also negative by competition ELISA to detect BTV specific antibodies and none of them developed clinical signs of infection. These studies have demonstrated the value of the assay for the rapid screening of field samples.

A duplex RT-PCR assay for detection of genome segment 7 (VP7 gene) from 24 BTV serotypes.

Since 1998, six distinct serotypes of Bluetongue virus (BTV) have invaded Southern and Central Europe, persisting in some regions for up to 6 years and resulting in the deaths of >1.8 million sheep. Rapid and reliable methods of virus detection and identification play an essential part in our fight against bluetongue disease (BT). We have therefore developed and evaluated a duplex, one-step RT-PCR assay that detects genome segment 7 (encoding the major serogroup (virus-species) specific antigen and outer-core-protein VP7) from any of the 24 BTV serotypes. Although Seg-7 is highly conserved, there are sequence differences in the near terminal regions that identify two distinct phylogenetic groups. Two sets of primers (targeting Seg-7 terminal regions of viruses from these two groups) were included in a duplex RT-PCR assay system. Assay sensitivity was evaluated using tissue culture derived virus, infected vector insects and clinical samples (blood and other tissues). The assay reliably amplified Seg-7 from any of the BTV strains tested, including isolates of the 24 BTV serotypes and isolates from different geographic origins. No cross-reactions were detected with members of closely related Orbivirus species (African horsesickness virus (AHSV), Epizootic haemorrhagic disease virus (EHDV), Equine encephalosis virus (EEV) and Palyam virus (PALV)).