Publications

Bluetongue virus (BTV) is the ‘type’ species of the genus Orbivirus within the family Reoviridae. The BTV genome is composed of ten linear segments of double-stranded RNA (dsRNA), each of which codes for one of ten distinct viral proteins. Previous phylogenetic comparisons have evaluated variations in genome segment 3 (Seg-3) nucleotide sequence as way to identify the geographical origin (different topotypes) of BTV isolates. The full-length nucleotide sequence of genome Seg-3 was determined for thirty BTV isolates recovered in the eastern Mediterranean region, the Balkans and other geographic areas (Spain, India, Malaysia and Africa). These data were compared, based on molecular variability, positive-selection-analysis and maximum-likelihood phylogenetic reconstructions (using appropriate substitution models) to 24 previously published sequences, revealing their evolutionary relationships. These analyses indicate that negative selection is a major force in the evolution of BTV, restricting nucleotide variability, reducing the evolutionary rate of Seg-3 and potentially of other regions of the BTV genome. Phylogenetic analysis of the BTV-4 strains isolated over a relatively long time interval (1979–2000), in a single geographic area (Greece), showed a low level of nucleotide diversity, indicating that the virus can circulate almost unchanged for many years. These analyses also show that the recent incursions into south-eastern Europe were caused by BTV strains belonging to two different major-lineages: representing an ‘eastern’ (BTV-9, -16 and -1) and a ‘western’ (BTV-4) group/topotype. Epidemiological and phylogenetic analyses indicate that these viruses originated from a geographic area to the east and southeast of Greece (including Cyprus and the Middle East), which appears to represent an important ecological niche for the virus that is likely to represent a continuing source of future BTV incursions into Europe.

Bluetongue virus serotype 8 (BTV-8) has caused a major outbreak of disease in cattle and sheep in several countries across northern and western Europe from 2006 to 2008. In 2008 the European Union instigated a mass-vaccination programme in affected countries using whole virus inactivated vaccines. We evaluated vaccinal responses in sheep and the ability of the vaccine to protect against experimental challenge. Sheep vaccinated 10 months previously under field conditions were challenged with BTV-8. One of 7 vaccinated sheep became infected, as evidenced by detection of viral RNA by real-time RT-PCR and by virus isolation. The remaining 6 sheep appeared fully protected from virus replication. None of the vaccinated sheep showed clinical signs of BTV and there was a good correlation between the presence of neutralising antibodies on challenge and protection. Commercially available ELISAs were evaluated for their ability to detect antibodies in sheep vaccinated on a single occasion. The sandwich (double antigen) ELISA assays were found to be more sensitive at detecting antibodies in vaccinated sheep than the competitive ELISAs.

In 2007, serological evidence for foot-and-mouth disease (FMD) infection was found as a result of differential diagnostic testing of Cypriot sheep suspected to be infected with bluetongue or contagious ecthyma. Seropositive sheep and goats were subsequently uncovered on ten geographically clustered flocks, while cattle and pigs in neighbouring herds were all seronegative. These antibodies were specific for serotype-O FMD virus, reacting with both structural and non-structural (NS) FMD viral proteins. However, no FMD virus could be recovered from the seropositive flocks. FMD had not been recorded in Cyprus since 1964 and there has been no vaccination programme since 1984. Since all the seropositive animals were at least 3 years old and home-bred, it was concluded that infection had occurred approximately 3 years previously had passed un-noticed and died out spontaneously. It therefore appears that antibodies to FMD virus NS proteins can still be detected around 3 years after infection of small ruminants, but that virus carriers cannot be detected at this time. This unusual situation of finding evidence of historical infection in a FMD-free country caused considerable disruption and alarm and posed questions about the definition of what constitutes a FMD outbreak.

Eight colostrum-deprived calves were inoculated intranasally with a non-cytopathic strain of bovine viral diarrhoea Virus (BVDV) genotype-1 and killed in batches of two at 3, 6, 9 and 14 days post-inoculation (dpi). Two non-inoculated animals with similar background served as controls. All infected calves developed mild pyrexia and transient leucopenia due primarily to lymphopenia. Viraemia was correlated with body temperature and inversely related to leucocyte count. Ileal Peyer's patches developed mild follicular lymphoid depletion from 3 dpi. This change was accompanied by cellular fragmentation and pyknosis, characteristic of apoptosis, which was most prominent from 6 dpi. Lymphocyte apoptosis was confirmed by ultrastructural examination. Stellate cells and macrophages located in the lymphoid follicles were identified as infected by virus From 3 dpi and the number of these infected cells increased until 9 dpi. Fewer lymphocytes expressed BVDV antigen. Macrophages had morphological features consistent with activation of secretory and phagocytic function from 3 dpi. These findings suggest that BVDV is only directly responsible for the destruction of a small number of lymphocytes. Although lymphocyte infection coincided with the onset of apoptosis, the intensity of infection was disproportionate to the marked depletion of gut-associated lymphoid tissue, particularly during the early stages of this process. Characterization of the indirect pathogenic mechanisms involved in the lymphoid depletion associated with BVDV infection will require additional study.