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Development of RT-PCR and phylogenetic sequence analyses for bluetongue virus serotype and strain identification - SE2612

British breeds of sheep, horses and other livestock species are among the most susceptible to diseases caused by the orbiviruses, including particularly Bluetongue (BT) and African horse sickness (AHS), wih mortality levels that may exceed 70% (BT in sheep) to 99% (AHS in horses). In the absence of any immunity in the serologically naive animal populations in the UK, any outbreak of these diseases here would cause a catastrophic mortality levels in Summer, which (particulary with global climate change) may not be curtailed by our cold winters. The primary strategy for control of Bluetongue and related orbivirus diseases is to prevent their entry into the UK. The work of this project underpins UK policy through maintenance of a diagnostic capability and the development of test systems that can identify virus type and strain more rapidly and precisely than before. These assays will generate epidemiological data that will provide a more accurate picture of the movement and persistence of BTV strains. The project will also evaluate the frequency and significance of genome segment reassortment in the field, and the role of genome segment 10 in recruitment of a novel European vector species.
The major aim of this research is to develop rapid nucleotide sequence based methods that can be used to conclusively identify individual bluetongue virus (BTV) isoltes. RT-PCR and sequencing methods will be established within our laboratory and used to generate a database of BTV genome segment 2 sequences (encoding he serotype specific outer capsid protein, VP2) for different BTV strains and serotypes. This will provide a resource that will allow us to analyse and identify new virus isolates by comparison to the nucleotide sequences of reference virus strains. This will specifically allow us to identify the serotype of new virus isolates ( essential for the design of vaccination strategies), more rapidly and reliably than before. However, the data provided by this approach will also distinguish between individual strains and lineages within a single BTV serotype, identifying the most likely origins of virus strains and thei global movements (including any vaccine strains whcih might persist in the field). These epidemiological data cannot be derived from the serological methods that are currently used for BTV detection and indentification. detailed comparisons of conserved and variable regions of genome segment 2 will help us to design and refine primers for use in RT-PCR based serotyping assays, making initial serotype identification possible within a matter of hours, rather than the weeks currently required for conclusive identification by serum neutralisation assays.
Live BTV vaccines originally developed for local use in South Africa, are currently being used to......(text missing from CSG7)...concerning the safety of these vaccines, and their effectiveness for use in a European eradication campaign, rather than simply for protection of the individual vaccinated animal. By analysing the electrophorectic migration patterns of individual BTV genome segments, using polyacrylamide gel electrophoresis and sequencing, we anticipate that it will be possible to identify genome segments derived either from vaccine strains or from different wild type viruses. Uisng these methods the project will examine the transmission and survival of BTV vaccine strains and the ability of such viruses to reassort (exchange genome segments) with wild type viruses in the field.
In most of Mediterranean Europe and north Africa, the current outbreaks of BT are thought to be transmitted by the known vector species Culicoides imicola. However, BTV has been transmitted and has even survived through the winter, in more northerly regions of Europe (Bulgaria, Serbia, Macedonia and Crotia) despite the absence of C. imicola, suggesting the involvement of a novel vector species. The predominanat Culicoides species in these regions are C. pulicaris and C. obsoletus, both of which are spread across much of northern Europe, including the UK. I appears likely that the use of a novel insect vector would allow BTV to continue its spread further north, posing a major threat to UK livestock. Genome segment 10 of BTV and related orbiviruses encodes a small non-structural membrane protein NS3 that is involved in the releasen of virus particles from insect cells. Our recent studies have indicated that variations in NS3 can affect the virus`s ability to cause a fully disseminated infection in the insect, and therefore both the efficiency of infection of the salivary gland and vector competence. The project will explore the hypothesis that variations in genome segment 10 (encoding NS3) are involved in the utilisation of a novel BTV vector species in Europe and attempt to identify the nature of these changes.
Project Documents
• Final Report : Development of RT-PCR and phylogenetic sequence analyses for bluetongue virus serotype and strain identification   (339k)
Time-Scale and Cost
From: 2003

To: 2006

Cost: £539,663
Contractor / Funded Organisations
Institute for Animal Health (BBSRC)
Animal Diseases              
Animal Health              
Molecular Biology              
Plants and Animals              
Fields of Study
Animal Health