Bluetongue Virus (BTV)
Bluetongue disease is a non-contagious, insect-borne, viral disease of ruminants, mainly sheep and less frequently cattle, goats, buffalo, deer, dromedaries and antelope. First identified in South Africa, the virus has spread rapidly throughout the world and is now found wherever its vector, the biting midge, thrives.
Bluetongue disease is particularly dangerous in sheep, with high morbidity and mortality. Over 27 different types (serotypes) of this virus are now recognised; unfortunately, recovery does not result in significant cross-protective immunity. Therefore, identification of the specific serotype is essential when diagnosing an outbreak.
In 2014, the BBSRC awarded a grant to Professor George Lomonossoff in collaboration with the Pirbright Institute to develop a Bluetongue diagnostic. The Hypertrans® system is an essential tool in reconstructing fully assembled and immunogenic BTV virus-like particles (VLPs) for use in the rapid screening of differential serotypes. These plant-produced particles have subsequently been developed into a full vaccine which has been shown to be able to protect sheep against Bluetongue disease. Work in this area has resulted in the filing of several patents.
The BTV particle consists of ten strands of double-stranded RNA surrounded by two outer capsid protein shells, VP2 and VP5, which mediate attachment and penetration of the virus into the target cell.
Co-expression in N. Benthamiana of the cowpea mosaic virus (CPMV) coat protein precursor along with the 24K proteinase using pEAQ vectors results in the efficient assembly and accumulation of empty virus-like particles (eVLPs) comprised of 60 copies each of the processed large and small coat proteins. (Fig. 2a)
In addition to the absence of RNA, the previous limitations on coat protein inserts imposed by compatibility with virus replication and movement have also been removed. Furthermore, the interior space is now available for encapsulation of various payloads and the synthetic reconstructions of CPMV eVLPs also revealed a hitherto unsuspected role of the small coat protein C-terminus in virus assembly.
In assembling the Bluetongue virus VLPs, the four structural proteins are simultaneously expressed and optimised by varying the expression levels between the proteins to favour the accumulation of fully assembled particles over assembly intermediates. (Fig. 2b).
This fine tuning is achieved by engineering the Hypertrans® system to reduce overexpression of BTV VP3 relative to the other coat proteins.
Expression cassettes derived from the Hypertrans® system have now been developed for modulating expression across a range of levels that will enable further fine tuning for the construction of complex macromolecular assemblies as well as for the synthetic reconstruction of metabolic pathways.