Bluetongue (BT) is a severe and economically important disease of ruminants that is widely distributed around the world, caused by the bluetongue virus (BTV). More than 28 different BTV serotypes have been identified in serum neutralisation tests (SNT), which, along with geographic variants (topotypes) within each serotype, reflect differences in BTV outer-capsid protein VP2. VP2 is the primary target for neutralising antibodies, although the basis for cross-reactions and serological variations between and within BTV serotypes is poorly understood. Recombinant BTV VP2 proteins (rVP2) were expressed in Nicotiana benthamiana, based on sequence data for isolates of thirteen BTV serotypes (primarily from Europe), including three ‘novel’ serotypes (BTV-25, -26 and -27) and alternative topotypes of four serotypes. Cross-reactions within and between these viruses were explored using rabbit anti-rVP2 sera and post BTV-infection sheep reference-antisera, in I-ELISA (with rVP2 target antigens) and SNT (with reference strains of BTV-1 to -24, -26 and -27). Strong reactions were generally detected with homologous rVP2 proteins or virus strains/serotypes. The sheep antisera were largely serotype-specific in SNT, but more cross-reactive by ELISA. Rabbit antisera were more cross-reactive in SNT, and showed widespread, high titre cross-reactions against homologous and heterologous rVP2 proteins in ELISA. Results were analysed and visualised by antigenic cartography, showing closer relationships in some, but not all cases, between VP2 topotypes within the same serotype, and between serotypes belonging to the same ‘VP2 nucleotype’.

Summary: This review discusses the varying roles that have been played by many plant‐viral regulatory sequences and proteins in the creation of plant‐based expression systems and virus particles for use in nanotechnology. Essentially, there are two ways of expressing an exogenous protein: the creation of transgenic plants possessing a stably integrated gene construction, or the transient expression of the desired gene following the infiltration of the gene construct. Both depend on disarmed strains of Agrobacterium tumefaciens to deliver the created gene construction into cell nuclei, usually through the deployment of virus‐derived components. The importance of efficient mRNA translation in the latter process is highlighted. Plant viruses replicate to sustain an infection to promote their survival. The major product of this, the virus particle, is finding increasing roles in the emerging field of bionanotechnology. One of the major products of plant‐viral expression is the virus‐like particle (VLP). These are increasingly playing a role in vaccine development. Similarly, many VLPs are suitable for the investigation of the many facets of the emerging field of synthetic biology, which encompasses the design and construction of new biological functions and systems not found in nature. Genetic and chemical modifications to plant‐generated VLPs serve as ideal starter templates for many downstream synthetic biology applications. Contents Summary 16 I. Transgenic gene expression 16 II. Transient gene expression 18 III. Exploitation of viral shells as templates for synthetic biological applications 20 IV. Conclusion and future prospects 23 Acknowledgements 24 References 24

The production of plant helical virus-like particles (VLPs) via plant-based expression has been problematic with previous studies suggesting that an RNA scaffold may be necessary for their efficient production. To examine this, we compared the accumulation of VLPs from two potexviruses, papaya mosaic virus and alternanthera mosaic virus (AltMV), when the coat proteins were expressed from a replicating potato virus X- based vector (pEff) and a non-replicating vector (pEAQ-HT). Significantly greater quantities of VLPs could be purified when pEff was used. The pEff system was also very efficient at producing VLPs of helical viruses from different virus families. Examination of the RNA content of AltMV and tobacco mosaic virus VLPs produced from pEff revealed the presence of vector-derived RNA sequences, suggesting that the replicating RNA acts as a scaffold for VLP assembly. Cryo-EM analysis of the AltMV VLPs showed they had a structure very similar to that of authentic potexvirus particles. Thus, we conclude that vectors generating replicating forms of RNA, such as pEff, are very efficient for producing helical VLPs.

Geminivirus particles, consisting of a pair of twinned isometric structures, have one of the most distinctive capsids in the virological world. Until recently, there was little information as to how these structures are generated. To address this, we developed a system to produce capsid structures following the delivery of geminivirus coat protein and replicating circular single-stranded DNA (cssDNA) by the infiltration of gene constructs into plant leaves. The transencapsidation of cssDNA of the Begomovirus genus by coat protein of different geminivirus genera was shown to occur with full-length but not half-length molecules. Double capsid structures, distinct from geminate capsid structures, were also generated in this expression system. By increasing the length of the encapsidated cssDNA, triple geminate capsid structures, consisting of straight, bent and condensed forms were generated. The straight geminate triple structures generated were similar in morphology to those recorded for a potato-infecting virus from Peru. These finding demonstrate that the length of encapsidated DNA controls both the size and stability of geminivirus particles.

Turnip crinkle virus (TCV), a member of the genus carmovirus of the Tombusviridae family, has a genome consisting of a single positive-sense RNA molecule that is encapsidated in an icosahedral particle composed of 180 copies of a single type of coat protein. We have employed the CPMV-HT transient expression system to investigate the formation of TCV-like particles following the expression of the wild-type coat protein or modified forms of it that contain either deletions and/or additions insertions. Transient expression of the coat protein in plants results in the formation of capsid structures that morphologically resemble TCV virions (T=3 structure) but encapsidate heterogeneous cellular RNAs, rather than the specific TCV coat protein messenger RNA. Expression of an amino-terminal deleted form of the coat protein resulted in the formation of smaller T=1 structures that are free of RNA. The possibility of utilising TCV as a carrier for the presentation of foreign proteins on the particle surface was also explored by fusing the sequence of GFP to the C-terminus of the coat protein. The expression of coat protein-GFP hybrids permitted the formation of VLPs but the yield of particles is diminished compared to the yield obtained with unmodified coat protein. Our results confirm the importance of the N-terminus of the coat protein for the encapsidation of RNA and show that the coat protein’s exterior P domain plays a key role in particle formation.

Bluetongue is an economically important disease of domesticated and wild ruminants caused by bluetongue virus (BTV). There are at least 36 different serotypes of BTV (the identity of which is determined by its outer-capsid protein VP2), most of which are transmitted by Culicoides biting midges. IFNAR^(−/−) mice immunised with plant-expressed outer-capsid protein VP2 (rVP2) of BTV serotypes -1, -4 or -8, or the smaller outer-capsid protein rVP5 of BTV-10, or mock-immunised with PBS, were subsequently challenged with virulent strains of BTV-4 or BTV-8, or with an attenuated clone of BTV-1 (BTV-1RG_C7). The mice that had received rVP2 generated a protective immune response against the homologous BTV serotype, reducing viraemia (as detected by qRT-PCR), the severity of clinical signs and mortality levels. No cross-serotype protection was observed after challenge with the heterologous BTV serotypes. However, the severity of clinical signs, viraemia and fatality levels after challenge with the attenuated strain of BTV-1 were all increased in mice immunised with rVP2 of BTV-4 and BTV-8, or with rVP5 of BTV10. The possibility is discussed that non-neutralising antibodies, reflecting serological relationships between the outer-capsid proteins of these different BTV serotypes, could lead to ‘antibody-dependent enhancement of infection’ (ADE). Such interactions could affect the epidemiology and emergence of different BTV strains in the field and would therefore be relevant to the design and implementation of vaccination campaigns.