Mark Boyce

Prospects for improved bluetongue vaccines.

Bluetongue has been recognized as a viral disease of livestock for more than 100 years. Repeated incursions of Bluetongue into Europe since 1998 have been particularly devastating for highly sensitive European fine-wool sheep breeds, and have resulted in a resurgence of interest in vaccine manufacture. Fortunately, the virus and its serology are well understood and vaccination prevents the disease. However, current vaccines are not without their problems, and many new approaches are being tested to improve the safety and breadth of protection afforded. This Review describes the leading technologies for improved bluetongue vaccines and looks ahead to how advances in other viral vaccines might be applied to this disease.

Generation of Replication-Defective Virus-Based Vaccines That Confer Full Protection in Sheep against Virulent Bluetongue Virus Challenge

ABSTRACT The reverse genetics technology for bluetongue virus (BTV) has been used in combination with complementing cell lines to recover defective BTV-1 mutants. To generate a potential disabled infectious single cycle (DISC) vaccine strain, we used a reverse genetics system to rescue defective virus strains with large deletions in an essential BTV gene that encodes the VP6 protein (segment S9) of the internal core. Four VP6-deficient BTV-1 mutants were generated by using a complementing cell line that provided the VP6 protein in trans. Characterization of the growth properties of mutant viruses showed that each mutant has the necessary characteristics for a potential vaccine strain: (i) viral protein expression in noncomplementing mammalian cells, (ii) no infectious virus generated in noncomplementing cells, and (iii) efficient replication in the complementing VP6 cell line. Further, a defective BTV-8 strain was made by reassorting the two RNA segments that encode the two outer capsid proteins (VP2 and VP5) of a highly pathogenic BTV-8 with the remaining eight RNA segments of one of the BTV-1 DISC viruses. The protective capabilities of BTV-1 and BTV-8 DISC viruses were assessed in sheep by challenge with specific virulent strains using several assay systems. The data obtained from these studies demonstrated that the DISC viruses are highly protective and could offer a promising alternative to the currently available attenuated and killed virus vaccines and are also compliant as DIVA (differentiating infected from vaccinated animals) vaccines.

Bluetongue virus non-structural protein 1 is a positive regulator of viral protein synthesis

BackgroundBluetongue virus (BTV) is a double-stranded RNA (dsRNA) virus of the Reoviridae family, which encodes its genes in ten linear dsRNA segments. BTV mRNAs are synthesised by the viral RNA-dependent RNA polymerase (RdRp) as exact plus sense copies of the genome segments. Infection of mammalian cells with BTV rapidly replaces cellular protein synthesis with viral protein synthesis, but the regulation of viral gene expression in the Orbivirus genus has not been investigated.ResultsUsing an mRNA reporter system based on genome segment 10 of BTV fused with GFP we identify the protein characteristic of this genus, non-structural protein 1 (NS1) as sufficient to upregulate translation. The wider applicability of this phenomenon among the viral genes is demonstrated using the untranslated regions (UTRs) of BTV genome segments flanking the quantifiable Renilla luciferase ORF in chimeric mRNAs. The UTRs of viral mRNAs are shown to be determinants of the amount of protein synthesised, with the pre-expression of NS1 increasing the quantity in each case. The increased expression induced by pre-expression of NS1 is confirmed in virus infected cells by generating a replicating virus which expresses the reporter fused with genome segment 10, using reverse genetics. Moreover, NS1-mediated upregulation of expression is restricted to mRNAs which lack the cellular 3′ poly(A) sequence identifying the 3′ end as a necessary determinant in specifically increasing the translation of viral mRNA in the presence of cellular mRNA.ConclusionsNS1 is identified as a positive regulator of viral protein synthesis. We propose a model of translational regulation where NS1 upregulates the synthesis of viral proteins, including itself, and creates a positive feedback loop of NS1 expression, which rapidly increases the expression of all the viral proteins. The efficient translation of viral reporter mRNAs among cellular mRNAs can account for the observed replacement of cellular protein synthesis with viral protein synthesis during infection.

Rapid Generation of Replication-Deficient Monovalent and Multivalent Vaccines for Bluetongue Virus: Protection against Virulent Virus Challenge in Cattle and Sheep

ABSTRACT Since 1998, 9 of the 26 serotypes of bluetongue virus (BTV) have spread throughout Europe, and serotype 8 has suddenly emerged in northern Europe, causing considerable economic losses, direct (mortality and morbidity) but also indirect, due to restriction in animal movements. Therefore, many new types of vaccines, particularly subunit vaccines, with improved safety and efficacy for a broad range of BTV serotypes are currently being developed by different laboratories. Here we exploited a reverse genetics-based replication-deficient BTV serotype 1 (BTV-1) (disabled infectious single cycle [DISC]) strain to generate a series of DISC vaccine strains. Cattle and sheep were vaccinated with these viruses either singly or in cocktail form as a multivalent vaccine candidate. All vaccinated animals were seroconverted and developed neutralizing antibody responses to their respective serotypes. After challenge with the virulent strains at 21 days postvaccination, vaccinated animals showed neither any clinical reaction nor viremia. Further, there was no interference with protection with a multivalent preparation of six distinct DISC viruses. These data indicate that a very-rapid-response vaccine could be developed based on which serotypes are circulating in the population at the time of an outbreak.

Multigene expression of protein complexes by iterative modification of genomic Bacmid DNA

BackgroundMany cellular multi-protein complexes are naturally present in cells at low abundance. Baculovirus expression offers one approach to produce milligram quantities of correctly folded and processed eukaryotic protein complexes. However, current strategies suffer from the need to produce large transfer vectors, and the use of repeated promoter sequences in baculovirus, which itself produces proteins that promote homologous recombination. One possible solution to these problems is to construct baculovirus genomes that express each protein in a complex from a separate locus within the viral DNA. However current methods for selecting such recombinant genomes are too inefficient to routinely modify the virus in this way.ResultsThis paper reports a method which combines the lambda red and bacteriophage P1 Cre-recombinase systems to efficiently generate baculoviruses in which protein complexes are expressed from multiple, single-locus insertions of foreign genes. This method is based on an 88 fold improvement in the selection of recombinant viruses generated by red recombination techniques through use of a bipartite selection cassette. Using this system, seven new genetic loci were identified in the AcMNPV genome suitable for the high level expression of recombinant proteins. These loci were used to allow the recovery two recombinant virus-like particles with potential biotechnological applications (influenza A virus HA/M1 particles and bluetongue virus VP2/VP3/VP5/VP7 particles) and the mammalian chaperone and cancer drug target CCT (16 subunits formed from 8 proteins).Conclusion1. Use of bipartite selections can significantly improve selection of modified bacterial artificial chromosomes carrying baculovirus DNA. Furthermore this approach is sufficiently robust to allow routine modification of the virus genome. 2. In addition to the commonly used p10 and polyhedrin loci, the ctx, egt, 39k, orf51, gp37, iap2 and odv-e56 loci in AcMNPV are all suitable for the high level expression of heterologous genes. 3. Two protein, four protein and eight protein complexes including virus-like particles and cellular chaperone complexes can be produced using the new approach.

Reconstitution of bluetongue virus polymerase activity from isolated domains based on a three-dimensional structural model.

Abstract Bluetongue virus (BTV) is a double‐stranded RNA virus of the Reoviridae family. The VP1 protein of BTV is the viral RNA‐dependent RNA polymerase (RdRp), which is responsible for the replication of the viral genome. Currently there is no structural information available for VP1. By manual alignment of BTV, Reovirus and other viral RdRps we have generated a model for the structure of VP1, the RdRp of BTV. The structure can be divided into three domains: an N‐terminal domain, a C‐terminal domain, and a central polymerase domain. Mutation of the putative catalytic site in the central polymerase domain by site‐directed mutagenesis abrogated in vitro replicase activity. Each of the domains was expressed individually and subsequently partially purified to obtain direct evidence for the location of polymerase activity and the nucleoside triphosphate binding site. The nucleoside triphosphate binding site was located by showing that CTP only bound to the full‐length protein or to the polymerase domain and not to either of the other two domains. None of the domains had catalytic activity when tested individually or in tandem but when all three domains were mixed together the RdRp activity was reconstituted. This is the first report of the reconstitution of a functional viral RdRp in vitro from individual domains.

Rapid mapping of functional cis-acting RNA elements by recovery of virus from a degenerate RNA population: application to genome segment 10 of bluetongue virus.

The regulatory elements which control the processes of virus replication and gene expression in the Orbivirus genus are uncharacterized in terms of both their locations within genome segments and their specific functions. The reverse genetics system for the type species, Bluetongue virus, has been used in combination with RNA secondary structure prediction to identify and map the positions of cis-acting regions within genome segment 10. Through the simultaneous introduction of variability at multiple nucleotide positions in the rescue RNA population, the functional contribution of these positions was used to map regions containing cis-acting elements essential for virus viability. Nucleotides that were individually lethal when varied mapped within a region of predicted secondary structure involving base pairing between the 5 and 3 ends of the transcript. An extended region of predicted perfect base pairing located within the 3 untranslated region of the genome segment was also found to be required for virus viability. In contrast to the identification of individually lethal mutations, gross alteration of the composition of this predicted stem region was possible, providing the base-pairing potential between the two strands was maintained, identifying a structural feature predicted to be conserved throughout the Orbivirus genus. The approach of identifying cis-acting sequences through sequencing the recovered virus following the rescue of a degenerate RNA population is broadly applicable to viruses where reverse genetics is available.

Inter-segment complementarity in orbiviruses: a driver for co-ordinated genome packaging in the Reoviridae?

The process by which eukaryotic viruses with segmented genomes select a complete set of genome segments for packaging into progeny virus particles is not understood. In this study a model based on the association of genome segments through specific RNA-RNA interactions driven by base pairing was formalized and tested in the Orbivirus genus of the Reoviridae family. A strategy combining screening of the genomic sequences for inter-segment complementarity with direct functional testing of inter-segment RNA-RNA interactions using reverse genetics is described in the type species of the Orbivirus genus, Bluetongue virus (BTV). Two examples, involving four of the ten BTV genomic segments, of specific inter-segment interaction motifs whose maintenance is essential for the generation of infectious virus, were identified. Equivalent inter-segment complementarities were found between the identified regions of the orthologous genome segments of all orbiviruses, including phylogenetically distant species. Specific interaction of the participating RNA segments was confirmed in vitro using electrophoretic mobility shift assays, with the interactions inhibited using oligonucleotides complementary to the interaction motif of one of the interacting partners, and also through mutagenesis of the motifs. In each example, the base pairing rather than the absolute sequence was critical to the formation of a functional inter-segment interaction, with mutations only being tolerated in rescued virus if compensating changes were made in the interacting partner to restore uninterrupted base pairing. The absolute sequence of the complementarity motifs varied between species, indicating that this newly identified phenomenon may contribute to the observed lack of reassortment between Orbivirus species.

Identification of the Genome Segments of Bluetongue Virus Serotype 26 (Isolate KUW2010/02) that Restrict Replication in a Culicoides sonorensis Cell Line (KC Cells)

Bluetongue virus (BTV) can infect most ruminant species and is usually transmitted by adult, vector-competent biting midges (Culicoides spp.). Infection with BTV can cause severe clinical signs and can be fatal, particularly in naïve sheep and some deer species. Although 24 distinct BTV serotypes were recognized for several decades, additional ‘types’ have recently been identified, including BTV-25 (from Switzerland), BTV-26 (from Kuwait) and BTV-27 from France (Corsica). Although BTV-25 has failed to grow in either insect or mammalian cell cultures, BTV-26 (isolate KUW2010/02), which can be transmitted horizontally between goats in the absence of vector insects, does not replicate in a Culicoides sonorensis cell line (KC cells) but can be propagated in mammalian cells (BSR cells). The BTV genome consists of ten segments of linear dsRNA. Mono-reassortant viruses were generated by reverse-genetics, each one containing a single BTV-26 genome segment in a BTV-1 genetic-background. However, attempts to recover a mono-reassortant containing genome-segment 2 (Seg-2) of BTV-26 (encoding VP2), were unsuccessful but a triple-reassortant was successfully generated containing Seg-2, Seg-6 and Seg-7 (encoding VP5 and VP7 respectively) of BTV-26. Reassortants were recovered and most replicated well in mammalian cells (BSR cells). However, mono-reassortants containing Seg-1 or Seg-3 of BTV-26 (encoding VP1, or VP3 respectively) and the triple reassortant failed to replicate, while a mono-reassortant containing Seg-7 of BTV-26 only replicated slowly in KC cells.