Meredith Stewart

Protective efficacy of Bluetongue virus-like and subvirus-like particles in sheep: presence of the serotype-specific VP2, independent of its geographic lineage, is essential for protection.

There have been multiple separate outbreaks of Bluetongue (BT) disease of ruminants in Europe since 1998, often entering via the Mediterranean countries of Italy, Spain and Greece. BT is caused by an orbivirus, Bluetongue virus (BTV), a member of the family Reoviridae. BTV is a non-enveloped double-capsid virus, which encodes 7 structural proteins (VP1-VP7) and several non-structural proteins (NS1, NS2, NS3/3a and NS4) from ten double-stranded RNA segments of the genome. In this report, we have prepared BTV virus-like particles (VLPs, composed of VP2, VP3, VP5 and VP7) and sub-viral, inner core-like particles (CLPs, VP3 and VP7) using a recombinant baculovirus expression system. We compared the protective efficacy of VLPs and CLPs in sheep and investigated the importance of geographical lineages of BTV in the development of vaccines. The Greek crossbred Karagouniko sheep, which display mild to sub-clinical BT, were vaccinated with VLPs or CLPs of BTV-1, derived from western lineage and were challenged with virulent BTV-1 from an eastern lineage. All VLP-vaccinated animals developed a neutralising antibody response to BTV-1 from both lineages prior to challenge. Moreover, post-challenged animals had no clinical manifestation or viraemia and the challenged virus replication was completely inhibited. In contrast, CLP-vaccinated animals did not induce any neutralising antibody response but developed the group specific VP7 antibodies. CLPs also failed to prevent the clinical manifestation and virus replication, but in comparison to controls, the severity of disease manifestation and viraemia was mitigated. The data demonstrated that the outer capsid was essential for complete protection, while the geographical origin of the BTV was not critical for development of a serotype specific vaccine.

Validation of a novel approach for the rapid production of immunogenic virus-like particles for bluetongue virus

Bluetongue virus causes an emerging disease of ruminants, principally affecting sheep and cattle. Since 1998, there have been multiple separate outbreaks of bluetongue disease in Europe that have highlighted the need for a safe, efficacious, DIVA compliant vaccine. We report here a new baculovirus expression strategy which allowed pre-integration of the genes encoding the BTV inner capsid proteins at one baculovirus locus and those encoding the outer capsid proteins at a different locus. A modified baculovirus with two marker proteins to facilitate the phenotypic selection of recombinant viruses was developed. The utility of this approach is demonstrated by the production of BTV VLPs to a number of serotypes. For a proof of concept, VLPs of one serotype was then tested for protective immune response. VLPs were demonstrated to be safe, highly effective, immunogens in sheep, reducing post-challenge viraemia to levels below the threshold detection limit of quantitative RT-PCR when vaccinated animals were challenged with virulent virus.

The Double-Stranded RNA Bluetongue Virus Induces Type I Interferon in Plasmacytoid Dendritic Cells via a MYD88-Dependent TLR7/8-Independent Signaling Pathway

ABSTRACT Dendritic cells (DCs), especially plasmacytoid DCs (pDCs), produce large amounts of alpha/beta interferon (IFN-α/β) upon infection with DNA or RNA viruses, which has impacts on the physiopathology of the viral infections and on the quality of the adaptive immunity. However, little is known about the IFN-α/β production by DCs during infections by double-stranded RNA (dsRNA) viruses. We present here novel information about the production of IFN-α/β induced by bluetongue virus (BTV), a vector-borne dsRNA Orbivirus of ruminants, in sheep primary DCs. We found that BTV induced IFN-α/β in skin lymph and in blood in vivo. Although BTV replicated in a substantial fraction of the conventional DCs (cDCs) and pDCs in vitro, only pDCs responded to BTV by producing a significant amount of IFN-α/β. BTV replication in pDCs was not mandatory for IFN-α/β production since it was still induced by UV-inactivated BTV (UV-BTV). Other inflammatory cytokines, including tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and IL-12p40, were also induced by UV-BTV in primary pDCs. The induction of IFN-α/β required endo-/lysosomal acidification and maturation. However, despite being an RNA virus, UV-BTV did not signal through Toll-like receptor 7 (TLR7) for IFN-α/β induction. In contrast, pathways involving the MyD88 adaptor and kinases dsRNA-activated protein kinase (PKR) and stress-activated protein kinase (SAPK)/Jun N-terminal protein kinase (JNK) were implicated. This work highlights the importance of pDCs for the production of innate immunity cytokines induced by a dsRNA virus, and it shows that a dsRNA virus can induce IFN-α/β in pDCs via a novel TLR-independent and Myd88-dependent pathway. These findings have implications for the design of efficient vaccines against dsRNA viruses.

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.

Characterization of a second open reading frame in genome segment 10 of bluetongue virus

Viruses have often evolved overlapping reading frames in order to maximize their coding capacity. Until recently, the segmented dsRNA genome of viruses of the Orbivirus genus was thought to be monocistronic, but the identification of the bluetongue virus (BTV) NS4 protein changed this assumption. A small ORF in segment 10, overlapping the NS3 ORF in the +1 position, is maintained in more than 300 strains of the 27 different BTV serotypes and in more than 200 strains of the phylogenetically related African horse sickness virus (AHSV). In BTV, this ORF (named S10-ORF2 in this study) encodes a putative protein 50–59 residues in length and appears to be under strong positive selection. HA- or GFP-tagged versions of S10-ORF2 expressed from transfected plasmids localized within the nucleoli of transfected cells, unless a putative nucleolar localization signal was mutated. S10-ORF2 inhibited gene expression, but not RNA translation, in transient transfection reporter assays. In both mammalian and insect cells, BTV S10-ORF2 deletion mutants (BTV8ΔS10-ORF2) displayed similar replication kinetics to wt virus. In vivo, S10-ORF2 deletion mutants were pathogenic in mouse models of disease. Although further evidence is required for S10-ORF2 expression during infection, the data presented provide an initial characterization of this ORF.

TaqMan Real-Time Reverse Transcription-PCR and JDVp26 Antigen Capture Enzyme-Linked Immunosorbent Assay To Quantify Jembrana Disease Virus Load during the Acute Phase of In Vivo Infection

ABSTRACT Jembrana disease virus (JDV) is an acutely pathogenic lentivirus that affects Bali cattle in Indonesia. The inability to propagate the virus in vitro has made it difficult to quantitate JDV and determine the kinetics of virus replication during the acute phase of the disease process. We report for the first time two techniques that enable quantification of the virus and the use of these techniques to quantify the virus load during the acute phase of the disease process. A one-step JDV pol TaqMan real-time reverse transcription-PCR (RT-PCR) assay was developed for the detection and quantification of JDV RNA in plasma. The limit of detection was 9.8 × 102 JDV viral RNA copies over 35 cycles, equivalent to 4.2 × 104 JDV genome copies/ml, and a peak virus load of 1.6 × 1012 during the acute febrile period. An antigen capture enzyme-linked immunosorbent assay (ELISA) was also developed to quantify the levels of JDV capsid (JDVp26) over a linear range of 10 to 200 ng/ml. Viral RNA and JDVp26 levels were correlated in 48 plasma samples obtained from experimentally infected cattle. A significant positive correlation (R = 0.860 and r2 = 0.740) was observed between the two techniques within the range of their detection limits. The relatively insensitive capture ELISA provides an economical and feasible method for monitoring of virus in the absence of more sensitive techniques.

Bluetongue virus serotype 8 virus-like particles protect sheep against virulent virus infection as a single or multi-serotype cocktail immunogen.

Since 1998, there have been multiple separate outbreaks of Bluetongue disease (BT) in Europe with the largest outbreak ever recorded in Northern Europe caused by Bluetongue virus serotype 8 (BTV-8). Coinciding with the BTV-8 outbreak, a virulent strain of BTV-1 emerged and co-infections of these two serotypes were reported. In response, we generated VLPs for BTV-8 and tested the efficacy of BTV-8 VLPs as a single immunogen and as a component of a multivalent vaccine, with VLPs of BTV-1 and BTV-2, in order to test if there was any interference between serotypes. All pre-Alps sheep vaccinated with BTV-8 VLPs developed a strong neutralising antibody response to BTV-8 and multivalent VLP vaccinated animals also developed neutralising antibodies to BTV-1 and BTV-2. There were no side effects observed due to the vaccination with either the single- or multivalent VLP cocktail. All VLP-vaccinated animals had no clinical manifestation of BT or viraemia after challenge with a virulent BTV-8 isolate. This data indicates that BTV-8 VLPs delivered as a single immunogen or as a component of a multivalent vaccine are highly efficacious. Moreover, there was no interference on the development of a strong protective immune response due to the combination of different phylogenetically unrelated BTV serotypes in the vaccinated animals. This report further highlights that BTV VLPs are safe and efficacious immunogens that are able to afford complete protection against a virulent virus challenge.

Role of cellular caspases, nuclear factor-kappa B and interferon regulatory factors in Bluetongue virus infection and cell fate.

BackgroundBluetongue virus (BTV) infection causes haemorrhagic disease in ruminants and induces cell death. The pathogenesis in animals and in cell culture has been linked to BTV-induced apoptosis.ResultsIn this report, we investigated BTV-induced apoptosis in cell culture in depth and show that both extrinsic (caspase-8 activation) and intrinsic (caspase-9 activation) pathways play roles in BTV apoptosis. Further, by using chemical inhibitors and knock-out cell lines, we show that these pathways act independently of each other in BTV infected cells. In addition to activation of caspase-8, -9 and executioner caspase-3, we also identified that BTV infection causes the activation of caspase-7, which results in the cleavage of poly (ADP-ribose) polymerase (PARP). BTV-induced cell death appears to be due to apoptosis rather than necrosis, as the HMBG-1 was not translocated from the nucleus. We also examined if NF-κB response is related to BTV-induced apoptosis as in reovirus. Our data suggests that NF-κB response is not linked to the induction of apoptosis. It is controlled by the degradation of only IκBα but not IκBβ, resulting in a rapid transient response during BTV infection. This was supported using an NF-κB dependent luciferase reporter gene assay, which demonstrated early response, that appeared to be suppressed by the late stage of BTV replication. Furthermore, virus titres were higher in the presence of NF-κB inhibitor (SN50), indicating that NF-κB has a role in initiating an antiviral environment. In addition, we show that BTV infection induces the translocation of interferon regulatory factors (IRF-3 and IRF-7) into the nucleus. The induction of IRF responses, when measured by IRF dependent luciferase reporter gene assay, revealed that the IRF responses, like NF-κB response, were also at early stage of infection and mirrored the timing of NF-κB induction.ConclusionBTV triggers a wide range of caspase activities resulting in cell apoptosis. Although both NF-κB and IRF responses are induced by BTV infection, they are not sustained.

Analysis of Jembrana disease virus replication dynamics in vivo reveals strain variation and atypical responses to infection

Jembrana disease virus (JDV) is an acute lentiviral infection of Bali cattle in Indonesia. Data generated during a series of cattle infection experiments was examined and significant differences were identified in the mean plasma viral load on the first and second days of the febrile response in cattle infected with JDV(TAB/87) compared to those infected with JDV(PUL/01). The peak and total viral loads >or=10(6) genome copies/ml during the acute stage of the disease were significantly higher in JDV(TAB/87) infected cattle. JDV(PUL/01) infected cattle developed peak rectal temperatures earlier than the JDV(TAB/87) cattle but there were no differences in the duration of the febrile responses observed for the 2 groups of animals. The plasma viremia was above 10(6) genome copies/ml for almost 3 days longer in JDV(TAB/87) compared to JDV(PUL/01) infected cattle. Atypical responses to infection occurred in approximately 15% of experimentally infected animals, characterized by reduced viral loads, lower or absent febrile responses and absence of p26-specific antibody responses. Most of these cattle developed normal Tm-specific antibody responses between 4-12 weeks post-infection.

Multiple large foreign protein expression by a single recombinant baculovirus: A system for production of multivalent vaccines

Baculovirus expression system offers the advantage of expression of several large proteins simultaneously by a single recombinant virus. To date, expression of multiple large (>100kDa) proteins has been hampered by the need to generate large constructs and repeat use of homologous sequence and promoter. The development of multi-loci baculovirus expression system overcomes these issues by enabling the recombination of large foreign sequences into different regions of the genome. In this paper, we have examined the co-expression of African horse sickness virus (AHSV) VP2 proteins from multiple serotypes in a single recombinant baculovirus. To this end, recombinant baculoviruses expressing multiple AHSV VP2 proteins were generated and it was found that up to six different AHSV serotypes (serotype 1, 3, 4, 5, 7 and 8) VP2 proteins (∼120kDa) could be expressed simultaneously from different loci of baculovirus genome. The expression of VP2 of one serotype was not significantly hindered by the presence of other serotypes, although there were slight differences in expression level between different serotypes. The expression of VP2 of further serotypes from additional loci resulted in a lesser expression level of VP2 proteins. Based on these findings, three additional recombinant baculoviruses encompassing all nine AHSV serotypes were constructed (serotypes 1, 7, 8 or serotypes 2, 4, 5 or serotypes 3, 6, 9) and each of the triple recombinant viruses exhibited similar expression level of each VP2. This system allows for the expression of a number of large proteins that has the potential to be exploited for multivalent vaccines production.