Publications

The Pirbright Institute publication directory contains details of selected publications written by our researchers.

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Abstract

Members of the genus Enterovirus (family Picornaviridae) are believed to be common and widespread among humans and different animal species, although only a few enteroviruses have been identified from animal sources. Intraspecies recombination among human enteroviruses is a well-known phenomenon, but only a few interspecies examples have been reported and, to our current knowledge, none of these have involved non-primate enteroviruses. In this study, we report the detection and complete genome characterization (using RT-PCR and long-range PCR) of a natural interspecies recombinant bovine/porcine enterovirus (ovine enterovirus type 1; OEV-1) in seven (44 %) of 16 faecal samples from 3-week-old domestic sheep (Ovis aries) collected in two consecutive years. Phylogenetic analysis of the complete coding region revealed that OEV-1 (ovine/TB4-OEV/2009/HUN; GenBank accession no. JQ277724) was a novel member of the species Porcine enterovirus B (PEV-B), implying the endemic presence of PEV-B viruses among sheep. However, the 5' UTR of OEV-1 showed a high degree of sequence and structural identity to bovine enteroviruses. The presumed recombination breakpoint was mapped to the end of the 5' UTR at nucleotide position 814 using sequence and SimPlot analyses. The interspecies-recombinant nature of OEV-1 suggests a closer relationship among bovine and porcine enteroviruses, enabling the exchange of at least some modular genetic elements that may evolve independently.

Abstract

Background: Bluetongue 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. Results: Using 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. Conclusions: NS1 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.

Abstract

The TLRs represent a family of pattern recognition receptors critical in the induction of vertebrate immune responses. Between 10 and 13 different TLR genes can be identified in each vertebrate species, with many represented as orthologous genes in different species. The agonist specificity of orthologous TLR is also highly conserved. In contrast, TLR15 can only be identified in avian and reptilian genomes, suggesting that this receptor arose similar to 320 million years ago after divergence of the bird/reptile and mammalian lineages. Transfection of a constitutively active form of chicken TLR15 led to NF-kappa B activation in HEK293 cells and induced cytokine mRNA upregulation in chicken cell lines. Full-length TLR15 mediated NF-kappa B induction in response to lysates from yeast, but not those derived from viral or bacterial pathogens, or a panel of well-characterized TLR agonists. TLR15 responses were induced by whole-cell lysates derived from Candida albicans, Saccharomyces cerevisiae, and Schizosaccharomyces pombe, but not zymosan preparations from S. cerevisiae. The ability of yeast lysate to activate TLR15-dependent NF-kappa B pathways (in transfection assays) or stimulate IL-1 beta mRNA upregulation in chicken macrophages was abrogated by heat inactivation or pre-exposure of the lysate to PMSF. Identification of yeast as an agonist source for TLR15 provides a functional framework for consideration of this TLR within the context of pattern recognition receptor evolution and may impact on the development of novel adjuvants. The Journal of Immunology, 2012, 189: 4930-4938.
Britton P (2012)

Recombinant vaccines against infectious bronchitis

Avian Pathology Newsletter (The Japanese Society of Veterinary Science) 20, 5-13
Publisher’s version:

Abstract

Infectious bronchitis virus (IBV) causes an infectious respiratory disease of domestic fowl that affects poultry of all ages causing economic problems for the poultry industry worldwide. Although IBV is controlled using live attenuated and inactivated vaccines it continues to be a major problem due to the existence of many serotypes, determined by the surface spike protein resulting in poor cross-protection, and loss of immunogenicity associated with vaccine production. Live attenuated IBV vaccines are produced by the repeated passage in embryonated eggs resulting in spontaneous mutations. As a consequence attenuated viruses have only a few mutations responsible for the loss of virulence, which will differ between vaccines affecting virulence and/or immunogenicity and can revert to virulence. A new generation of vaccines is called for and one means of controlling IBV involves the development of new and safer vaccines by precisely modifying the IBV genome using reverse genetics for the production of rationally attenuated IBVs in order to obtain an optimum balance between loss of virulence and capacity to induce immunity.

Abstract

Meq is the major Marek's disease virus (MDV)-encoded oncoprotein and is essential for T-cell lymphomagenesis. Meq and several noncoding RNAs, including three microRNA (MiR) clusters, are expressed from the repeats of the MDV genome during latent infection of T cells. To investigate the state of the chromatin in this and flanking regions, we carried out chromatin immunoprecipitation (ChIP) analysis of covalent histone modifications and associated bound proteins. T-cell lines and a lymphoma were compared. The chromatin around the promoters for Meq and the noncoding RNAs in both cell lines and the lymphoma were associated with H3K9 acetylation and H3K4 trimethylation, which are marks of transcriptionally active chromatin. These correlated with bound Meq–c-Jun heterodimers. The only binding site for Meq homodimers is located at the lytic origin of replication (OriLyt), next to the lytic gene pp38. This region lacked active marks and was associated with repressive histone modifications (H3K27 and H3K9 trimethylation). DNA CpG methylation was investigated using methylated DNA precipitation (MeDP). In cell lines, DNA methylation was abundant across the repeats but noticeably reduced or absent around the active promoters. In primary tumors, CpG methylation occurred less than 2 months after infection, focused within the ICP4 gene. These data suggest that nonrandom de novo DNA methylation occurs early in lymphomagenesis. In addition, the histone data indicate a role for Meq in the epigenetic regulation of the MDV genome repeats in transformed T cells and suggest that the OriLyt region and the Meq/MiR region might be separated by chromatin boundary elements, and preliminary data on CTCF binding are consistent with this.

Abstract

The eradication of rinderpest virus (RPV) from the globe was possible through the availability of a safe and effective live attenuated vaccine and a suitable companion diagnostic test. However, the inability to serologically 'Differentiate between naturally Infected and Vaccinated Animals' (DIVA) meant that both the time taken to complete the eradication programme and the economic burden on countries involved was significantly greater than if a vaccine and companion diagnostic test that fulfilled the DIVA concept had been available. During the RPV eradication campaign serosurveillance for RPV was primarily based on a competitive ELISA using a RPV specific (C1) monoclonal antibody (mAb) directed against the viral haemagglutinin (H) protein but this test was not able to meet DIVA requirements. To provide proof of concept for the generation of novel morbillivirus DIVA vaccines we have identified, by phage display, and mutated residues critical for C1 mAb binding and assessed the functionality of mutants in an in vitro fusion assay. Finally we have incorporated mutated epitopes into a full length clone and rescued recombinant RPV using reverse genetics techniques. Here we describe a novel mechanism of marking morbillivirus vaccines, using RPV as a proof of concept, and discuss the applicability of this method to the development of marked vaccines for peste des petits ruminants virus (PPRV).

Bumbarov V, Brenner J, Rotenberg D, Batten C, Sharir B, Gorohov A, Golender N, Shainin T, Kanigswald G, Asis I, Oura C (2012)

The presence and possible effects of bluetongue virus in goat herds in Israel

Israel Journal of Veterinary Medicine 67 (4), 237-243

Abstract

In spite of the many reports on bluetongue (BT) in cattle and sheep, publications on BT-attributed manifestations in goats naturally infected with bluetong-ue virus (BTV) are scarce, and up to now there have been no reports on BT-attributed manifestations in goats in Israel. In this retrospective study, which was carried out on 8 goat farms in Israel, high levels of neonatal morbidity or mortality, as well as abortions, were observed in goats which were concurrently infected with four different BTV serotypes (BTV-2, BTV-4, BTV-8, and BTV-16). It is not however clear whether BTV was the primary cause of disease in the goats, or whether it may have contributed as a secondary pathogen in predisposing the goats to disease caused by other pathogens.
Chu J-Q, Kim S-M, Lee K-N, Oem J-K, Ko Y-J, Lee H-S, Kim Y-J, Park J-Y, Kim K-J, Parida S, Oh Y, Paton D J, Joo Y-S, Kim B, Park J-H (2012)

Adenovirus expressing human interferon inhibits replication of foot and mouth disease virus and reduces fatal rate in mice

Journal of Bacteriology and Virology 42 (3), 224-231

Abstract

Interferon is an important cytokine that plays a critical role in the initial host defense against viral infection. Recombinant human adenoviruses expressing human interferon-? (Ad-HIFN?) or pig interferon-? fused with interleukin-18 (Ad-PIFN?-IL18) were constructed and used to induce an early protective response against foot and mouth disease (FMD). To analyze the antiviral effect, bovine thyroid and porcine kidney IBRS-2 cells and ICR mice were treated with Ad-HIFN?, Ad-PIFN?-IL18, and cocktail of Ad-HIFN? and Ad-PIFN?-IL18. The survival rate of suckling mice was monitored after foot and mouth disease virus (FMDV) challenge following intra-peritoneal (IP) administration of appropriate adenovirus. Indirect antigen ELISA was performed to evaluate inhibition of FMDV replication following challenge with the FMDV O, A, or Asia 1 serotypes in vitro. These recombinant adenoviruses reduced the replication of FMDV in susceptible cells, thereby decreasing the fatality in mice, suggesting that they can be a useful control method for the early protection against FMD infection in livestock after field trial.

Abstract

Vaccination of poultry against coccidiosis caused by the Eimeria species is almost entirely based upon varied formulations of live parasites. The recent development of a series of protocols that support genetic complementation by transfection in Eimeria now provides an opportunity to utilise live anticoccidial vaccines to deliver additional vaccinal antigens. The capacity of Eimeria tenella to express an exogenous antigen and induce an immune response during in vivo infection which is protective against subsequent bacterial challenge has been tested here using the anti-Campylobacter jejuni vaccine candidate CjaA. Using restriction enzyme mediated integration (REMI) a transgenic E. tenella population expressing CjaA and the fluorescent reporter mCitrine has been developed. Vaccination of specific pathogen free chickens by single or multiple oral inoculation of E. tenella-CjaA oocysts induced 91% and 86% immune protection against C. jejuni challenge compared with unvaccinated and wild-type E. tenella vaccinated controls (p

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