Publications

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

There were a total of 2599 results for your search.

Abstract

The translation of flaviviral RNA genomes yields a single polyprotein that is processed into the mature proteins by viral and host cell proteases. Mature capsid protein C is freed from the polyprotein by the viral NS2B/3 protease, cleaving in the C-terminal region of protein C in front of the signal sequence for prM. Protein C has been shown to be involved in viral assembly and RNA packaging. To examine further the role of protein C and its production by proteolysis, we replaced the NS2B/3 capsid cleavage site in tick-borne encephalitis virus (TBEV) and West Nile virus (WNV) by the 2A protein of foot-and-mouth disease virus (TBEV-2A and WNV-2A). This obviated the need for NS2B/3 processing at the C terminus of mature protein C while simultaneously producing a 19-amino-acid extension on protein C. Infectious virions were generated with both viruses; the phenotype depended on the host cell. TBEV-2A replicated well in BHK-21 cells but was essentially incapable of replication in tick cells. In contrast, WNV-2A replicated well in mosquito cells but showed a small-plaque phenotype in Vero cells, with frequent production of larger plaques. Sequencing of viral RNA from the larger plaques showed substitutions in the signal sequence for prM, presumably improving coordinated protein processing at the C-prM junction. Furthermore, both TBEV-2A and WNV-2A were also defective in unpackaging and/or early RNA synthesis. Together, these results indicate a role for flavivirus protein C in both viral assembly and RNA replication, possibly by interacting with host cell factors required to set up the cell for RNA replication.

Abstract

We demonstrate the presence of a functional internal ribosome entry site (IRES) within the 5? leader (designated 5L) from a variant of bicistronic mRNAs that encode the pp14 and RLORF9 proteins from Marek's disease virus (MDV) serotype 1. Transcribed as a 1.8-kb family of immediate-early genes, the mature bicistronic mRNAs have variable 5? leader sequences due to alternative splicing or promoter usage. Consequently, the presence or absence of the 5L IRES in the mRNA dictates the mode of pp14 translation and leads to the production of two pp14 isoforms that differ in their N-terminal sequences. Real-time reverse transcription-quantitative PCR indicates that the mRNA variants with the 5L IRES is two to three times more abundant in MDV-infected and transformed cells than the mRNA variants lacking the 5L IRES. A common feature to all members of the 1.8-kb family of transcripts is the presence of an intercistronic IRES that we have previously shown to control the translation of the second open reading frame (i.e., RLORF9). Investigation of the two IRESs residing in the same bicistronic reporter mRNA revealed functional synergism for translation efficiency. In analogy with allosteric models in proteins, we propose IRES allostery to describe such a novel phenomenon. The functional implications of our findings are discussed in relation to host-virus interactions and translational control.

Abstract

In the light of the recent emergence of multidrug-resistant and extensively drug-resistant strains of Mycobacterium tuberculosis, the epidemic of tuberculosis (TB) in populations coinfected with human immunodeficiency virus, and the failure of Mycobacterium bovis bacillus Calmette-Guerin (BCG) to protect against disease, new vaccines against TB are urgently needed. Two promising new vaccine candidates are the recombinant Delta ureC hly(+) BCG (recBCG), which has been developed to replace the current BCG vaccine strain, and modified vaccinia virus Ankara (MVA) expressing M. tuberculosis antigen 85A (MVA85A), which is a leading candidate vaccine designed to boost the protective efficacy of BCG. In the present study, we examined the effect of MVA85A boosting on the protection afforded at 12 weeks postchallenge by BCG and recBCG by using bacterial CFU as an efficacy readout. recBCG-immunized mice were significantly better protected against aerosol challenge with M. tuberculosis than mice immunized with the parental strain of BCG. Intradermal boosting with MVA85A did not reduce the bacterial burden any further. In order to identify a marker for the development of a protective immune response against M. tuberculosis challenge, we analyzed splenocytes after priming or prime-boosting by using intracytoplasmic cytokine staining and assays for cytokine secretion. Boosting with MVA85A, but not priming with BCG or recBCG, greatly increased the antigen 85A-specific T-cell response, suggesting that the mechanism of protection may differ from that against BCG or recBCG. We show that the numbers of systemic multifunctional cytokine-producing cells did not correlate with protection against aerosol challenge in BALB/c mice. This emphasizes the need for new biomarkers for the evaluation of TB vaccine efficacy.
Tuppurainen E S M, Stoltsz H W, Troskie M, Wallace D B, Oura C, Mellor P S, Coetzer K, Venter E H (2009)

Determination of the role of hard (ixodid) ticks in the transmission of lumpy skin disease virus in cattle

8th International Congress of Veterinary Virology, Budapest, Hungary, 23-26 August, 2009. 20 years of ESVV: integrating classical and molecular virology, programme and proceedings, 64-64
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Abstract

Lumpy skin disease virus (LSDV) belongs to the genus Capripoxvirus within the family Poxviridae and subfamily Chordopoxvirinae. The virus infects cattle and occurs in most African countries and in the Middle East. Lumpy skin disease (LSD) is an economically important disease and therefore it is included in the list of Notifiable Diseases by the World Organization of Animal Health (OIE). The mode of transmission of LSDV has not been fully established. In general, poxviruses have been shown to enter the host through the skin or respiratory tract. Direct contact between infected and susceptible animals is considered to be a relatively inefficient route of the transmission of LSDV. The virus is secreted in saliva, ocular and nasal discharge and semen of infected animals and therefore contaminated food or water and artificial insemination may serve as a source of infection. Several researchers have observed that the occurrence of the disease is closely connected to warm and wet weather conditions and the abundance of insects. However, little is known about the importance of the different insect vectors in the transmission of LSDV during the natural outbreaks. So far no studies on the potential role of hard ticks in the transmission of LSDV have been carried out. The aim of this project was to investigate the potential role of laboratory-bred ixodid ticks: Rhipicephalus appendiculatus, Amblyomma hebraeum and Boophilus decoloratus in the transmission of LSDV between cattle. To evaluate the mode of transmission (mechanical, transstadial, transovarial) the presence of live virus or viral antigen was studied in mouthparts, salivary glands, and gut cells of nymphs and adult ticks post feeding on the skin of experimentally infected cattle and in eggs laid by a female ticks previously fed on the skin lesions.

Abstract

Equine rhinitis A virus (ERAV) is closely related to foot-and-mouth disease virus (FMDV), belonging to the genus Aphthovirus of the Picornaviridae. How picornaviruses introduce their RNA genome into the cytoplasm of the host cell to initiate replication is unclear since they have no lipid envelope to facilitate fusion with cellular membranes. It has been thought that the dissociation of the FMDV particle into pentameric subunits at acidic pH is the mechanism for genome release during cell entry, but this raises the problem of how transfer across the endosome membrane of the genome might be facilitated. In contrast, most other picornaviruses form 'altered' particle intermediates (not reported for aphthoviruses) thought to induce membrane pores through which the genome can be transferred. Here we show that ERAV, like FMDV, dissociates into pentamers at mildly acidic pH but demonstrate that dissociation is preceded by the transient formation of empty 80S particles which have released their genome and may represent novel biologically relevant intermediates in the aphthovirus cell entry process. The crystal structures of the native ERAV virus and a low pH form have been determined via highly efficient crystallization and data collection strategies, required due to low virus yields. ERAV is closely similar to FMDV for VP2, VP3 and part of VP4 but VP1 diverges, to give a particle with a pitted surface, as seen in cardioviruses. The low pH particle has internal structure consistent with it representing a pre-dissociation cell entry intermediate. These results suggest a unified mechanism of picornavirus cell entry.
Webb P R, Powell L, Denyer M, Marsh S, Weaver C, Simmons M M, Johns E, Sheehan J, Horsfield P, Lyth C, Wilson C, Long A, Cawthraw S, Saunders G C, Spencer Y I (2009)

A retrospective immunohistochemical study reveals atypical scrapie has existed in the United Kingdom since at least 1987

Journal of Veterinary Diagnostic Investigation 21 (6), 826-829

Abstract

Atypical scrapie is a relatively recent discovery, and it was unknown whether it was a new phenomenon or whether it had existed undetected in the United Kingdom national flock. Before 1998, the routine statutory diagnosis of transmissible spongiform encephalopathy (TSE) in sheep relied on the presence of TSE vacuolation in the brainstem. This method would not have been effective for the detection of atypical scrapie. Currently, immunohistochemistry (IHC) and Western blot are commonly used for the differential diagnosis of classical and atypical scrapie. The IHC pattern of PrP(d) deposition in atypical scrapie is very different from that in classical scrapie using the same antibody. It is thus possible that because of a lack of suitable diagnostic techniques and awareness of this form of the disease, historic cases of atypical scrapie remain undiagnosed. Immunohistochemistry was performed on selected formalin-fixed, paraffin-embedded (FFPE) blocks of ovine brain from the Veterinary Laboratories Agency archives that were submitted for various reasons, including suspect neurological disorders, between 1980 and 1989. It was found that PrP(d) deposits in a single case were consistent with atypical scrapie. A method was developed to obtain a PrP genotype from FFPE tissues and was applied to material from this single case, which was shown to be AHQ/AHQ. This animal was a scrapie suspect from 1987, but diagnosis was not confirmed by the available techniques at that time.

Abstract

African horse sickness virus (AHSV) is an orbivirus that is usually transmitted between its equid hosts by adult Culicoides midges. In this article, we review the ways in which AHSV may have adapted to this mode of transmission. The AHSV particle can be modified by the pH or proteolytic enzymes of its immediate environment, altering its ability to infect different cell types. The degree of pathogenesis in the host and vector may also represent adaptations maximising the likelihood of successful vectorial transmission. However, speculation upon several adaptations for vectorial transmission is based upon research on related viruses such as bluetongue virus (BTV), and further direct studies of AHSV are required in order to improve our understanding of this important virus.
Wilson A J, Mellor P S (2009)

Bluetongue in Europe: past, present and future

Philosophical Transactions of the Royal Society B-Biological Sciences 364 (1530), 2669-2681

Abstract

The recent arrival in Northern and Western (NW) Europe of bluetongue virus (BTV), which causes the ruminant disease ‘bluetongue’, has raised the profile of this vector-borne ruminant disease and sparked discussions on the reasons for its sudden emergence so far north. This expansion has not happened in isolation and the disease has been expanding into Southern and Eastern Europe for the last decade. This shifting disease distribution is being facilitated by a number of different introduction mechanisms including the movement of infected livestock, the passive movement of infected Culicoides on the wind and, in NW Europe, an unknown route of introduction. The expansion of BTV in Europe has forced a re-evaluation of the importance of Palaearctic Culicoides species in transmission, as well as the importance of secondary transmission routes, such as transplacental transmission, in facilitating the persistence of the virus. The current European outbreak of BTV-8 is believed to have caused greater economic damage than any previous single-serotype outbreak. Although attempts are being made to improve the capacity of European countries to cope with future BTV incursions, the options available are limited by a lack of basic entomological data and limited virological surveillance.

Abstract

Herpesviruses account for 134 out of the 140 virus-encoded microRNAs (miRNAs) known today. Here we report the identification of 11 novel miRNAs encoded by herpesvirus of turkey (HVT), a virus used as a live vaccine in poultry against the highly oncogenic Marek's disease virus type 1. Ten of these miRNAs were clustered together within the repeat long region of the viral genome, demonstrating some degree of positional conservation with other mardiviruses. Close sequence and phylogenetic relationships of some miRNAs in this cluster indicate evolution by duplication. HVT miRNAs represent the first example of virus-encoded miRNAs that show evolution by duplication.

Abstract

CD4 is a molecule commonly expressed on the surface of T-helper lymphocytes with a recognized critical role in the antigen presentation process that has also been reported in monocytes and macrophages, although its role in these cells remains unknown. The objective of the present study was to analyze whether experimental conditions involving a potent acquired immune component, as occurs in experimental autoimmune encephalomyelitis (EAE), are able to induce CD4 expression in the population of microglia/macrophages. Myelin Basic Protein (MBP) immunized female Lewis rats, were examined at different phases during the course of EAE according to their clinical score. Spinal cords were analyzed by flow cytometry for CD11b, CD4 and CD45, by histochemistry for NDPase and by immunohistochemistry for ED2, Iba1, CD45 and CD4. Flow cytometry analysis showed that EAE induced CD4 expression in macrophages (CD11b+/CD45(high)) and microglia (in both CD11b+/CD45(intermediate) and CD11b+/CD45(low) phenotypes). Noticeably, microglial CD4 expression was found during the recovery phase and was maintained until 40 days post-induction. In agreement, immunolabelled sections revealed CD4 expression in microglial cells with ramified morphology during the recovery and post-recovery phases. In conclusion, our results indicate that, in this EAE model, perivascular cells, microglia and macrophages showed different dynamics during the course of the disease in close relation with symptomatology and that microglial cells expressed CD4 interestingly during the recovery phase, suggesting a role of microglial CD4 expression in the resolution of the immune response.

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