Publications

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

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Zweygarth E, Josemans A I, Spickett A M, Steyn H C, Putterill J, Troskie P C, Mtshali M S, Bell-Sakyi L, Shkap V, Fish L, Kocan K M, Blouin E F (2006)

In vitro cultivation of a South African isolate of an Anaplasma sp in tick cell cultures

Onderstepoort Journal of Veterinary Research 73 (4), 251-255

Abstract

This paper describes the first successful in vitro cultivation of a South African isolate of an Anaplasma sp., initially thought to be Anaplasma marginale, in the continuous tick cell line IDE8. Blood from a bovine naturally infected with A. marginale kept on the farm Kaalplaas (28 degrees 08'E, 25 degrees 38'S) was collected, frozen, thawed and used as inoculum on confluent IDE8 cell cultures. Twenty days after culture initiation small intracellular colonies were detected in a Cytospin smear prepared from culture supernatant. Cultures were passaged on Day 34. Attempts to infect IRE/CTVM18 cell cultures with the Kaalplaas isolate derived from IDE8 cultures failed, whereas a reference stock of A. marginale from Israel infected IRE/CTVM18 tick cell cultures. Attempts to infect various mammalian cell lines (BA 886, SBE 189, Vero, L 929, MDBK) and bovine erythrocytes, kept under various atmospheric conditions, with tick cell-derived Anaplasma sp. or the Israeli strain of A. marginale failed. Molecular characterization revealed that the blood inoculum used to initiate the culture contained both A. marginale and Anaplasma sp. (Omatjenne) whereas the organisms from established cultures were only Anaplasma sp. (Omatjenne).

Abstract

Swine vesicular disease virus (SVDV) is a picornavirus closely related to the human pathogen coxsackievirus B5. In common with other picornaviruses, the 5' untranslated region (5' UTR) of SVDV contains an internal ribosomal entry site (IRES) that plays an important role in cap-independent translation. The aim of this study was to use RT-PCR and sequencing to characterize a fragment of the 5' UTR encompassing the entire IRES. Sequence analysis demonstrated high nucleotide identities within the IRES between 33 representative SVDV isolates. These data support the choice of this region as a diagnostic target and provide information for the improvement of laboratory-based molecular assays to detect SVDV. In contrast to the relative conservation of the IRES element, there was considerable nucleotide variability in the spacer region located between the cryptic AUG at the 3' end of the IRES and the initiation codon of the polyprotein. Interestingly, 11 SVDV isolates had block deletions of between 6 and 125'nt in this region. Nine of these isolates were of recent European origin and were phylogenetically closely related. In vitro growth studies showed that selected isolates with these deletions had a significantly reduced plaque diameter and grew to a significantly lower titre relative to an isolate with a full-length 5' UTR. Further work is required to define the significance of these deletions and to assess whether they impact on the pathogenesis of SVD.

Abstract

The discovery of Toll-like receptors (TLR) has revolutionised our understanding of innate immunity. Numerous reviews have been written on the subject in the past few years. Here, we review the evidence that TLRs are involved in sensing and initiating anti-viral responses. There are now three strong lines of evidence that support such a role for TLRs. Firstly, TLRs 'recognise' virally derived molecules and are required for various virus-induced cellular effects. Secondly, TLRs trigger anti-viral signalling pathways leading to the induction of the interferon response. Thirdly, viral immune strategies employed against TLRs have been identified.

Abstract

Vaccinia virus, a member of the Poxviridae, expresses many proteins involved in immune evasion. In this review, we present a brief characterisation of the virus and its effects on host cells and discuss representative secreted and intracellular proteins expressed by vaccinia virus that are involved in modulation of innate immunity. These proteins target different aspects of the innate response by binding cytokines and interferons, inhibiting cytokine synthesis, opposing apoptosis or interfering with different signalling pathways, including those triggered by interferons and toll-like receptors.

Stack J, Haga I R, Schröder M, Bartlett N W, Maloney G, Reading P C, Fitzgerald K A, Smith G L, Bowie A G (2005)

Vaccinia virus protein A46R targets multiple Toll-like-interleukin-1 receptor adaptors and contributes to virulence

Journal of Experimental Medicine 201 (6), 1007-1018

Abstract

Viral immune evasion strategies target key aspects of the host antiviral response. Recently, it has been recognized that Toll-like receptors (TLRs) have a role in innate defense against viruses. Here, we define the function of the vaccinia virus (VV) protein A46R and show it inhibits intracellular signalling by a range of TLRs. TLR signalling is triggered by homotypic interactions between the Toll-like-interleukin-1 resistance (TIR) domains of the receptors and adaptor molecules. A46R contains a TIR domain and is the only viral TIR domain–containing protein identified to date. We demonstrate that A46R targets the host TIR adaptors myeloid differentiation factor 88 (MyD88), MyD88 adaptor-like, TIR domain-containing adaptor inducing IFN-β (TRIF), and the TRIF-related adaptor molecule and thereby interferes with downstream activation of mitogen-activated protein kinases and nuclear factor βB. TRIF mediates activation of interferon (IFN) regulatory factor 3 (IRF3) and induction of IFN-β by TLR3 and TLR4 and suppresses VV replication in macrophages. Here, A46R disrupted TRIF-induced IRF3 activation and induction of the TRIF-dependent gene regulated on activation, normal T cell expressed and secreted. Furthermore, we show that A46R is functionally distinct from another described VV TLR inhibitor, A52R. Importantly, VV lacking the A46R gene was attenuated in a murine intranasal model, demonstrating the importance of A46R for VV virulence.

Abstract

The RNA segment (Gene 10) from a human group B rotavirus which encodes the homologue of the rotavirus enterotoxin (NSP4) has been cloned and sequenced. The gene is of the same length (751 nucleotides) as its better-characterized group A rotavirus counterpart but shows minimal homology (?10%) to it at the primary sequence level. Despite this low level of sequence homology, secondary structure predictions for the group B protein (ADRV-NSP4) showed a close similarity of structural features with the group A protein. Full-length ADRV-NSP4 was expressed in Escherichia coli with an amino terminal 6xHis tag that was used to purify it to homogeneity. The cytotoxicity of the purified protein was examined in a rapid dye-uptake assay that assesses membrane permeability and was found to be comparable to its group A counterpart.

Abstract

A fluorescence-based assay is presented for measuring the cytoxicity of viral proteins added exogenously to cells. The assay is based on the use of two fluorescent dyes, calcein-AM and ethidium homodimer (EtD-1) to specifically stain living and dead cells respectively and employs fluorescence activated cells sorting (FACS) to achieve a rapid and accurate measurement of the cytotoxic capacity of a potential viral toxin. The assay has been developed using the group B homologue (ADRV-NSP4) of the NSP4 enterotoxin encoded by Group A rotaviruses but should be applicable to assaying any viral protein exhibiting cytotoxic activity.

Abstract

Field strains of Foot-and-mouth disease virus (FMDV) use a number of alpha v-integrins as receptors to initiate infection on cultured cells, and integrins are believed to be the receptors used to target epithelial cells in animals. In this study, immunofluorescence confocal microscopy and real-time RT-PCR were used to investigate expression of two of the integrin receptors of FMDV, alpha v beta 6 and alpha v beta 3, within various epithelia targeted by this virus in cattle. These studies show that alpha v beta 6 is expressed constitutively on the surfaces of epithelial cells at sites where infectious lesions occur during a natural infection, but not at sites where lesions are not normally formed. Expression of alpha v beta 6 protein at these sites showed a good correlation with the relative abundance of beta 6 mRNA. In contrast, alpha v beta 3 protein was only detected at low levels on the vasculature and not on the epithelial cells of any of the tissues investigated. Together, these data suggest that in cattle, alpha v beta 6, rather than alpha v beta 3, serves as the major receptor that determines the tropism of FMDV for the epithelia normally targeted by this virus.

Abstract

In this study, we report the dynamic changes in activation and functions that occur in spleen dendritic cell (sDC) subsets following infection of mice with a natural murine pathogen, lymphocytic choriomeningitis virus (LCMV). Within 24 h postinfection (pi), sDCs acquired the ability to stimulate naive LCMV-specific CD8(+) T cells ex vivo. Conventional (CD11c(high) CD8(+) and CD4(+)) sDC subsets rapidly up-regulated expression of costimulatory molecules and began to produce proinflammatory cytokines. Their tendency to undergo apoptosis ex vivo simultaneously increased, and in vivo the number of conventional DCs in the spleen decreased markedly, dropping similar to2-fold by day 3 pi. Conversely, the number of plasmacytoid (CD11c(low)B220(+)) DCs in the spleen increased, so that they constituted almost 40% of sDCs by day 3 pi. Type 1 IFN production was up-regulated in plasmacytoid DCs by 24 h pi. Analysis of DC activation and maturation in mice unable to respond to type 1 IFNs implicated these cytokines in driving infection-associated phenotypic activation of conventional DCs and their enhanced tendency to undergo apoptosis, but also indicated the existence of type I IFN-independent pathways for the functional maturation of DCs during LCMV infection.
Nunez A, Gomez-Villamandos J C, Sanchez-Cordon P J, de Marco M F, Pedrera M, Salguero F J, Carrasco L (2005)

Expression of proinflammatory cytokines by hepatic macrophages in acute classical swine fever

Journal of Comparative Pathology 133 (1), 23-32

Abstract

Fourteen pigs were inoculated with the 'Alfort 187' strain of classical swine fever (CSF) virus and killed in pairs at 2, 4, 7, 9, 11, 14 or 17 days post-inoculation for histopathological, ultrastructural and immunohistochemical examination. For the latter method, the antibodies used were those against viral antigen Gp55, porcine myeloid marker SWC3, IL-1 alpha, IL-6, TNF-alpha and Factor VIII-related antigen. Activation and increase in the number of hepatic macrophages was observed following viral detection in liver, as well as an increase in IL-1 alpha and IL-6 production, mainly by Kupffer cells. Maximum detection of viral antigen was observed in the middle stage of the experiment coinciding with overexpression of the three cytokines studied, with IL-6 production by interstitial macrophages prominent at the end. Additionally, the labelling of platelets for Factor VIII-related antigen and the ultrastructural study of the sinusoids revealed activation and aggregation of thrombocytes close to Kupffer cells at the beginning of the infection. The liver seems to play a prominent role in the origin of the thrombocytopenia that occurs in CSF and contributes to the overexpression of proinflammatory cytokines considered responsible for the disorders observed during the course of the disease.

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