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

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The establishment of persistent infections with non-cytopathic bovine virus diarrhoea virus (ncpBVDV) is crucial for the maintenance of BVDV in cattle populations. Also, super-infection of persistently infected individuals with antigenically homologous cytopathic BVDV (cpBVDV) results in fatal mucosal disease. Persistent infection with ncpBVDV is established by infection of the foetus during the first trimester of pregnancy. It has been shown previously that foetal infection with cpBVDV does not result in persistent infection. Infection of cells in vitro has demonstrated that cpBVDV induces type I interferon (IFN), whereas ncpBVDV fails to induce IFN. In this study we demonstrate that foetal challenge with cpBVDV results in IFN production, whereas ncpBVDV does not. These findings strongly suggest that the ability of ncpBVDV to inhibit the induction of type I IFN has evolved to enable the virus to establish persistent infection in the early foetus.


Acute infection of calves, previously vaccinated with bacille Calmette-Guerin (BCG), with non-cytopathic viral diarrhoea virus (BVDV) resulted in the temporary suppression of two in vitro assays used to monitor Mycobacterium bovis infection. Lymphocyte proliferation and interferon-gamma production by whole blood cultures containing purified protein derivatives prepared from Mycobacterium avium (PPD-A) and M bovis (PPD-B) were markedly suppressed. The implication is that acute infections of cattle with non-cytopathic BVDV may temporarily compromise diagnostic tests for M bovis infections and result in a failure to identify cattle with tuberculosis.
Chernukhin I V, Seago J E, Newbury S F (2001)

Drosophila 5′ → 3′-exoribonuclease Pacman

Methods in Enzymology: Ribonucleases, Pt B edited by A. W. Nicholson 342, 293-302


This chapter concentrates on the methods used to express a Drosophila recombinat 5? ? 3?-exoribonuclease, purify the protein, and analyze its activity in vitro. Analysis of early development in Drosophila has shown that RNA localization, control of translation, and mRNA stability are intimately linked. Generally, translational repression leads to degradation of an RNA, and failure of an RNA to localize correctly also leads to its degradation. Work on the yeast Saccharomyces cerevisiae has identified many ribonucleases and associated factors that control mRNA decay, RNA splicing, and rRNA processing. In yeast, it has been shown that 3? ? 5? degradation/processing of RNA requires the exosome, which is a complex of at least 10 proteins. Degradation of RNA in a 5? ? 3? direction occurs by initial decapping of the mRNA, followed by 5? ? 3? degradation of the RNA by Xrn1p. The two decapping proteins (Dcp1p and Dcp2p) and Xrnlp have been shown to be complexed to seven Lsm proteins, which are likely to form a ring encircling the RNA. To understand the role of RNA stability in development, a number of approaches can be used. Once the genes encoding particular ribonucleases or associated factors have been identified, then expression of the RNA during development can be determined. These techniques have been used to show that the 5? ? 3?-exoribonuclease Pacman is differentially expressed during development.

Fernandez-Fernandez M R, Lucini C, Lopez-Moya J J, Guo H, Martinez-Torrecuadrada J, Casal J I, Mourino M, Plana-Duran J, Rivera J, Rodriguez J F, Montoya M, Del Val M, Garcia J A (2001)

Use of plum pox potyvirus as an expression vector in plants

Molecular Farming. Proceedings of the OECD Workshop held in La Grande Motte, France, 3-6 September, 2000., 161-172
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Plum pox potyvirus (PPV) is the causal agent of a devastating disease that affects stone fruit trees of the Prunus genus. However, it is also able to infect herbaceous hosts from several different families. This broad host range confers PPV an added value regarding its interest as a putative expression vector in plants. Two strategies have been used to express foreign sequences of interest using vectors based on PPV. The N-terminal region of the capsid protein (CP) has been used to express epitopes on the surface of virion particles. Four different insertion sites were evaluated, but only three of them have rendered viable chimeras. These vectors have shown notable differences in tolerance to sequence insertions. Foreign antigenic peptides expressed in them were easily recognized by specific antibodies. Moreover, sequences cloned at one of these vectors (position 12 of CP) were able to elicit an efficient B-cell response in experimental hosts. Epitope mapping by pepscan analysis and fine tuning of cloning positions are being conducted in an attempt to select optimal sites for epitope insertion. The second approach that we have followed is the use of PPV as an expression vector of whole independent proteins. Two insertion sites have been selected, one between P1 and HC proteins (PPV-XS) and the other between NIb and CP proteins (PPV-NK). A double expression vector has been constructed, which allows production of two foreign proteins with a single vector. ifferent reporter genes have been cloned in both insertion sites. The VP60 structural protein of rabbit hemorrhagic disease virus (RHDV) was also successfully expressed making use of PPV-NK vector. Extracts from the VP60-expressing plants were able to induce a remarkable immune response against RHDV in its natural hosts, rabbits. Moreover, these animals were protected against a lethal challenge with RHDV.
Gomez-Villamandos J C, Ruiz-Villamor E, Bautista M J, Sanchez C P, Sanchez-Cordon P J, Salguero F J, Jover A (2001)

Morphological and immunohistochemical changes in splenic macrophages of pigs infected with classical swine fever

Journal of Comparative Pathology 125 (2-3), 98-109


Classical swine fever (CSF) was induced in 20 pigs by inoculation with a virulent strain of CSF virus to determine sequential changes (2, 4, 7, 10 and 14 days post-inoculation) in the number and morphology of splenic macrophages (red pulp and lymphoid marginal zone) and thus to assess the role of these cells in the pathogenesis of the disease. The first splenic cells to be infected with CSF virus were macrophages in the marginal zone followed by other macrophage populations. The initial phase of CSF was associated with an increase in splenic macrophage numbers in the marginal zone and a decrease in the red pulp. Subsequently, the numbers in the red pulp increased. The study suggested that infection, mobilization and apoptosis of splenic macrophages play an important role in the spread of CSF virus in vivo. Moreover, the secretory changes that occurred in macrophages in the initial phase of the infection suggested that macrophages release chemical mediators capable of modulating pathogenesis, (C) 2001 Harcourt Publishers Ltd.


Control of Theileria annulata is currently best achieved by the use of live attenuated cell line vaccines. However, the mechanisms underlying attenuation are unclear and there is a need to rapidly produce new cell line vaccines, which could safely and effectively vaccinate cattle against tropical theileriosis. There is increasing evidence to suggest that proinflammatory cytokines produced by T. annulata infected cells play a central role in both pathology and immune evasion. This study aimed to test this hypothesis and to evaluate cytokine expression as a marker of virulence. The pathogenicity and protective efficacy of cloned T. annulata cell lines that expressed different levels of proinflammatory cytokines were compared. In two independent trials using different stocks of T. annulata, cell lines that expressed higher levels of proinflammatory cytokines induced severe reactions, and in some cases death, when used to vaccinate groups of cattle. In contrast, low cytokine expressing lines induced low post-vaccinal reactions. The results clearly demonstrated that cytokine expression by T. annulata infected cells could be used as a marker of virulence and provided strong evidence to support a role for cytokines in the induction of pathology. Both high and low cytokine expressing cell lines protected cattle against heterologous challenge infection, offering the possibility of using cytokine expression to rapidly select new safe, potent vaccines against tropical theileriosis without the need for culture attenuation.


Onchocerciasis is a debilitating parasitic infection caused by the filarial nematode Onchocerca volvulus. Infections are chronic, and persistence of the parasites for several years argues for highly adapted mechanisms of immune evasion. Due to the restricted host repertoire of O. volvulus, we have used the cattle parasite Onchocerca ochengi to investigate the nature of immunomodulation underpinning these long-term infections. Cattle were infected with a single inoculation of 350 infective-stage larvae under laboratory conditions (n = 6). Intradermal nodules containing immature adult worms were detected from 110 days postinfection, and microfilariae in skin were detected from day 280 postinfection. Parasite-specific responses during early infection were nonpolarized with respect to the major Th cytokines (interleukin-4 [IL-4], IL-2, and gamma interferon [IFN-gamma]) produced by antigen-stimulated peripheral blood mononuclear cells (PBMC) or serum antibody isotypes. Antigen-induced proliferation of PBMC peaked shortly after exposure and remained high during the prepatent infection. As the parasites matured and animals developed patent infections, there was a profound down-regulation of lymphoproliferation, accompanied by sharp falls in the expression of both IL-4 and IFN-gamma and a gradual decline in IL-2. Levels of immunoglobulin G2 (IgG2) fell, while those of IgG1 remained high. We conclude that neither a classical Th2 response nor a simple Th1-to-Th2 switch is sufficient to explain the immunomodulation associated with patent Onchocerca infections. Instead, there is an initial Th0 response, which matures into a response with some, but not all of the features of a Th2 response. The natural host-parasite relationship of O. ochengi in cattle may be useful as both a descriptive and predictive tool to test more refined models of immunomodulation in onchocerciasis.


The coronavirus nucleoprotein (N) has been reported to be involved in various aspects of virus replication. We examined by confocal microscopy the subcellular localization of the avian infectious bronchitis virus N protein both in the absence and in the context of an infected cell and found that N protein localizes both to the cytoplasmic and nucleolar compartments.
Salguero F J, Mekonnen T, Ruiz-Villamor E, Sanchez-Cordon P J, Gomez-Villamandos J C (2001)

Detection of monokines in paraffin-embedded tissues of pigs using polyclonal antibodies

Veterinary Research 32 (6), 601-609


Monokines are glycoproteins, synthesised by macrophages, which exert various effects on the organism. The most important monokines are interleukin (IL)-1 alpha, IL-1 beta, tumor necrosis factor (TNF)-alpha and IL-6. This paper reports on immunohistochemical techniques developed for the detection of IL-alpha, IL-1 beta, IL-6 and TNF-alpha in fixed and paraffin-embedded pig tissues (spleen, lymph nodes, thymus, liver and kidney). Different fixatives (buffered formalin, acetic formalin, paraformadehyde-lysine-periodate and Bouin solution), and antigen unmasking techniques (permeabilisation with Tween 20, pronase enzymatic digestion and microwave-citrate buffer) were used. We describe different protocols for detection of monokines using polyclonal antibodies against the studied monokines. No signal was obtained with monoclonal antibodies against pig-TNF-alpha and human IL-1 alpha. Bouin solution was shown to be the best fixative for immunohistochemical detection of IL-1 alpha, TNF-alpha, and IL-6, using permeabilisation with Tween 20 as an unmasking antigen method. Acetic formalin was shown to be the best fixative for IL-1 beta detection, not needing antigen retrieval techniques. Macrophages were identified as the main cytokine-producing cells, although other types of cells also stained positively to some cytokines. These techniques represent valuable tools for studies of the pathogenesis of viral and bacterial diseases, and of the immune system of the pigs.


We have identified and characterized a Drosophila orthologue of SK12, which, in Saccharomyces cerevisiae. is one of the key components in the cytoplasmic 3 ' -5 ' decay of mRNA. The Drosophila orthologue (twister, tst), is expressed as two transcripts which differ in the lengths of their 3 ' -UTRs, with the smaller transcript being particularly abundant in 0-2 h embryos and the larger transcript reaching its highest levels in 6-8 h embryos. TST protein is expressed in two forms which are differentially expressed in adult tissues and throughout development. Differential expression of TST may modulate activity of the mRNA turnover pathway and could have a major impact on the expression of target RNAs.


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