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

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Goodwin S, Tuthill T J, Arias A, Killington R A, Rowlands D J (2009)

Foot-and-mouth disease virus assembly: Processing of recombinant capsid precursor by exogenous protease induces self-assembly of pentamers in vitro in a myristoylation-dependent manner

Journal of Virology 83 (21), 11275-11282
Publisher’s version: http://dx.doi.org/10.1128/jvi.01263-09

Abstract

The assembly of foot-and-mouth disease virus (FMDV) particles is poorly understood. In addition, there are important differences in the antigenic and receptor binding properties of virus assembly and dissociation intermediates, and these also remain unexplained. We have established an experimental model in which the antigenicity, receptor binding characteristics, and in vitro assembly of capsid precursor can be studied entirely from purified components. Recombinant capsid precursor protein (P1 region) was expressed in Escherichia coli as myristoylated or unmyristoylated protein. The protein sedimented in sucrose gradients at 5S and reacted with monoclonal antibodies which recognize conformational or linear antigen determinants on the virion surface. In addition, it bound the integrin ?v?6, a cellular receptor for FMDV, indicating that unprocessed recombinant capsid precursor is both structurally and antigenically similar to native virus capsid. These characteristics were not dependent on the presence of 2A at the C terminus but were altered by N-terminal myristoylation and in mutant precursors which lacked VP4. Proteolytic processing of myristoylated precursor by recombinant FMDV 3Cpro in vitro induced a shift in sedimentation from 5S to 12S, indicating assembly into pentameric capsid subunits. Nonmyristoylated precursor still assembled into higher-order structures after processing with 3Cpro, but these particles sedimented in sucrose gradients at approximately 17S. In contrast, mutant precursors lacking VP4 were antigenically distinct, were unable to form pentamers, and had reduced capacity for binding integrin receptor. These studies demonstrate the utility of recombinant capsid precursor protein for investigating the initial stages of assembly of FMDV and other picornaviruses.
Graef T, Moesta A K, Norman P J, Abi-Rached L, Vago L, Older Aguilar A M, Gleimer M, Hammond J A, Guethlein L A, Bushnell D A, Robinson P J, Parham P (2009)

KIR2DS4 is a product of gene conversion with KIR3DL2 that introduced specificity for HLA-A*11 while diminishing avidity for HLA-C

Journal of Experimental Medicine 206 (11), 2557-2572
Publisher’s version: http://dx.doi.org/10.1084/jem.20091010

Abstract

Human killer cell immunoglobulin-like receptors (KIRs) are distinguished by expansion of activating KIR2DS, whose ligands and functions remain poorly understood. The oldest, most prevalent KIR2DS is KIR2DS4, which is represented by a variable balance between "full-length" and "deleted" forms. We find that full-length 2DS4 is a human histocompatibility leukocyte antigen (HLA) class I receptor that binds specifically to subsets of C1+ and C2+ HLA-C and to HLA-A*11, whereas deleted 2DS4 is nonfunctional. Activation of 2DS4+ NKL cells was achieved with A*1102 as ligand, which differs from A*1101 by unique substitution of lysine 19 for glutamate, but not with A*1101 or HLA-C. Distinguishing KIR2DS4 from other KIR2DS is the proline-valine motif at positions 71-72, which is shared with KIR3DL2 and was introduced by gene conversion before separation of the human and chimpanzee lineages. Site-directed swap mutagenesis shows that these two residues are largely responsible for the unique HLA class I specificity of KIR2DS4. Determination of the crystallographic structure of KIR2DS4 shows two major differences from KIR2DL: displacement of contact loop L2 and altered bonding potential because of the substitutions at positions 71 and 72. Correlation between the worldwide distributions of functional KIR2DS4 and HLA-A*11 points to the physiological importance of their mutual interaction.
Graham E M, Thom M L, Howard C J, Boysen P, Storset A K, Sopp P, Hope J C (2009)

Natural killer cell number and phenotype in bovine peripheral blood is influenced by age

Veterinary Immunology and Immunopathology 132 (2-4), 101-108
Publisher’s version: http://dx.doi.org/10.1016/j.vetimm.2009.05.002

Abstract

Natural killer (NK) cells are critical to the innate defence against intracellular infection. High NK cell frequencies have been detected in human neonates, which may compensate for the relative immaturity of the specific immune response. Additionally, phenotypic subsets of NK cells have been identified in humans with different functional properties. In this study, we examined the age distribution and phenotype of NK populations in bovine peripheral blood, including neonatal animals. We found that the NK cell populations defined by the phenotypes CD3?CD2+ and NKp46+ largely overlapped, so that the majority of NK cells in bovine peripheral blood were CD3?CD2+NKp46+. The remainder of the NK-like cells comprised two minor populations, CD3?CD2+NKp46? and CD3?CD2?NKp46+; the relative proportions of these varied with age. The lowest frequency of NK cells was recorded in 1-day-old calves, with the highest frequency in day 0 calves. The phenotypic characteristics of CD3?CD2+ and NKp46+ NK populations were similar; both populations expressed CD45RO, CD45RB, CD11b, CC84, CD8?? and CD8?? and did not express CD21, WC1, CD14 or ?? TCR. Age-related phenotypic differences were apparent. The phenotypic characteristics of three NK subpopulations were described; a significantly greater proportion of the CD3?CD2?NKp46+ population expressed CD8? compared to CD3?CD2+NKp46+ cells. Furthermore, a significantly greater proportion of the CD3?CD2+NKp46? population expressed CD8 compared to total CD3?CD2+ cells. Adult cattle had a significantly higher proportion of perforin+ cells compared to calves aged ?6 weeks. In this age group, the majority of perforin+ cells expressed NKp46, while in adults the majority of perforin+ cells were NKp46?. However, the proportion of NKp46+ and CD3?CD2+ cells that expressed perforin was not significantly different in any age group tested.
Hammond J A, Guethlein L A, Abi-Rached L, Moesta A K, Parham P (2009)

Evolution and survival of marine carnivores did not require a diversity of killer Cell Ig-Like receptors or Ly49 NK cell receptors

Journal of Immunology 182 (6), 3618-3627
Publisher’s version: http://dx.doi.org/10.4049/jimmunol.0803026

Abstract

Ly49 lectin-like receptors and killer cell Ig-like receptors (KIR) are structurally unrelated cell surface glycoproteins that evolved independently to function as diverse NK cell receptors for MHC class I molecules. Comparison of primates and various domesticated animals has shown that species have either a diverse Ly49 or KIR gene family, but not both. In four pinniped species of wild marine carnivore, three seals and one sea lion, we find that Ly49 and KIR are each represented by single, orthologous genes that exhibit little polymorphism and are transcribed to express cell surface protein. Pinnipeds are therefore species in which neither Ly49 nor KIR are polygenic, but retain the ancestral single-copy state. Whereas pinniped Ly49 has been subject to purifying selection, we find evidence for positive selection on KIR3DL during pinniped evolution. This selection, which focused on the D0 domain and the stem, points to the functionality of the KIR and most likely led to the sea lion’s loss of D0. In contrast to the dynamic and rapid evolution of the KIR and Ly49 genes in other species, the pinniped KIR and Ly49 have been remarkably stable during the >33 million years since the last common ancestor of seals and sea lions. These results demonstrate that long-term survival of placental mammal species need not require a diverse system of either Ly49 or KIR NK cell receptors.
Iqbal M, Xiao H, Baillie G, Warry A, Essen S C, Londt B, Brookes S M, Brown I H, McCauley J W (2009)

Within-host variation of avian influenza viruses

Philosophical Transactions of The Royal Society B-Biological Sciences 364 (1530), 2739-2747
Publisher’s version: http://dx.doi.org/10.1098/rstb.2009.0088

Abstract

The emergence and spread of H5N1 avian influenza viruses from Asia through to Europe and Africa pose a significant animal disease problem and have raised concerns that the virus may pose a pandemic threat to humans. The epizootological factors that have influenced the wide distribution of the virus are complex, and the variety of viruses currently circulating reflects these factors. Sequence analysis of the virus genes sheds light on the H5N1 virus evolution during its emergence and spread, but the degree of virus variation at the level of an individual infected bird has been described in only a few studies. Here, we describe some results of a study in which turkeys, ducks and chickens were infected with either one of two H5N1 or one of three H7N1 viruses, and the degree of sequence variation within an individual infected avian host was examined. We developed ‘deep amplicon’ sequence analysis for this work, and the methods and results provide a background framework for application to disease outbreaks in the field.
Iqbal M, Yaqub T, Reddy K, McCauley J W (2009)

Novel genotypes of H9N2 influenza A viruses isolated from poultry in Pakistan containing NS genes similar to highly pathogenic H7N3 and H5N1 viruses

PLoS One 4 (6), e5788
Publisher’s version: http://dx.doi.org/10.1371/journal.pone.0005788

Abstract

The impact of avian influenza caused by H9N2 viruses in Pakistan is now significantly more severe than in previous years. Since all gene segments contribute towards the virulence of avian influenza virus, it was imperative to investigate the molecular features and genetic relationships of H9N2 viruses prevalent in this region. Analysis of the gene sequences of all eight RNA segments from 12 viruses isolated between 2005 and 2008 was undertaken. The hemagglutinin (HA) sequences of all isolates were closely related to H9N2 viruses isolated from Iran between 2004 and 2007 and contained leucine instead of glutamine at position 226 in the receptor binding pocket, a recognised marker for the recognition of sialic acids linked ?2–6 to galactose. The neuraminidase (NA) of two isolates contained a unique five residue deletion in the stalk (from residues 80 to 84), a possible indication of greater adaptation of these viruses to the chicken host. The HA, NA, nucleoprotein (NP), and matrix (M) genes showed close identity with H9N2 viruses isolated during 1999 in Pakistan and clustered in the A/Quail/Hong Kong/G1/97 virus lineage. In contrast, the polymerase genes clustered with H9N2 viruses from India, Iran and Dubai. The NS gene segment showed greater genetic diversity and shared a high level of similarity with NS genes from either H5 or H7 subtypes rather than with established H9N2 Eurasian lineages. These results indicate that during recent years the H9N2 viruses have undergone extensive genetic reassortment which has led to the generation of H9N2 viruses of novel genotypes in the Indian sub-continent. The novel genotypes of H9N2 viruses may play a role in the increased problems observed by H9N2 to poultry and reinforce the continued need to monitor H9N2 infections for their zoonotic potential.
Cottam E M, Wadsworth J, Shaw A E, Rowlands R J, Goatley L, Maan S, Maan N S, Mertens P P C, Ebert K, Li Y, Ryan E D, Juleff N, Ferris N P, Wilesmith J W, Haydon D, King D P, Paton D J, Knowles N J (2008)

Transmission pathways of foot-and-mouth disease virus in the United Kingdom in 2007

PLoS Pathogens 4 (4), e1000050
Publisher’s version: https://dx.doi.org/10.1371/journal.ppat.1000050

Abstract

Foot-and-mouth disease (FMD) virus causes an acute vesicular disease of domesticated and wild ruminants and pigs. Identifying sources of FMD outbreaks is often confounded by incomplete epidemiological evidence and the numerous routes by which virus can spread (movements of infected animals or their products, contaminated persons, objects, and aerosols). Here, we show that the outbreaks of FMD in the United Kingdom in August 2007 were caused by a derivative of FMDV O1 BFS 1860, a virus strain handled at two FMD laboratories located on a single site at Pirbright in Surrey. Genetic analysis of complete viral genomes generated in real-time reveals a probable chain of transmission events, predicting undisclosed infected premises, and connecting the second cluster of outbreaks in September to those in August. Complete genome sequence analysis of FMD viruses conducted in real-time have identified the initial and intermediate sources of these outbreaks and demonstrate the value of such techniques in providing information useful to contemporary disease control programmes.

Langner K F A, Darpel K E, Drolet B S, Fischer A, Hampel S, Heselhaus J E, Mellor P S, Mertens P P C, Leibold W (2008)

Comparison of cellular and humoral immunoassays for the assessment of summer eczema in horses

Veterinary Immunology and Immunopathology 122 (01-Feb), 126-137
Publisher’s version: http://dx.doi.org/10.1016/j.vetimm.2007.11.001

Abstract

The objective of this study was to compare and analyze three common diagnostic methods for summer eczema (SE) in horses, an allergic dermatitis caused by bites of Culicoides spp. Nine horses with a medical history of SE and nine control animals were intradermally challenged with whole body extracts (WBE) and the saliva of a native (C. nubeculosus) and exotic (C. sonorensis) Culicoides species. Blood and serum samples of the horses were examined for basophil reactivity by a histamine release test (HRT) and for Culicoides-specific serum immunoglobulin E (IgE) and G (IgG) by enzyme-linked immunosorbent assay (ELISA). The results of intradermal testing (IDT) at 30 min (immediate reactivity) and 4 h (late-phase reactivity) post challenge with most insect preparations revealed significant differences between horses with and without SE. Overall, the HRT showed the most accurate results with a sensitivity of 1.00 for all Culicoides preparations and specificities of 0.78 (WBE) and 1.00 (saliva). By contrast, delayed reactions of the IDT (24 h), and levels of Culicoides-specific IgE and IgG in the native serum showed little or no distinction between allergic and non-allergic horses. However, the use of purified serum IgE and IgG indicated the possibility for elevated titers of insect-specific serum immunoglobulins in horses with SE. The IDT and HRT did not reveal obvious differences in onset and intensity of positive reactions for the native verses exotic Culicoides species, whereas the ELISA showed slightly higher numbers of positive reactions for serum IgG with the indigenous species. Saliva, as compared to WBE, was found to have improved sensitivity and/or specificity for the HRT and for the late-phase immune reactions as measured by the IDT. Overall, the results indicate that allergy tests utilizing effector cells (mast cells, basophils) are more accurate in diagnosing SE in horses than serological analysis by ELISA.

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