You are here

Home » Publications

Publications

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

There were a total of 2609 results for your search.
Zunszain P A, Knox S R, Sweeney T R, Yang J, Roque-Rosell N, Belsham G J, Leatherbarrow R J, Curry S (2010)

Insights into cleavage specificity from the crystal structure of foot-and-mouth disease virus 3C protease complexed with a peptide substrate

Journal of Molecular Biology 395 (2), 375-389
Publisher’s version: https://doi.org/10.1016/j.jmb.2009.10.048

Abstract

Picornavirus replication is critically dependent on the correct processing of a polyprotein precursor by 3C protease(s) (3Cpro) at multiple specific sites with related but non-identical sequences. To investigate the structural basis of its cleavage specificity, we performed the first crystallographic structural analysis of non-covalent complexes of a picornavirus 3Cpro with peptide substrates. The X-ray crystal structure of the foot-and-mouth disease virus 3Cpro, mutated to replace the catalytic Cys by Ala and bound to a peptide (APAKQ|LLNFD) corresponding to the P5–P5′ region of the VP1-2A cleavage junction in the viral polyprotein, was determined up to 2.5 Å resolution. Comparison with free enzyme reveals significant conformational changes in 3Cpro on substrate binding that lead to the formation of an extended interface of contact primarily involving the P4–P2′ positions of the peptide. Strikingly, the deep S1′ specificity pocket needed to accommodate P1′-Leu only forms when the peptide binds. Substrate specificity was investigated using peptide cleavage assays to show the impact of amino acid substitutions within the P5–P4′ region of synthetic substrates. The structure of the enzyme–peptide complex explains the marked substrate preferences for particular P4, P2 and P1 residue types, as well as the relative promiscuity at P3 and on the P′ side of the scissile bond. Furthermore, crystallographic analysis of the complex with a modified VP1-2A peptide (APAKE|LLNFD) containing a Gln-to-Glu substitution reveals an identical mode of peptide binding and explains the ability of foot-and-mouth disease virus 3Cpro to cleave sequences containing either P1-Gln or P1-Glu. Structure-based mutagenesis was used to probe interactions within the S1′ specificity pocket and to provide direct evidence of the important contribution made by Asp84 of the Cys-His-Asp catalytic triad to proteolytic activity. Our results provide a new level of detail in our understanding of the structural basis of polyprotein cleavage by 3Cpro.

Maringer K, Elliott G (2010)

Recruitment of Herpes simplex virus type 1 immediate-early protein ICP0 to the virus particle

Journal of Virology 84 (9), 4682-4696
Publisher’s version: https://doi.org/10.1128/JVI.00126-10

Abstract

Although the herpes simplex virus type 1 (HSV-1) tegument is comprised of a large number of viral and cellular proteins, how and where in the cell these proteins are recruited into the virus structure is poorly understood. We have shown previously that the immediate-early gene product ICP0 is packaged by a mechanism dependent on the major tegument protein VP22, while others have shown a requirement for ICP27. We now extend our studies to show that ICP0 packaging correlates directly with the ability of ICP0 to complex with VP22 in infected cells. ICP27 is not, however, present in this VP22-ICP0 complex but is packaged into the virion in a VP22- and ICP0-independent manner. Biochemical fractionation of virions indicated that ICP0 associates tightly with the virus capsid, but intranuclear capsids contained no detectable ICP0. The RING finger domain of ICP0 and the N terminus of VP22 were both shown to be essential but not sufficient for ICP0 packaging and complex formation. Strikingly, however, the N-terminal region of VP22, while unable to form a complex with ICP0, inhibited its translocation from the nucleus to the cytoplasm. PML degradation by ICP0 was efficient in cells infected with this VP22 mutant virus, confirming that ICP0 retains activity. Hence, we would suggest that VP22 is an important molecular partner of ICP0 that controls at least one of its activities: its assembly into the virion. Moreover, we propose that the pathway by which VP22 recruits ICP0 to the virion may begin in the nucleus prior to ICP0 translocation to its final site of assembly in the cytoplasm.

Dietrich I, Macintyre A, McMonagle E, Price A J, James L C, McEwan W A, Hosie M J, Willett B J (2010)

Potent lentiviral restriction by a synthetic feline TRIM5 cyclophilin A fusion

Journal of Virology 84 (17), 8980-8985
Publisher’s version: https://doi.org/10.1128/jvi.00858-10

Abstract

A synthetic feline TRIM5-cyclophilin A fusion protein (feTRIMCyp) was generated and transduced into feline cells. feTRIMCyp was highly efficient at preventing infection with human (HIV) and feline (FIV) immunodeficiency virus pseudotypes, and feTRIMCyp-expressing cells resisted productive infection with either FIV-Fca or FIV-Pco. The restriction of FIV infection by feTRIMCyp was reversed by the cyclosporine (Cs) derivatives NIM811 and Debio-025 but less so by Cs itself. FeTRIMCyp and FIV infections of the cat offer a unique opportunity to evaluate TRIMCyp-based approaches to genetic therapy for HIV infection and the treatment of AIDS.

Limon G, Guitian J, Gregory N G (2010)

An evaluation of the humaneness of puntilla in cattle

Meat Science 84 (3), 352-355
Publisher’s version: https://doi.org/10.1016/j.meatsci.2009.09.001

Abstract

Slaughter by puntilla followed by neck sticking was examined in 309 cattle, to assess the humaneness of this method. After the neck stab, brain and spinal function as well as presence of selected cognitive responses were measured. In addition breed, sex, live weight, body condition score, number of stabs given and level of experience of the slaughterman were recorded. Repeat stabbing was needed to penetrate the foramen ovale in 24% of the animals, and was significantly less frequent in slaughtermen who were experienced, and more frequent in heavy weight animals (>380 kg). Prevalence of brain and spinal function was 91%. When animals attempted to stand after the neck stab they were more likely to have rhythmic breathing, positive palpebral response and responsiveness to threat, noise and short air stimulus. These findings indicate that nerve pathways are often functional after neck stab and therefore it is highly likely that the animals are still conscious.

Banyard A C, Simpson J, Monaghan P, Barrett T (2010)

Rinderpest virus expressing enhanced green fluorescent protein as a separate transcription unit retains pathogenicity for cattle

Journal of General Virology 91 (12), 2918-2927
Publisher’s version: http://dx.doi.org/10.1099/vir.0.023598-0

Abstract

A full-length DNA clone of a virulent strain of rinderpest virus was constructed with the gene for the enhanced green fluorescent protein (eGFP) inserted as a separate transcription unit between the P and M genes. Rescue of the virus from the modified clone using reverse genetics generated a virus that grew to the same levels as the virus rescued from the unmodified DNA clone in cell culture. The recombinant virus expressed eGFP to a high level and was used to follow virus replication in real-time using live-cell imaging. Cattle infected with both the recombinant wild-type virus and the recombinant eGFP expressing virus developed clinical disease similar to that of the wild-type natural virus isolate. Detection of virus in circulating peripheral blood leukocytes was equivalent to that of the animals infected with the wild-type virus. The high level of expression of soluble eGFP by this virus allowed us to detect viral replication in infected animals by confocal microscopy. Imaging vibrating microtome sections by confocal microscopy provided good preservation of tissue and cellular architecture as well as revealing the sites of replication of the virus in different tissues of infected animals.
Gale P, Estrada-Peña A, Martinez M, Ulrich R G, Wilson A, Capelli G, Phipps P, De La Torre A, Muñoz M J, Dottori M, Mioulet V, Fooks A R (2010)

The feasibility of developing a risk assessment for the impact of climate change on the emergence of Crimean-Congo haemorrhagic fever in livestock in Europe: a review

Journal of Applied Microbiology 108 (6), 1859-1870
Publisher’s version: http://dx.doi.org/10.1111/j.1365-2672.2009.04638.x

Abstract

Crimean-Congo haemorrhagic fever virus (CCHFV) is one of the most widespread of all medically important arboviruses with ticks of the Hyalomma spp. serving as the main vectors. Infection of livestock by CCHFV serves as a route of exposure to humans, as a reservoir of disease and as a route of importation. This study discusses the pathways and data requirements for a qualitative risk assessment for the emergence of CCHFV in livestock in Europe. A risk map approach is proposed based on layers that include the potential routes of release (e.g. by migrating birds carrying infected ticks) together with the main components for exposure, namely the distributions of the tick vectors, the small vertebrate host reservoirs and the livestock. A layer on landscape fragmentation serves as a surrogate for proximity of livestock to the tick cycle. Although the impact of climate change on the emergence of CCHF is not clear, comparing the distribution of risk factors in each layer currently with those predicted in the 2080s with climate change can be used to speculate how potential high-risk areas may shift. According to the risk pathway, transstadial and/or transovarial transmission in the tick vector are crucial for CCHFV spread. Vector competence and tick vector switching, however, remain critical factors for CCHFV colonization of new regions in Europe. The species of migratory bird is also an important consideration in the release assessment with greater abundance and biodiversity of ground-dwelling birds in southern Europe than in northern Europe.

Tarradas J, Argilaguet J M, Rosell R, Nofrarias M, Crisci E, Cordoba L, Perez-Martin E, Diaz I, Rodriguez F, Domingo M, Montoya M, Ganges L (2010)

Interferon-gamma induction correlates with protection by DNA vaccine expressing E2 glycoprotein against classical swine fever virus infection in domestic pigs

Veterinary Microbiology 142 (1-2), 51-58
Publisher’s version: http://dx.doi.org/10.1016/j.vetmic.2009.09.043

Abstract

Classical swine fever (CSF) is a highly contagious viral infection affecting domestic and wild pigs. For classical swine fever virus (CSFV), immunization with plasmids expressing different versions of glycoprotein E2 has proven an effective way to induce protection. Previously, we have also shown that immunization with DNA vaccine expressing glycoprotein E2 (DNA-E2) induced specific T helper cell responses in the absence of neutralizing antibodies. However, the role of T cell responses in protection against CSFV is largely unknown. Here we have extended these studies to deeply characterize the role of T cell responses by a DNA-E2 and their correlation with protection against CSFV infection. Thus, pigs vaccinated with the DNA vaccine induced a strong cellular immune response, characterized by the specific induction IFN-gamma expressing T cells after vaccination without any detectable levels of CSFV neutralizing antibodies. Constant levels of CSFV-specific IFN-gamma producing cells observed from the beginning of the infection until 7 days after challenge in vaccinated animals might contribute to early control of CSFV replication, at least until neutralizing antibodies are developed. Severe clinical signs of the disease, including high titers of viremia, pyrexia and virus spread to different organs, were recorded in the non-vaccinated challenged animals, in comparison to the vaccinated animals where only one animal showed mild clinical signs and a short peak of viremia. Lack of complete protection in this animal correlated with a delay on the induction of neutralizing antibodies, detectable only from day 11 post-CSFV challenge. Conversely, the rest of the pigs within the group developed neutralizing antibodies as early as at day two post-challenge, correlating with sterile protection. Finally, an inverse correlation seemed to exist between early induction of IFN-alpha and the protection observed, while IL-10 seemed to be differentially regulated in vaccinated and non-vaccinated animals. Our results support the relevance of the induction of a strong T cellular response to confer a solid protection upon DNA vaccination against CSFV. Further experiments are needed to be done in order to clarify the key cytokines playing a role in CSFV-protection and to obtain emergency vaccines capable to confer robust and fast protection.
Tuthill T J, Groppelli E, Hogle J M, Rowlands D J (2010)

Picornaviruses.

Current Topics in Microbiology and Immunology (Cell Entry by Non-Enveloped Viruses edited by J E Johnson, Springer) 343, 43-89
Publisher’s version: http://dx.doi.org/10.1007/82_2010_37

Abstract

The picornavirus family consists of a large number of small RNA viruses, many of which are significant pathogens of humans and livestock. They are amongst the simplest of vertebrate viruses comprising a single stranded positive sense RNA genome within a T = 1 (quasi T = 3) icosahedral protein capsid of approximately 30 nm diameter. The structures of a number of picornaviruses have been determined at close to atomic resolution by X-ray crystallography. The structures of cell entry intermediate particles and complexes of virus particles with receptor molecules or antibodies have also been obtained by X-ray crystallography or at a lower resolution by cryo-electron microscopy. Many of the receptors used by different picornaviruses have been identified, and it is becoming increasingly apparent that many use co-receptors and alternative receptors to bind to and infect cells. However, the mechanisms by which these viruses release their genomes and transport them across a cellular membrane to gain access to the cytoplasm are still poorly understood. Indeed, detailed studies of cell entry mechanisms have been made only on a few members of the family, and it is yet to be established how broadly the results of these are applicable across the full spectrum of picornaviruses. Working models of the cell entry process are being developed for the best studied picornaviruses, the enteroviruses. These viruses maintain particle integrity throughout the infection process and function as genome delivery modules. However, there is currently no model to explain how viruses such as cardio- and aphthoviruses that appear to simply dissociate into subunits during uncoating deliver their genomes into the cytoplasm.
Uttenthal A, Parida S, Rasmussen T B, Paton D J, Haas B, Dundon W G (2010)

Strategies for differentiating infection in vaccinated animals (DIVA) for foot-and-mouth disease, classical swine fever and avian influenza

Expert Review of Vaccines 9 (1), 73-87
Publisher’s version: http://dx.doi.org/10.1586/ERV.09.130

Abstract

The prophylactic use of vaccines against exotic viral infections in production animals is undertaken exclusively in regions where the disease concerned is endemic. In such areas, the infection pressure is very high and so, to assure optimal protection, the most efficient vaccines are used. However, in areas considered to be free from these diseases and in which there is the possibility of only limited outbreaks, the use of Differentiation of infected from Vaccinated Animals (DIVA) or marker vaccines allows for vaccination while still retaining the possibility of serological surveillance for the presence of infection. This literature review describes the current knowledge on the use of DIVA diagnostic strategies for three important transboundary animal diseases: foot-and-mouth disease in cloven-hoofed animals, classical swine fever in pigs and avian influenza in poultry.
Velkers F C, Blake D P, Graat E A M, Vernooij J C M, Bouma A, de J M C M, Stegeman J A (2010)

Quantification of Eimeria acervulina in faeces of broilers: Comparison of McMaster oocyst counts from 24 h faecal collections and single droppings to real-time PCR from cloacal swabs

Veterinary Parasitology 169 (1-2), 1-7
Publisher’s version: http://dx.doi.org/10.1016/j.vetpar.2010.01.001

Abstract

Coccidiosis is an economically important disease in chickens, caused by infection with Eimeria species parasites. Diagnosis of coccidiosis is frequently based on oocyst enumeration in pooled faecal samples or litter. In studies on infection dynamics and for monitoring in the field, samples from individual chickens may be more appropriate as these support the determination of infection status of individual birds and more accurately reflect oocyst output at time of sampling. Faecal samples from individual birds can be collected, but the counting procedure limits the number of samples that can be processed and unequivocal microscopic differentiation between Eimeria species is very difficult. A test that overcomes these drawbacks would improve efficiency and quality of the diagnosis. The aim of this study was to compare two methods for Eimeria oocyst quantification in samples from individual birds. A real-time PCR that quantifies oocysts in cloacal swabs (qPCR) and oocyst counts in single droppings were compared to the standard procedure of oocyst counts in bulked 24 h faeces. Faecal samples were collected daily from 30 broiler chickens, inoculated with different doses of Eimeria acervulina. The three techniques produced comparable oocyst counts for all inoculation doses. Single dropping counts are applicable for small sample sizes and when a single Eimeria species is used. For larger sample sizes qPCR is preferable as it can be carried out on samples that have been frozen for storage. Furthermore, qPCR can identify and quantify different Eimeria species, which makes it a valuable diagnostic tool for field or experimental work.

Pages

Filter Publications

Trim content

® The Pirbright Institute 2024 | A company limited by guarantee, registered in England no. 559784. The Institute is also a registered charity.