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

Equine rhinitis A virus (ERAV) shares many features with foot-and-mouth disease virus (FMDV) and both are classified within the genus Aphthovirus of the family Picornaviridae. ERAV is used as a surrogate for FMDV research as it does not require high-level biosecurity. In contrast to FMDV, which uses integrins as cellular receptors, the receptor for ERAV has been reported to involve the sugar moiety sialic acid. This study confirmed the importance of sialic acid for cell entry by ERAV and reports the crystal structure of ERAV particles complexed with the receptor analogue 3?-sialyllactose. The receptor is attached to the rim of a capsid pit adjacent to the major immunogenic site and distinct from the sialic acid binding site used by a related picornavirus, the cardiovirus Theiler's murine encephalitis virus. The structure of the major antigenic determinant of the virus, previously identified from antibody escape mutations, is also described as the EF loop of VP1, which forms a hairpin stretching across the capsid surface close to the icosahedral fivefold axis, neighbouring the receptor-binding site, and spanning two protomeric units.
Fu G L, Lees R S, Nimmo D, Aw D, Jin L, Gray P, Berendonk T U, White-Cooper H, Scaife S, Phuc H K, Marinotti O, Jasinskiene N, James A A, Alphey L (2010)

Female-specific flightless phenotype for mosquito control

Proceedings of the National Academy of Sciences of the United States of America 107 (10), 4550-4554

Abstract

Dengue and dengue hemorrhagic fever are increasing public health problems with an estimated 50-100 million new infections each year. Aedes aegypti is the major vector of dengue viruses in its range and control of this mosquito would reduce significantly human morbidity and mortality. Present mosquito control methods are not sufficiently effective and new approaches are needed urgently. A "sterile-male-release" strategy based on the release of mosquitoes carrying a conditional dominant lethal gene is an attractive new control methodology. Transgenic strains of Aedes aegypti were engineered to have a repressible female-specific flightless phenotype using either two separate transgenes or a single transgene, based on the use of a female-specific indirect flight muscle promoter from the Aedes aegypti Actin-4 gene. These strains eliminate the need for sterilization by irradiation, permit male-only release ("genetic sexing"), and enable the release of eggs instead of adults. Furthermore, these strains are expected to facilitate area-wide control or elimination of dengue if adopted as part of an integrated pest management strategy.

Abstract

For many viruses, endocytosis and exposure to the low pH within acidic endosomes is essential for infection. It has previously been reported that feline calicivirus uses clathrin-mediated endocytosis for entry into mammalian cells. Here, we report that infection of RAW264 7 macrophages by the closely related murine norovirus-1 (MNV-1) does not require the clathrin pathway, as infection was not inhibited by expression of dominant-negative Eps15 or by knockdown of the adaptin-2 complex. Further, infection was not inhibited by reagents that raise endosomal pH RAW264.7 macrophages were shown not to express caveolin, and flotillin depletion did not inhibit infection, suggesting that caveolae and the flotillin pathway are not required for cell entry However, MNV-1 infection was inhibited by methyl-beta-cyclodextrin and the dynamin inhibitor, dynasore. Addition of these drugs to the cells after a period of virus internalization did not inhibit infection, suggesting the involvement of cholesterol-sensitive lipid rafts and dynamin in the entry mechanism. Macropinocytosis (MPC) was shown to be active in RAW264 7 macrophages (as indicated by uptake of dextran) and could be blocked by 5-(N-ethyl-N-isopropyl) amiloride (EIPA), which is reported to inhibit this pathway. However, infection was enhanced in the presence of EIPA Similarly, actin disruption, which also inhibits MPC, resulted in enhanced infection These results suggest that MPC could contribute to virus degradation or that inhibition of MPC could lead to the upregulation of other endocytic pathways of virus uptake.
Gloster J, Jones A, Redington A, Burgin L, Sorensen J H, Turner R, Dillon M, Hullinger P, Simpson M, Astrup P, Garner G, Stewart P, D'Amours R, Sellers R, Paton D (2010)

Airborne spread of foot-and-mouth disease – model intercomparison

Veterinary Journal 183 (3), 278-286

Abstract

Foot-and-mouth disease virus (FMDV) spreads by direct contact between animals, by animal products (milk, meat and semen), by mechanical transfer on people or fomites and by the airborne route, with the relative importance of each mechanism depending on the particular outbreak characteristics. Atmospheric dispersion models have been developed to assess airborne spread of FMDV in a number of countries, including the UK, Denmark, Australia, New Zealand, USA and Canada. These models were compared at a Workshop hosted by the Institute for Animal Health/Met Office in 2008. Each modeller was provided with data relating to the 1967 outbreak of FMD in Hampshire, UK, and asked to predict the spread of FMDV by the airborne route. A number of key issues emerged from the Workshop and subsequent modelling work: (1) in general all models predicted similar directions for livestock at risk, with much of the remaining differences strongly related to differences in the meteorological data used; (2) determination of an accurate sequence of events on the infected premises is highly important, especially if the meteorological conditions vary substantially during the virus emission period; (3) differences in assumptions made about virus release, environmental fate and susceptibility to airborne infection can substantially modify the size and location of the downwind risk area. All of the atmospheric dispersion models compared at the Workshop can be used to assess windborne spread of FMDV and provide scientific advice to those responsible for making control and eradication decisions in the event of an outbreak of disease.

Abstract

Acid dependent infection of Hela and Vero cells by BTV-10 occurs from within early-endosomes following virus uptake by clathrin-mediated endocytosis (Forzan et al., 2007: J Virol 81: 4819-4827). Here we report that BTV-1 infection of BHK cells is also dependent on a low endosomal pH; however, virus entry and infection were not inhibited by dominant-negative mutants of Eps15, AP180 or the 'aa' splice variant of dynamin-2, which were shown to inhibit clathrin-mediated endocytosis. In addition, infection was not inhibited by depletion of cellular cholesterol, which suggests that virus entry is not mediated by a lipid-raft dependent process such as caveolae-mediated endocytosis. Although virus entry and infection were not inhibited by the dominant-negative dynamin-2 mutant, entry was inhibited by the general dynamin inhibitor, dynasore, indicating that virus entry is dynamin dependent. During entry, BTV-1 co-localised with LAMP-1 but not with transferrin, suggesting that virus is delivered to late-endosomal compartments without first passing through early-endosomes. BTV-1 entry and infection were inhibited by EIPA and cytochalasin-D, known macropinocytosis inhibitors, and during entry virus co-localised with dextran, a known marker for macropinocytosis/fluid-phase uptake. Our results extend earlier observations with BTV-10, and show that BTV-1 can infect BHK cells via an entry mechanism that is clathrin and cholesterol-independent, but requires dynamin, and shares certain characteristics in common with macropinocytosis.

Abstract

The American grass carp reovirus (AGCRV) Aquareovirus G is not strongly associated with disease in fish, but it is often detected by cell culture during routine inspections of healthy fish. The cytopathic effect of AGCRV does not involve the typical syncytia associated with most aquareoviruses. Instead, the AGCRV produces a pattern of cell rounding that is very similar to that produced by rhabdoviruses, including those that are highly regulated. We have developed a quantitative polymerase chain reaction assay that can be used to identify AGCRV in cell cultures or directly on fish tissues. The assay detects as few as two copies of the plasmid template, has a coefficient of variation of 15% among assays performed on different days, and does not cross-react with any other aquareoviruses tested. Assays performed on tissues of cultured golden shiners Notemigonus crysoleucas and fathead minnow Pimephales promelas revealed a high prevalence of infection among healthy fish but no association with disease.
Graham J F, Agarwal S, Kurian D, Kirby L, Pinheiro T J T, Gill A C (2010)

Low density subcellular fractions enhance disease-specific prion protein misfolding

Journal of Biological Chemistry 285 (13), 9868-9880

Abstract

The production of prion particles in vitro by amplification with or without exogenous seed typically results in infectivity titers less than those associated with PrPSc isolated ex vivo and highlights the potential role of co-factors that can catalyze disease-specific prion protein misfolding in vivo. We used a cell-free conversion assay previously shown to replicate many aspects of transmissible spongiform encephalopathy disease to investigate the cellular location of disease-specific co-factors using fractions derived from gradient centrifugation of a scrapie-susceptible cell line. Fractions from the low density region of the gradient doubled the efficiency of conversion of recombinant PrP. These fractions contain plasma membrane and cytoplasmic proteins, and conversion enhancement can be achieved using PrPSc derived from two different strains of mouse-passaged scrapie as seed. Equivalent fractions from a second scrapie-susceptible cell line also stimulate conversion. We also show that subcellular fractions enhancing disease-specific prion protein conversion prevent in vitro fibrillization of recombinant prion protein, suggesting the existence of separate, competing mechanisms of disease-specific and nonspecific misfolding in vivo.

Abstract

Existing live attenuated classical swine fever virus (CSFV) vaccines provide a rapid onset of complete protection but pose problems in discriminating infected amongst vaccinated animals. With a view to providing additional information on the cellular mechanisms that may contribute to protection, which in turn may aid the development of the next generation of CSFV vaccines, we explored the kinetics of the cytokine responses from peripheral blood cells of pigs vaccinated with an attenuated C-strain vaccine strain and/or infected with a recent CSFV isolate. Peripheral blood cells were isolated over the course of vaccination/infection and stimulated in vitro with C-strain or UK2000/7.1 viruses. Virus-specific responses of peripheral blood cells isolated from C-strain vaccinated pigs were dominated by the production of IFN-?. IFN-? production in response to the C-strain virus was first detected in vaccinates 9 days post-vaccination and was sustained over the period of observation. In contrast, cells from challenge control animals did not secrete IFN-? in response to stimulation with C-strain or UK2000/7.1 viruses. Supernatants from UK2000/7.1 infected animals contained significant levels of pro-inflammatory cytokines from day 8 post-infection and these cytokines were present in both virus and mock stimulated cultures. The results suggest that the C-strain virus is a potent inducer of a type-1 T cell response, which may play a role in the protection afforded by such vaccines, whereas the pro-inflammatory cytokine responses observed in cultures from infected pigs may reflect a pathological pro-inflammatory cascade initiated in vivo following the replication and spread of CSFV.

Abstract

The nucleocapsid (N) protein of dolphin morbillivirus (DMV) was expressed from a baculovirus (Autographa californica nuclear polyhedrosis virus) vector and shown by SDS-PAGE and Western blot analysis to be about 57 kDa. Transmission electron microscopy revealed fully assembled nucleocapsid-like particles (NLPs) exhibiting the typical helical herringbone morphology. These NLPs were approximately 20-22 nm in diameter and varied in length from 50 to 100 nm. Purified DMV-N protein was used as antigen in an indirect ELISA (iELISA) and shown to react with rabbit and human antisera to measles virus (MV) and dog sera with antibodies to canine distemper virus (CDV). The iELISA was used for the demonstration of morbillivirus antibodies in the serum of cetaceans and manatees, showing potential as a serological tool for the mass screening of morbillivirus antibodies in marine mammals.

Abstract

Equine rhinitis A virus (ERAV) is genetically closely related to foot-and-mouth disease virus (FMDV), and both are now classified within the genus Aphthovirus of the family Picornaviridae. For disease security reasons, FMDV can be handled only in high-containment facilities, but these constraints do not apply to ERAV, making it an attractive alternative for the study of aphthovirus biology. Here, we show, using immunofluorescence, pharmacological agents, and dominant negative inhibitors, that ERAV entry occurs (as for FMDV) via clathrin-mediated endocytosis and acidification of early endosomes. This validates the use of ERAV as a model system to study the mechanism of cell entry by FMDV.

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