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

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Chengat Prakashbabu B, Thenmozhi V, Limon G, Kundu K, Kumar S, Garg R, Clark E L, Srinivasa Rao A S, Raj D G, Raman M, Banerjee P S, Tomley F M, Guitian J, Blake D P (2017)

Eimeria species occurrence varies between geographic regions and poultry production systems and may influence parasite genetic diversity

Veterinary Parasitology 233, 62-72


Coccidiosis is one of the biggest challenges faced by the global poultry industry. Recent studies have highlighted the ubiquitous distribution of all Eimeria species which can cause this disease in chickens, but intriguingly revealed a regional divide in genetic diversity and population structure for at least one species, Eimeria tenella. The drivers associated with such distinct geographic variation are unclear, but may impact on the occurrence and extent of resistance to anticoccidial drugs and future subunit vaccines. India is one of the largest poultry producers in the world and includes a transition between E. tenella populations defined by high and low genetic diversity. The aim of this study was to identify risk factors associated with the prevalence of Eimeria species defined by high and low pathogenicity in northern and southern states of India, and seek to understand factors which vary between the regions as possible drivers for differential genetic variation. Faecal samples and data relating to farm characteristics and management were collected from 107 farms from northern India and 133 farms from southern India. Faecal samples were analysed using microscopy and PCR to identify Eimeria occurrence. Multiple correspondence analysis was applied to transform correlated putative risk factors into a smaller number of synthetic uncorrelated factors. Hierarchical cluster analysis was used to identify poultry farm typologies, revealing three distinct clusters in the studied regions. The association between clusters and presence of Eimeria species was assessed by logistic regression. The study found that large-scale broiler farms in the north were at greatest risk of harbouring any Eimeria species and a larger proportion of such farms were positive for E. necatrix, the most pathogenic species. Comparison revealed a more even distribution for E. tenella across production systems in south India, but with a lower overall occurrence. Such a polarised region- and system-specific distribution may contribute to the different levels of genetic diversity observed previously in India and may influence parasite population structure across much of Asia and Africa. The findings of the study can be used to prioritise target farms to launch and optimise appropriate anticoccidial strategies for long-term control.

Limon G, Beauvais W, Dadios N, Villena I, Cockle C, Blaga R, Guitian J (2017)

Cross-sectional study of Toxoplasma gondii infection in pig farms in England

Foodborne Pathogens and Diseases 14 (5), 269-281


Ingestion of undercooked meat has been proposed as an important source of human Toxoplasma gondii infection. To ascertain the contribution of meat consumption to the risk of human infection, estimates of the prevalence of infection in meat-producing animals are required. A cross-sectional study was conducted to assess T. gondii infection in pigs raised in England, to identify risk factors for infection, and to compare performance of two serological tests: modified agglutination test (MAT) and enzyme-linked immunosorbent assay (ELISA). Blood samples from 2071 slaughter pigs originating from 131 farms were collected and 75 (3.6%) were found to be positive by MAT. Positive pigs originated from 24 farms. A subset of samples (n = 492) were tested using ELISA, and a significant disagreement (p < 0.001) was found between the two tests. An empirical Bayes approach was used to estimate the farm-level prevalence and the probability of each individual farm having at least one positive animal, considering the uncertainty arising from the sampling strategy and the imperfect test performance. The adjusted farm-level prevalence was 11.5% (95% credible interval of positive farms 8.4-16.0%). Two different criteria were used for classifying farms as infected: (1) >/=50% probability of having at least one infected pig (n = 5, 6.8%) and (2) >/=10% probability (n = 15, 20.5%). Data on putative risk factors were obtained for 73 farms. Using a 10% cutoff, the relative risk (RR) of infection was higher in farms where cats have direct access to pigs' food (RR = 2.6; p = 0.04), pigs have outdoor access (RR = 3.0; p = 0.04), and farms keeping

Crotta M, Limon G, Blake D P, Guitian J (2017)

Knowledge gaps in host-parasite interaction preclude accurate assessment of meat-borne exposure to Toxoplasma gondii

International Journal of Food Microbiology 261, 95-101


Toxoplasma gondii is recognized as a widely prevalent zoonotic parasite worldwide. Although several studies clearly identified meat products as an important source of T. gondii infections in humans, quantitative understanding of the risk posed to humans through the food chain is surprisingly scant. While probabilistic risk assessments for pathogens such as Campylobacter jejuni, Listeria monocytogenes or Escherichia coli have been well established, attempts to quantify the probability of human exposure to T. gondii through consumption of food products of animal origin are at early stages. The biological complexity of the life cycle of T. gondii and limited understanding of several fundamental aspects of the host/parasite interaction, require the adoption of numerous critical assumptions and significant simplifications. In this study, we present a hypothetical quantitative model for the assessment of human exposure to T. gondii through meat products. The model has been conceptualized to capture the dynamics leading to the presence of parasite in meat and, for illustrative purposes, used to estimate the probability of at least one viable cyst occurring in 100g of fresh pork meat in England. Available data, including the results of a serological survey in pigs raised in England were used as a starting point to implement a probabilistic model and assess the fate of the parasite along the food chain. Uncertainty distributions were included to describe and account for the lack of knowledge where necessary. To quantify the impact of the key model inputs, sensitivity and scenario analyses were performed. The overall probability of 100g of a hypothetical edible tissue containing at least 1 cyst was 5.54%. Sensitivity analysis indicated that the variables exerting the greater effect on the output mean were the number of cysts and number of bradyzoites per cyst. Under the best and the worst scenarios, the probability of a single portion of fresh pork meat containing at least 1 viable cyst resulted 1.14% and 9.97% indicating that the uncertainty and lack of data surrounding key input parameters of the model preclude accurate estimation of T. gondii exposure through consumption of meat products. The hypothetical model conceptualized here is coherent with current knowledge of the biology of the parasite. Simulation outputs clearly identify the key gaps in our knowledge of the host-parasite interaction that, when filled, will support quantitative assessments and much needed accurate estimates of the risk of human exposure.

Gonçalves-Carneiro D, McKeating J A, Bailey D (2017)

The measles virus receptor SLAMF1 can mediate particle endocytosis

Journal of Virology 91 (7),


The signaling lymphocyte activation molecule F1 (SLAMF1) is both a microbial sensor and entry receptor for measles virus (MeV). Herein, we describe a new role for SLAMF1 to mediate MeV endocytosis that is in contrast with the alternative, and generally accepted, model that MeV genome enters cells only after fusion at the cell surface. We demonstrated that MeV engagement of SLAMF1 induces dramatic but transient morphological changes, most prominently in the formation of membrane blebs, which were shown to colocalize with incoming viral particles, and rearrangement of the actin cytoskeleton in infected cells. MeV infection was dependent on these dynamic cytoskeletal changes as well as fluid uptake through a macropinocytosis-like pathway as chemical inhibition of these processes inhibited entry. Moreover, we identified a role for the RhoA-ROCK-myosin II signaling axis in this MeV internalization process, highlighting a novel role for this recently characterized pathway in virus entry. Our study shows that MeV can hijack a microbial sensor normally involved in bacterial phagocytosis to drive endocytosis using a complex pathway that shares features with canonical viral macropinocytosis, phagocytosis, and mechanotransduction. This uptake pathway is specific to SLAMF1-positive cells and occurs within 60 min of viral attachment. Measles virus remains a significant cause of mortality in human populations, and this research sheds new light on the very first steps of infection of this important pathogen.

Measles is a significant disease in humans and is estimated to have killed over 200 million people since records began. According to current World Health Organization statistics, it still kills over 100,000 people a year, mostly children in the developing world. The causative agent, measles virus, is a small enveloped RNA virus that infects a broad range of cells during infection. In particular, immune cells are infected via interactions between glycoproteins found on the surface of the virus and SLAMF1, the immune cell receptor. In this study, we have investigated the steps governing entry of measles virus into SLAMF1-positive cells and identified endocytic uptake of viral particles. This research will impact our understanding of morbillivirus-related immunosuppression as well as the application of measles virus as an oncolytic therapeutic.

Patten D A, Wilson G K, Bailey D, Shaw R K, Jalkanen S, Salmi M, Rot A, Weston C J, Adams D H, Shetty S (2017)

Human liver sinusoidal endothelial cells promote intracellular crawling of lymphocytes during recruitment: A new step in migration

Hepatology 65 (1), 294-309


The recruitment of lymphocytes via the hepatic sinusoidal channels and positioning within liver tissue is a critical event in the development and persistence of chronic inflammatory liver diseases. The hepatic sinusoid is a unique vascular bed lined by hepatic sinusoidal endothelial cells (HSECs), a functionally and phenotypically distinct subpopulation of endothelial cells. Using flow-based adhesion assays to study the migration of lymphocytes across primary human HSECs, we found that lymphocytes enter into HSECs, confirmed by electron microscopy demonstrating clear intracellular localization of lymphocytes in vitro and by studies in human liver tissues. Stimulation by interferon-gamma increased intracellular localization of lymphocytes within HSECs. Furthermore, using confocal imaging and time-lapse recordings, we demonstrated "intracellular crawling" of lymphocytes entering into one endothelial cell from another. This required the expression of intracellular adhesion molecule-1 and stabilin-1 and was facilitated by the junctional complexes between HSECs.

CONCLUSION: Lymphocyte migration is facilitated by the unique structure of HSECs. Intracellular crawling may contribute to optimal lymphocyte positioning in liver tissue during chronic hepatitis. (Hepatology 2017;65:294-309).

Malik N, Kotecha A, Gold S, Asfor A, Ren J, Huiskonen J T, Tuthill T J, Fry E E, Stuart D I (2017)

Structures of foot and mouth disease virus pentamers: Insight into capsid dissociation and unexpected pentamer reassociation

PLOS Pathogens 13 (9), e1006607


This is the first structure of dissociated pentamers for FMDV, and indeed for any picornavirus. We also present the first visualization of a picornavirus particle assembled from pentamers comprising VP1, 2 and 3. The formation of regular assemblies rather than random aggregates has enabled us to solve the structure to relatively high resolution using cryo-electron microscopy. Overall there are remarkably few large-scale conformational changes in the dis-assembled pentamer from its structure in the intact virion, suggesting that the mature virus pentamer is essentially a stable endpoint. However, a small rotation in VP3 abrogates the usual pentamer interface and together with the lack of VP4 drives the abnormal inside-out association of these pentameric subunits, rather than the correct capsid assembly. This change in conformation of the dis-assembled pentamer changes the antigenic surface and presumably underlies, in part, the loss of efficacy of vaccines if native particle integrity is destroyed.

Herod M R, Gold S, Lasecka-Dykes L, Wright C, Ward J C, McLean T C, Forrest S, Jackson T, Tuthill T J, Rowlands D J, Stonehouse N J (2017)

Genetic economy in picornaviruses: foot-and-mouth disease virus replication exploits alternative precursor cleavage pathways

PLoS Pathogens 13 (10), e1006666


The RNA genomes of picornaviruses are translated into single polyproteins which are subsequently cleaved that into structural and non-structural protein products. For genetic economy, proteins and processing intermediates have evolved to perform distinct functions. The picornavirus precursor protein, P3, is cleaved to produce membrane-associated 3A, primer peptide 3B, protease 3Cpro and polymerase 3Dpol. Uniquely, foot-and-mouth disease virus (FMDV) encodes three similar copies of 3B (3B1-3), thus providing a convenient natural system to explore the role(s) of 3B in the processing cascade. Using a replicon system, we confirmed by genetic deletion or functional inactivation that each copy of 3B appears to function independently to prime FMDV RNA replication. However, we also show that deletion of 3B3 prevents replication and that this could be reversed by introducing mutations at the C-terminus of 3B2 that restored the natural sequence at the 3B3-3C cleavage site. In vitro translation studies showed that precursors with 3B3 deleted were rapidly cleaved to produce 3CD but that no polymerase, 3Dpol, was detected. Complementation assays, using distinguishable replicons bearing different inactivating mutations, showed that replicons with mutations within 3Dpol could be recovered by 3Dpol derived from "helper" replicons (incorporating inactivation mutations in all three copies of 3B). However, complementation was not observed when the natural 3B-3C cleavage site was altered in the "helper" replicon, again suggesting that a processing abnormality at this position prevented the production of 3Dpol. When mutations affecting polyprotein processing were introduced into an infectious clone, viable viruses were recovered but these had acquired compensatory mutations in the 3B-3C cleavage site. These mutations were shown to restore the wild-type processing characteristics when analysed in an in vitro processing assay. Overall, this study demonstrates a dual functional role of the small primer peptide 3B3, further highlighting how picornaviruses increase genetic economy.

Reis A L, Goatley L C, Jabbar T, Sanchez-Cordon P J, Netherton C L, Chapman D G, Dixon L K (2017)

Deletion of the African swine fever virus gene DP148R does not reduce virus replication in culture but reduces virus virulence in pigs and induces high levels of protection against challenge

Journal of Virology early view,


Many of the approximately 165 proteins encoded by African swine fever virus do not have significant similarity to known proteins and have not been studied experimentally. One such protein is DP148R. We showed that the DP148R gene is transcribed at early times post-infection. Deletion of this gene did not reduce virus replication in macrophages showing that is not essential for replication in these cells. However deleting this gene from a virulent isolate, Benin 97/1, dramatically reduced the virulence of the virus in vivo. All pigs infected with the BeninΔDP148R virus survived infection showing only transient mild clinical signs soon after immunisation. Following challenge with the parental virulent virus all pigs immunised by the intramuscular route (11/11) and all except one immunised by the intranasal route (5/6) survived. Mild or no clinical signs were observed after challenge. As expected control non-immune pigs developed signs of acute ASF. Virus genome and infectious virus were observed soon after immunisation coincident with the onset of clinical signs (∼106 genome copies or TCID50/ml). Levels of virus genome declined over an extended period of up to 60 days post-immunisation. In contrast infectious virus was no longer detectable by days 30 to 35. IFN-γ was detected in serum between days 4 and 7 post-immunisation, and IFN-γ producing cells were detected in all pigs analysed following stimulation of immune lymphocytes with whole virus. ASFV specific antibodies were first detected from day 10 post-immunisation.

IMPORTANCE: African swine fever (ASF) is endemic in Africa, parts of the Trans Caucasus, Russian Federation and several European countries. The lack of a vaccine hinders control. Many of the ASF virus genes lack similarity to known genes and have not been characterised. We have shown that one of these, DP148R, is transcribed early during virus replication in cells and can be deleted from the virus genome without reducing virus replication. The gene deleted virus, BeninΔDP148R caused mild clinical signs in pigs and induced high levels of protection against challenge with parental virulent virus. Therefore deletion of this gene can provide a target for rational development of vaccines.

Soria I, Quattrocchi V, Langellotti C, Gammella M, Digiacomo S, Garcia de la Torre B, Andreu D, Montoya M, Sobrino F, Blanco E, Zamorano P (2017)

Dendrimeric peptides can confer protection against foot-and-mouth disease virus in cattle

PLoS One 12 (9), e0185184


Foot-and-mouth disease virus (FMDV) causes a highly contagious disease in cloven-hoofed animals. A synthetic vaccine candidate consisting of dendrimeric peptides harbouring two copies of a B-epitope [VP1(136–154)] linked to a T-cell epitope [3A(21–35)] of FMDV confers protection to type O FMDV challenge in pigs. Herein we show in cattle that novel dendrimeric peptides bearing a T-cell epitope [VP1(21–40] and two or four copies of a B-cell epitope [VP1(135–160)] from type O1 Campos FMDV (termed B2T and B4T, respectively) elicited FMDV specific immune responses to similar levels to a commercial vaccine. Animals were challenged with FMDV and 100% of vaccinated cattle with B2T or B4T were protected to podal generalization. Moreover, bovines immunized with B4T were completely protected (with no clinical signs) against FMDV challenge after three vaccine doses, which was associated with titers of viral neutralizing antibodies in serum higher than those of B2T group (p< 0.05) and levels of opsonic antibodies similar to those of animals immunized with one dose of FMDV commercial vaccine. Bovines vaccinated with both dendrimeric peptides presented high levels of IgG1 anti FMDV in sera and in mucosa. When IgA in nasal secretions was measured, 20% or 40% of the animals in B2T or B4T groups respectively, showed anti-FMDV IgA titers. In addition, B2T and B4T peptides evoked similar consistent T cell responses, being recognized in vitro by lymphocytes from most of the immunized cattle in the proliferation assay, and from all animals in the IFN -γ production assay. Taken together, these results support the potential of dendrimers B2T or B4T in cattle as a highly valuable, cost-effective FMDV candidate vaccine with DIVA potential.

Xu J, Zhang X, Zhou S, Shen J, Yang D, Wu J, Li X, Li M, Huang X, Sealy J E, Iqbal M, Li Y (2017)

A DNA aptamer efficiently inhibits the infectivity of bovine herpesvirus 1 by blocking viral entry

Scientific Reports 7 (1), 11796


Bovine herpesvirus 1 (BoHV-1) is an important pathogen of domestic and wild cattle responsible for major economic losses in dairy and beef industries throughout the world. Inhibition of viral entry plays a crucial role in the control of BoHV-1 infection and aptamers have been reported to inhibit viral replication. In this study, nine DNA aptamers that target BoHV-1 were generated using systemic evolution of ligands by exponential enrichment. Of the nine candidates, aptamer IBRV-A4 exhibited the highest affinity and specificity for BoHV-1, which bound to BoHV-1 with a Kd value of 3.519?nM and demonstrated the greatest virus binding as shown by fluorescence imaging. The neutralizing ability of aptamer IBRV-A4 was determined using neutralization assays and real time PCR in BoHV-1 infected Madin-darby bovine kidney cells. Virus titration, immunofluorescence and confocal laser scanning microscopy showed virus replication significantly decreased when aptamer IBRV-A4 was added to BoHV-1 infected MDBK cells at 0 and 0.5?hours post-infection, whereas no change was seen when IBRV-A4 was added 2?hours post-infection. This concludes that aptamer IBRV-A4 efficiently inhibits viral entry of BoHV-1 in MDBK cells and is therefore a novel tool for diagnosis and treatment of BoHV-1 infection in cattle.


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