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

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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.

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.

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 91 (24), e01428-17


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.

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.

Lee H J, Lee K Y, Park Y H, Choi H J, Yao Y, Nair V, Han J Y (2017)

Acquisition of resistance to avian leukosis virus subgroup B through mutations on tvb cysteine-rich domains in DF-1 chicken fibroblasts

Veterinary Research 48 (1), 48


Avian leukosis virus (ALV) is a retrovirus that causes tumors in avian species, and its vertical and horizontal transmission in poultry flocks results in enormous economic losses. Despite the discovery of specific host receptors, there have been few reports on the modulation of viral susceptibility via genetic modification. We therefore engineered acquired resistance to ALV subgroup B using CRISPR/Cas9-mediated genome editing technology in DF-1 chicken fibroblasts. Using this method, we efficiently modified the tumor virus locus B (tvb) gene, encoding the TVB receptor, which is essential for ALV subgroup B entry into host cells. By expanding individual DF-1 clones, we established that artificially generated premature stop codons in the cysteine-rich domain (CRD) of TVB receptor confer resistance to ALV subgroup B. Furthermore, we found that a cysteine residue (C80) of CRD2 plays a crucial role in ALV subgroup B entry. These results suggest that CRISPR/Cas9-mediated genome editing can be used to efficiently modify avian cells and establish novel chicken cell lines with resistance to viral infection.

Lee H J, Lee K Y, Jung K M, Park K J, Lee K O, Suh J-Y, Yao Y, Nair V, Han J Y (2017)

Precise gene editing of chicken Na+/H+ exchange type 1 (chNHE1) confers resistance to avian leukosis virus subgroup J (ALV-J)

Developmental and Comparative Immunology 77 (Supp C), 340-349


Avian leukosis virus subgroup J (ALV-J), first isolated in the late 1980s, has caused economic losses to the poultry industry in many countries. As all chicken lines studied to date are susceptible to ALV infection, there is enormous interest in developing resistant chicken lines. The ALV-J receptor, chicken Na+/H+ exchange 1 (chNHE1) and the critical amino acid sequences involved in viral attachment and entry have already been characterized. However, there are no reported attempts to induce resistance to the virus by targeted genome modification of the receptor sequences. In an attempt to induce resistance to ALV-J infection, we used clustered regularly interspaced short palindromic repeats (CRISPR)-associated (CRISPR/Cas9)-based genome editing approaches to modify critical residues of the chNHE1 receptor in chicken cells. The susceptibility of the modified cell lines to ALV-J infection was examined using enhanced green fluorescent protein (EGFP)-expressing marker viruses. We showed that modifying the chNHE1 receptor by artificially generating a premature stop codon induced absolute resistance to viral infection, with mutations of the tryptophan residue at position 38 (Trp38) being very critical. Single-stranded oligodeoxynucleotide (ssODN)-mediated targeted recombination of the Trp38 region revealed that deletions involving the Trp38 residue were most effective in conferring resistance to ALV-J. Moreover, protein structure analysis of the chNHE1 receptor sequence suggested that its intrinsically disordered region undergoes local conformational changes through genetic alteration. Collectively, these results demonstrate that targeted mutations on chNHE1 alter the susceptibility to ALV-J and the technique is expected to contribute to develop disease-resistant chicken lines.

Howson E L A, Kurosaki Y, Yasuda J, Takahashi M, Goto H, Gray A R, Mioulet V, King D P, Fowler V L (2017)

Defining the relative performance of isothermal assays that can be used for rapid and sensitive detection of foot-and-mouth disease virus

Journal of Virological Methods 249, 102-110


This study describes the first multiway comparison of portable isothermal assays for the detection of foot-and-mouth disease virus (FMDV), benchmarked against real-time reverse transcription RT-PCR (rRT-PCR). The selected isothermal chemistries included reverse transcription loop-mediated isothermal amplification (RT-LAMP) and reverse transcription recombinase polymerase amplification (RT-RPA). The analytical sensitivity of RT-LAMP was comparable to rRT-PCR (101 RNA copies), while RT-RPA was one log10 less sensitive (102 RNA copies). Diagnostic performance was evaluated using a panel of 35 samples from FMDV-positive cattle and eight samples from cattle infected with other vesicular viruses. Assay concordance for RT-LAMP and RT-RPA was 86–98% and 67–77%, respectively, when compared to rRT-PCR, with discordant samples consistently having high rRT-PCR cycle threshold values (no false-positives were detected for any assay). In addition, a hierarchy of sample preparation methods, from robotic extraction to simple dilution of samples, for epithelial suspensions, serum and oesophageal-pharyngeal (OP) fluid were evaluated. Results obtained for RT-LAMP confirmed that FMDV RNA can be detected in the absence of RNA extraction. However, simple sample preparation methods were less encouraging for RT-RPA, with accurate results only obtained when using RNA extraction. Although the evaluation of assay performance is specific to the conditions tested in this study, the compatibility of RT-LAMP chemistry with multiple sample types, both in the presence and absence of nucleic acid extraction, provides advantages over alternative isothermal chemistries and alternative pen-side diagnostics such as antigen-detection lateral-flow devices. These characteristics of RT-LAMP enable the assay to be performed over a large diagnostic detection window, providing a realistic means to rapidly confirm positive FMD cases close to the point of sampling.

Harmsen M M, Seago J, Perez E, Charleston B, Eblé P L, Dekker A (2017)

Isolation of single-domain antibody fragments that preferentially detect intact (146s) particles of foot-and-mouth disease virus for use in vaccine quality control

Frontiers in Immunology 8, 960


Intact (146S) foot-and-mouth disease virions (FMDVs) can dissociate into specific (12S) viral capsid degradation products. FMD vaccines normally consist of inactivated virions. Vaccine quality is dependent on 146S virus particles rather than 12S particles. We earlier isolated two llama single-domain antibody fragments (VHHs) that specifically recognize 146S particles of FMDV strain O1 Manisa and shown their potential use in quality control of FMD vaccines during manufacturing. These 146S specific VHHs were specific for particular O serotype strains and did not bind strains from other FMDV serotypes. Here we describe the isolation of 146S specific VHHs against FMDV SAT2 and Asia 1 strains by phage display selection from llama immune libraries. VHHs that bind both 12S and 146S particles were readily isolated but VHHs that bind specifically to 146S particles could only be isolated by phage display selection using prior depletion for 12S particles. We obtained one 146S specific VHH – M332F – that binds to strain Asia 1 Shamir and several VHHs that preferentially bind 146S particles of SAT2 strain SAU/2/00, from which we selected VHH M379F for further characterization. Both M332F and M379F did not bind FMDV strains from other serotypes. In a sandwich ELISA employing unlabeled and biotinylated versions of the same VHH M332F showed high specificity for 146S particles but M379F showed lower 146S-specificity with some cross-reaction with 12S particles. These ELISAs could detect 146S particle concentrations as low as 2.3-4.6 µg/l. They can be used for FMD vaccine quality control and research and development, for example to identify virion stabilizing excipients.


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