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

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Jacquot M, Rao P P, Yadav S, Nomikou K, Maan S, Jyothi Y K, Reddy N, Putty K, Hemadri D, Singh K P, Maan N S, Hegde N R, Mertens P, Biek R (2019)

Contrasting selective patterns across the segmented genome of bluetongue virus in a global reassortment hotspot

Virus Evolution 5 (2), vez027
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For segmented viruses, rapid genomic and phenotypic changes can occur through the process of reassortment, whereby co-infecting strains exchange entire segments creating novel progeny virus genotypes. However, for many viruses with segmented genomes, this process and its effect on transmission dynamics remain poorly understood. Here, we assessed the consequences of reassortment for selection on viral diversity through time using bluetongue virus (BTV), a segmented arbovirus that is the causative agent of a major disease of ruminants. We analysed ninety-two BTV genomes isolated across four decades from India, where BTV diversity, and thus opportunities for reassortment, are among the highest in the world. Our results point to frequent reassortment and segment turnover, some of which appear to be driven by selective sweeps and serial hitchhiking. Particularly, we found evidence for a recent selective sweep affecting segment 5 and its encoded NS1 protein that has allowed a single variant to essentially invade the full range of BTV genomic backgrounds and serotypes currently circulating in India. In contrast, diversifying selection was found to play an important role in maintaining genetic diversity in genes encoding outer surface proteins involved in virus interactions (VP2 and VP5, encoded by segments 2 and 6, respectively). Our results support the role of reassortment in driving rapid phenotypic change in segmented viruses and generate testable hypotheses for in vitro experiments aiming at understanding the specific mechanisms underlying differences in fitness and selection across viral genomes.

Ferretti L, Tennakoon C, Silesian A, Freimanis G, Ribeca P (2019)

SiNPle: fast and sensitive variant calling for deep sequencing data

Genes 10 (8), 561


Current high-throughput sequencing technologies can generate sequence data and provide information on the genetic composition of samples at very high coverage. Deep sequencing approaches enable the detection of rare variants in heterogeneous samples, such as viral quasi-species, but also have the undesired effect of amplifying sequencing errors and artefacts. Distinguishing real variants from such noise is not straightforward. Variant callers that can handle pooled samples can be in trouble at extremely high read depths, while at lower depths sensitivity is often sacrificed to specificity. In this paper, we propose SiNPle (Simplified Inference of Novel Polymorphisms from Large coveragE), a fast and effective software for variant calling. SiNPle is based on a simplified Bayesian approach to compute the posterior probability that a variant is not generated by sequencing errors or PCR artefacts. The Bayesian model takes into consideration individual base qualities as well as their distribution, the baseline error rates during both the sequencing and the PCR stage, the prior distribution of variant frequencies and their strandedness. Our approach leads to an approximate but extremely fast computation of posterior probabilities even for very high coverage data, since the expression for the posterior distribution is a simple analytical formula in terms of summary statistics for the variants appearing at each site in the genome. These statistics can be used to filter out putative SNPs and indels according to the required level of sensitivity. We tested SiNPle on several simulated and real-life viral datasets to show that it is faster and more sensitive than existing methods. The source code for SiNPle is freely available to download and compile, or as a Conda/Bioconda package.

Cortey M, Ferretti L, Perez-Martin E, Zhang F, de Klerk-Lorist L M, Scott K, Freimanis G, Seago J, Ribeca P, van Schalkwyk L, Juleff N D, Maree F F, Charleston B (2019)

Persistent infection of African buffalo (Syncerus caffer) with foot-and-mouth disease virus: limited viral evolution and no evidence of antibody neutralization escape

Journal of Virology 93 (15), e00563-19


African buffaloes (Syncerus caffer) are the principal "carrier" hosts of foot-and-mouth disease virus (FMDV). Currently, the epithelia and lymphoid germinal centres of the oropharynx have been identified as sites for FMDV persistence. We carried out studies in FMDV SAT-1 persistently infected buffaloes to characterize the diversity of viruses in oropharyngeal epithelia, germinal centres, probang (oropharyngeal scrapings) and tonsil swabs, to determine if sufficient virus variation is generated during persistence for immune escape. Most sequencing reads of the VP1-coding region of the SAT 1 virus inoculum clustered around two subpopulations differing by 22 single nucleotide variants of intermediate frequency. Similarly, most sequences from oropharynx tissue clustered into two subpopulations, albeit with different proportions depending on the days post-infection (dpi). There was a significant difference between the population of viruses in the inoculum and in lymphoid tissue taken at 35 dpi. Thereafter, until 400 dpi, no significant variation was detected in the viral population in samples from individual animals, germinal centres and epithelia tissues. Deep sequencing of virus from probang or tonsil swab samples harvested prior to post mortem showed less within-sample variability of VP1 compared to tissue sample sequences analysed at the same time. Importantly, there was no significant difference in the ability of sera collected between 14 and 400 dpi to neutralise the inoculum or viruses isolated at later time points in the study from the same animal. Therefore, based on this study there is no evidence of escape from antibody neutralization contributing to FMDV persistent infection in African buffalo. Foot-and-mouth disease virus (FMDV) is a highly contagious virus of cloven hooved animals and is recognised as the most important constraint to international trade in animals and animal products. African buffaloes (Syncerus caffer) are efficient carriers of FMDV and it has been proposed that new virus variants are produced in buffalo during the prolonged carriage after acute infection, which may spread to cause disease in livestock populations. Here, we show that despite an accumulation of low frequency sequence variants over time there is no evidence of significant antigenic variation leading to immune escape. Therefore, carrier buffalo are unlikely to be a major source of new virus variants.

Odon V, Fros J J, Goonawardane N, Dietrich I, Ibrahim A, Alshaikhahmed K, Nguyen D, Simmonds P (2019)

The role of ZAP and OAS3/RNAseL pathways in the attenuation of an RNA virus with elevated frequencies of CpG and UpA dinucleotides

Nucleic Acids Research 45 (15), 8061-8083
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Zinc finger antiviral protein (ZAP) is a powerful restriction factor for viruses with elevated CpG dinucleotide frequencies. We report that ZAP similarly mediates antiviral restriction against echovirus 7 (E7) mutants with elevated frequencies of UpA dinucleotides. Attenuation of both CpG- and UpA-high viruses and replicon mutants was reversed in ZAP k/o cell lines, and restored by plasmid-derived reconstitution of expression in k/o cells. In pull-down assays, ZAP bound to viral RNA transcripts with either CpG- and UpA-high sequences inserted in the R2 region. We found no evidence that attenuation of CpG- or UpA-high mutants was mediated through either translation inhibition or accelerated RNA degradation. Reversal of the attenuation of CpG-high, and UpA-high E7 viruses and replicons was also achieved through knockout of RNAseL and oligodenylate synthetase 3 (OAS3), but not OAS1. WT levels of replication of CpG- and UpA-high mutants were observed in OAS3 k/o cells despite abundant expression of ZAP, indicative of synergy or complementation of these hitherto unconnected pathways. The dependence on expression of ZAP, OAS3 and RNAseL for CpG/UpA-mediated attenuation and the variable and often low level expression of these pathway proteins in certain cell types, such as those of the central nervous system, has implications for the use of CpG-elevated mutants as attenuated live vaccines against neurotropic viruses.

Netherton C L, Goatley L C, Reis A L, Portugal R, Nash R H, Morgan S B, Gault L, Nieto R, Norlin V, Gallardo C, Ho C-S, Sánchez-Cordón P J, Taylor G, Dixon L K (2019)

Identification and immunogenicity of African swine fever virus antigens

Frontiers in Immunology 10, 1318


African swine fever (ASF) is a lethal haemorrhagic disease of domestic pigs for which there is no vaccine. Strains of the virus with reduced virulence can provide protection against related virulent strains of ASFV, but protection is not 100% and there are concerns about the safety profile of such viruses. However, they provide a useful tool for understanding the immune response to ASFV and previous studies using the low virulent isolate OUR T88/3 have shown that CD8+ cells are crucial for protection. In order to develop a vaccine that stimulates an effective anti-ASFV T-cell response we need to know which of the greater than 150 viral proteins are recognised by the cellular immune response. Therefore we used a gamma interferon ELIspot assay to screen for viral proteins recognised by lymphocytes from ASF-immune pigs using peptides corresponding to 133 proteins predicted to be encoded by OUR T88/3. Eighteen antigens that were recognised by ASFV-specific lymphocytes were then incorporated into adenovirus and MVA vectors, which were used in immunisation and challenge experiments in pigs. We present a systematic characterisation of the cellular immune response to this devastating disease and identify proteins capable of inducing ASFV-specific cellular and humoral immune responses in pigs. Pools of viral vectors expressing these genes did not protect animals from severe disease, but did reduce viremia in a proportion of pigs following ASFV challenge.

McCarthy R R, Everett H E, Graham S P, Steinbach F, Crooke H R (2019)

Head start immunity: characterizing the early protection of C strain vaccine against subsequent classical swine fever virus infection

Frontiers in Immunology 10, 1584


Classical Swine Fever Virus (CSFV) is an ongoing threat to the pig industry due to its high transmission and mortality rates associated with infection. Live attenuated vaccines such as the CSFV C strain vaccine are capable of protecting against infection within 5 days of vaccination, but the molecular mechanisms through which this early protection is mediated have yet to be established. In this study, we compared the response of pigs vaccinated with the C strain to non-vaccinated pigs both challenged with a pathogenic strain of CSFV. Analysis of transcriptomic data from the tonsils of these animals during the early stages after vaccination and challenge reveals a set of regulated genes that appear throughout the analysis. Many of these are linked to the ISG15 antiviral pathway suggesting it plays a key role in the rapid and early protection conferred by C strain vaccination.

Boodhoo, N; Kamble, N; Kaufer, B; Behboudi, S (2019)

Replication of Marek's disease virus is dependent on synthesis of de novo fatty acid and prostaglandin E2

Journal of Virology Volume 93 (Issue 13), JVI.00352-19


Marek's disease virus (MDV) causes deadly lymphoma and induces an imbalance of the lipid metabolism in infected chickens. Here, we discovered that MDV activates fatty acid synthesis (FAS) pathway in primary chicken embryo fibroblasts (CEF). In addition, MDV infected cells contained high levels of fatty acids and showed an increased numbers of lipid droplets (LDs). Chemical inhibitors of the FAS pathway (TOFA and C75) reduced MDV titre by approximately 30 folds. Addition of the downstream metabolites including malonyl-CoA or palmitic acid completely restored the inhibitory effects of the FAS inhibitors. Furthermore, we could demonstrate that MDV infection activates COX-2/PGE2 pathway as evident by increased levels of arachidonic acid, COX-2 expression and PGE2 synthesis. Inhibition of COX-2/PGE2 pathway by chemical inhibitors or knockdown of COX2 using shRNA reduced MDV titres, suggesting that COX-2 promotes virus replication. Exogenous PGE2 completely restored the inhibition of the COX-2/PGE2 pathway in MDV replication. Unexpectedly, exogenous PGE2 also partially rescued the inhibitory effects of FAS inhibitors on MDV replication, suggesting that there is link between these two pathways in MDV infection. Taken together, our data demonstrate that the FAS and COX-2/PGE2 pathways play an important role in the replication of this deadly pathogen.Importance Disturbances of the lipid metabolism in chickens infected with Marek's disease virus (MDV) contribute to the pathogenesis of disease. However, the role of lipid metabolism in MDV replication remained unknown. Here, we demonstrate that MDV infection activates fatty acid synthesis (FAS) and induces lipid droplet (LD) formation. Moreover, our results demonstrate that MDV replication is highly dependent on the FAS pathway and the downstream metabolites. Finally, our results reveal that MDV also activates the COX-2/PGE2 pathway which supports MDV replication by activating PGE2/EP2 and PGE2/EP4 signalling pathways.

Benedikz E K, Bailey D, Cook C N L, Gonçalves-Carneiro D, Buckner M M C, Blair J M A, Wells T J, Fletcher N F, Goodall M, Flores-Langarica A, Kingsley R A, Madsen J, Teeling J, Johnston S L, MacLennan C A, Balfe P, Henderson I R, Piddock L J V, Cunningham A F, McKeating J A (2019)

Bacterial flagellin promotes viral entry via an NF-kB and Toll Like Receptor 5 dependent pathway

Scientific Reports 9 (1), 7903


Viruses and bacteria colonize hosts by invading epithelial barriers. Recent studies have shown that interactions between the microbiota, pathogens and the host can potentiate infection through poorly understood mechanisms. Here, we investigated whether diverse bacterial species could modulate virus internalization into host cells, often a rate-limiting step in establishing infections. Lentiviral pseudoviruses expressing influenza, measles, Ebola, Lassa or vesicular stomatitis virus envelope glycoproteins enabled us to study entry of viruses that exploit diverse internalization pathways. Salmonella Typhimurium, Escherichia coli and Pseudomonas aeruginosa significantly increased viral uptake, even at low bacterial frequencies. This did not require bacterial contact with or invasion of host cells. Studies determined that the bacterial antigen responsible for this pro-viral activity was the Toll-Like Receptor 5 (TLR5) agonist flagellin. Exposure to flagellin increased virus attachment to epithelial cells in a temperature-dependent manner via TLR5-dependent activation of NF-κB. Importantly, this phenotype was both long lasting and detectable at low multiplicities of infection. Flagellin is shed from bacteria and our studies uncover a new bystander role for this protein in regulating virus entry. This highlights a new aspect of viral-bacterial interplay with significant implications for our understanding of polymicrobial-associated pathogenesis.

Ren J, Nettleship J E, Harris G, Mwangi W, Rhaman N, Grant C, Kotecha A, Fry E, Charleston B, Stuart D I, Hammond J, Owens R J (2019)

The role of the light chain in the structure and binding activity of two cattle antibodies that neutralize bovine respiratory syncytial virus

Molecular Immunology 112, 123-130


Cattle antibodies have unusually long CDR3 loops in their heavy chains (HCs), and limited light chain (LC) diversity, raising the question of whether these mask the effect of LC variation on antigen recognition. We have investigated the role of the LC in the structure and activity of two neutralizing cattle antibodies (B4 and B13) that bind the F protein of bovine respiratory syncytial virus (bRSV). Recombinant Fab fragments of B4 and B13 bound bRSV infected cells and showed similar affinities for purified bRSV F protein. Exchanging the LCs between the Fab fragments produced hybrid Fabs: B13* (B13 HC/B4 LC) and B4* (B4 HC/B13 LC). The affinity of B13* to the F protein was found to be two-fold lower than B13 whilst the binding affinity of B4* was reduced at least a hundred-fold compared to B4 such that it no longer bound to bRSV infected cells. Comparison of the structures of B4 and B13 with their LC exchanged counterparts B4* and B13* showed that paratope of the HC variable domain (VH) of B4 was disrupted on pairing with the B13 LC, consistent with the loss of binding activity. By contrast, B13 H3 adopts a similar conformation when paired with either B13 or B4 LCs. These observations confirm the expected key role of the extended H3 loop in antigen-binding by cattle antibodies but also show that the quaternary LC/HC subunit interaction can be crucial for its presentation and thus the LC variable domain (VL) is also important for antigen recognition.

Long J S, Idoko-Akoh A, Mistry B, Goldhill D, Staller E, Schreyer J, Ross C, Goodbourn S, Shelton H, Skinner M A, Sang H, McGrew M J, Barclay W (2019)

Species specific differences in use of ANP32 proteins by influenza A virus

Elife 8, e45066


Influenza A viruses (IAV) are subject to species barriers that prevent frequent zoonotic transmission and pandemics. One of these barriers is the poor activity of avian IAV polymerases in human cells. Differences between avian and mammalian ANP32 proteins underlie this host range barrier. Human ANP32A and ANP32B homologues both support function of human-adapted influenza polymerase but do not support efficient activity of avian IAV polymerase which requires avian ANP32A. We show here that the gene currently designated as avian ANP32B is evolutionarily distinct from mammalian ANP32B, and that chicken ANP32B does not support IAV polymerase activity even of human-adapted viruses. Consequently, IAV relies solely on chicken ANP32A to support its replication in chicken cells. Amino acids 129I and 130N, accounted for the inactivity of chicken ANP32B. Transfer of these residues to chicken ANP32A abolished support of IAV polymerase. Understanding ANP32 function will help develop antiviral strategies and aid the design of influenza virus resilient genome edited chickens.


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