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

There were a total of 1912 results for your search.
Yao Y, Ding C, Nair V (2019)

Herpesvirus of turkeys (Meleagridis herpesvirus 1) encodes a functional microRNA-221 homolog with high sequence conservation

Advances in Microbiology 9 (8), 728-736


Herpesviruses account for most of the known virus-encoded miRNAs. Herpesvirus of turkey (HVT), a non-pathogenic avian herpesvirus used as an avian vaccine and viral vector, encodes 28 mature miRNAs. This included HVT-miR-H14-3p that showed almost identical sequence to gga-miR-221, suggesting that it is pirated from the avian host. Although the functional homolog between the two miRNAs has been proposed based on the sequence similarity, the direct experimental evidence is still lacking. In this report, we provide the evidence for the first time that HVT-miR-H14-3p is indeed a gga-miR-221 homolog through modulating the expression of p27Kip1, a known target of miR-221 by binding to its 3’UTR. We also created an HVT-miR-H14-3p deletion virus and show that this miRNA is not essential for in vitro replication.

Sealy J E, Fournie G, Trang P H, Dang N H, Sadeyen J-R, Thanh T L, van Doorn H R, Bryant J E, Iqbal M (2019)

Poultry trading behaviours in Vietnamese live bird markets as risk factors for avian influenza infection in chickens

Transboundary and Emerging Diseases Early View,
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Vietnamese poultry are host to co-circulating subtypes of avian influenza viruses, including H5N1 and H9N2, which pose a great risk to poultry productivity and to human health. AIVs circulate throughout the poultry trade network in Vietnam, with live bird markets being an integral component to this network. Traders at LBMs exhibit a variety of trading practices, which may influence the transmission of AIVs. We identified trading practices that impacted on AIV prevalence in chickens marketed in northern Vietnamese LBMs. We generated sequencing data for 31 H9N2 and 2 H5N6 viruses. Viruses isolated in the same LBM or from chickens sourced from the same province were genetically closer than viruses isolated in different LBMs or from chickens sourced in different provinces. The position of a vendor in the trading network impacted on their odds of having AIV infected chickens. Being a retailer and purchasing chickens from middlemen was associated with increased odds of infection, whereas odds decreased if vendors purchased chickens directly from large farms. Odds of infection were also higher for vendors having a greater volume of ducks unsold per day. These results indicate how the spread of AIVs is influenced by the structure of the live poultry trading network.

Ren C, Xie R, Yao Y, Yu M, Chang F, Xing L, Zhang Y, Liu Y, Wang S, Farooque M, Wang Y, Qi X, Liu C, Zhang Y, Cui H, Li K, Gao L, Pan Q, Nair V, Wang X, Gao Y (2019)

MiR-125b suppression inhibits apoptosis and negatively regulates Sema4D in avian leukosis virus-transformed cells

Viruses 11 (8), 728
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Subgroup J avian leukosis virus (ALV-J), an oncogenic retrovirus, causes hemangiomas and myeloid tumors in chickens. We previously showed that miR-125b is down-regulated in ALV-J-induced tumors. This study aimed to investigate the possible role of miR-125b in ALV-J-mediated infection and tumorigenesis. Knockdown of miR-125b expression in HP45 cells reduced, whereas over-expression induced late-stage apoptosis. Bioinformatics analysis and luciferase activity assays indicate that miR-125b targets Semaphorin 4D/CD100 (Sema4D) by binding the 3'-untranslated region of messenger RNA (mRNA). Up-regulation of miR-125b in the DF1 cell line suppressed Sema4D expression, whereas miR-125 down-regulation increased Sema4D expression levels. To uncover the function of Sema4D during ALV-J infection, animal infection experiments and in vitro assays were performed and show that Sema4D mRNA levels were up-regulated in ALV-J-infected tissues and cells. Finally, functional experiments show that miR-125 down-regulation and Sema4D over-expression inhibited apoptosis in HP45 cells. These results suggest that miR-125b and its target Sema4D might play an important role in the aggressive growth of HP45 cells induced by avian leukosis viruses (ALVs). These findings improve our understanding of the underlying mechanism of ALV-J infection and tumorigenesis. 

Mahapatra M, Howson E, Fowler V, Batten C, Flannery J, Selvaraj M, Parida S (2019)

Rapid detection of peste des petits ruminants virus (PPRV) nucleic acid using a novel low-cost reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for future use in nascent PPR eradication programme

Viruses 11 (8), 699
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Peste des petits ruminants (PPR) is a disease of small ruminants caused by peste des petits ruminants virus (PPRV), and is endemic in Asia, the Middle East and Africa. Effective control combines the application of early warning systems, accurate laboratory diagnosis and reporting, animal movement restrictions, suitable vaccination and surveillance programs, and the coordination of all these measures by efficient veterinary services. Molecular assays, including conventional reverse transcription-polymerase chain reaction (RT-PCR) and real-time RT-PCR (RT-qPCR) have improved the sensitivity and rapidity of diagnosing PPR. However, currently these assays are only performed within laboratory settings; therefore, the development of field diagnostics for PPR would improve the fast implementation of control policies, particularly when PPR has been targeted to be eradicated by 2030. Loop-mediated isothermal amplification (LAMP) assays are simple to use, rapid, and have sensitivity and specificity within the range of RT-qPCR; and can be performed in the field using disposable consumables and portable equipment. This study describes the development of a novel RT-LAMP assay for the detection of PPRV nucleic acid by targeting the N-protein gene. The RT-LAMP assay was evaluated using cell culture propagated PPRVs, field samples from clinically infected animals and samples from experimentally infected animals encompassing all four lineages (I-IV) of PPRV. The test displayed 100% concordance with RT-qPCR when considering an RT-qPCR cut-off value of CT >40. Further, the RT-LAMP assay was evaluated using experimental and outbreak samples without prior RNA extraction making it more time and cost-effective. This assay provides a solution for a pen-side, rapid and inexpensive PPR diagnostic for use in the field in nascent PPR eradication programme.

Kelly J T, Human S, Alderman J, Jobe F, Logan L, Rix T, Gonçalves-Carneiro D, Leung C, Thakur N, Birch J, Bailey D (2019)

BST2/Tetherin overexpression modulates Morbillivirus glycoprotein production to inhibit cell–cell fusion

Viruses 11 (8), 692
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The measles virus (MeV), a member of the genus Morbillivirus, is an established pathogen of humans. A key feature of morbilliviruses is their ability to spread by virus–cell and cell–cell fusion. The latter process, which leads to syncytia formation in vitro and in vivo, is driven by the viral fusion (F) and haemagglutinin (H) glycoproteins. In this study, we demonstrate that MeV glycoproteins are sensitive to inhibition by bone marrow stromal antigen 2 (BST2/Tetherin/CD317) proteins. BST2 overexpression causes a large reduction in MeV syncytia expansion. Using quantitative cell–cell fusion assays, immunolabeling, and biochemistry we further demonstrate that ectopically expressed BST2 directly inhibits MeV cell–cell fusion. This restriction is mediated by the targeting of the MeV H glycoprotein, but not other MeV proteins. Using truncation mutants, we further establish that the C-terminal glycosyl-phosphatidylinositol (GPI) anchor of BST2 is required for the restriction of MeV replication in vitro and cell–cell fusion. By extending our study to the ruminant morbillivirus peste des petits ruminants virus (PPRV) and its natural host, sheep, we also confirm this is a broad and cross-species specific phenotype. 

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 early view,
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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.


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