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

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Marek’s disease is a major scourge challenging poultry health worldwide. It is caused by the highly contagious Marek’s disease virus (MDV), an alphaherpesvirus. Here, we showed that, similar to other members of its Herpesviridae family, MDV also presents a complex landscape of splicing events, most of which are uncharacterised and/or not annotated. Quite strikingly, and although the biological relevance of this fact is unknown, we found that a number of viral splicing isoforms are strain-specific, despite the close sequence similarity of the strains considered: very virulent RB-1B and vaccine CVI-988. We validated our findings by devising an assay that discriminated infections caused by the two strains in chicken embryonic fibroblasts on the basis of the presence of some RNA species. To our knowledge, this study is the first to accomplish such a result, emphasizing how relevant a comprehensive picture of the viral transcriptome is to fully understand viral pathogenesis.

Rosen B D, Bickhart D M, Schnabel R D, Koren S, Elsik C G, Tseng E, Rowan T N, Low W Y, Zimin A, Couldrey C, Hall R, Li W, Rhie A, Ghurye J, McKay S D, Thibaud-Nissen F, Hoffman J, Murdoch B M, Snelling W M, McDaneld T G, Hammond J A, Schwartz J C, Nandolo W, Hagen D E, Dreischer C, Schultheiss S J, Schroeder S G, Phillippy A M, Cole J B, Van Tassell C P, Liu G, Smith T P L, Medrano J F (2020)

De novo assembly of the cattle reference genome with single-molecule sequencing

GigaScience 19 (3), giaa021


Major advances in selection progress for cattle have been made following the introduction of genomic tools over the past 10-12 years. These tools depend upon the Bos taurus reference genome (UMD3.1.1), which was created using now-outdated technologies and is hindered by a variety of deficiencies and inaccuracies. We present the new reference genome for cattle, ARS-UCD1.2, based on the same animal as the original to facilitate transfer and interpretation of results obtained from the earlier version, but applying a combination of modern technologies in a de novo assembly to increase continuity, accuracy, and completeness. The assembly includes 2.7 Gb and is >250x more continuous than the original assembly, with contig N50 >25 Mb and L50 of 32. We also greatly expanded supporting RNA-based data for annotation that identifies 30,396 total genes (21,039 protein coding). The new reference assembly is accessible in annotated form for public use. We demonstrate that improved continuity of assembled sequence warrants the adoption of ARS-UCD1.2 as the new cattle reference genome and that increased assembly accuracy will benefit future research on this species.


Peste des Petits Ruminants (PPR) is a highly contagious viral disease of both domestic (goats and sheep) and wild ruminants. Caused by a morbillivirus, that belongs to the family Paramyxoviridae. The disease is clinically and pathologically similar to rinderpest of cattle and human measles. PPR is one of the most economically devastating viral diseases of small ruminants. In April 2015, the Food and Agriculture Organization of the United Nations (FAO) and the World Organisation for Animal Health (OIE) launched the PPR Global Control and Eradication Strategy (PPR GCES) with the vision for global eradication by 2030. There is a strong and lasting international consensus to eradicate the disease in order to protect the livelihoods of the world's poorest populations. As with any disease, eradication is feasible when, policy, scientific and technical challenges are addressed. Ten majors challenges are described in this paper namely: understanding small ruminant production, facilitating research to support eradication, refining laboratory testing, improving epidemiological understanding of the virus, defining infection of wildlife and other species, optimizing vaccine delivery and novel vaccines, developing better control of animal movement, heightening serological monitoring, understanding socio-economic impact, and garnering funding and political will.


Diagnostic tests for foot-and-mouth disease (FMD) include the detection of antibodies against either the viral non-structural proteins or the capsid. The detection of antibodies against the structural proteins (SP) of the capsid can be used to monitor seroconversion in both infected and vaccinated animals. However, SP tests need to be tailored to the individual FMD virus serotype and their sensitivity performances may be affected by antigenic variability within each serotype and mismatching between tests reagents. As a consequence, FMD Reference Laboratories are required to maintain multiple type-specific SP assays and reagents. A universal SP test would simplify frontline diagnostics and facilitate large-scale serological surveillance and post-vaccination monitoring. In this study, a highly conserved region in the N terminus of FMDV capsid protein VP2 (VP2N) was characterised using a panel of intertypic-reactive monoclonal antibodies. This revealed a universal epitope in VP2N which could be used as a peptide antigen to detect FMDV-specific antibodies against all types of the virus. A VP2-peptide ELISA (VP2-ELISA) was optimised using experimental and reference antisera from immunized, convalescent and negative animals (n=172). The VP2-ELISA is universal, simple and provided sensitive (99 %) and specific (93%) detection of antibodies to all FMDV strains used in this study. We anticipate that this SP test could have utility for sero-surveillance during virus incursions in FMD-free countries and as an additional screening tool to assess FMD virus circulation in endemic countries.

Anderson M A E, Purcell J, Verkuijl S A N, Norman V C, Leftwich P T, Harvey-Samuel T, Alphey L S (2020)

Expanding the CRISPR toolbox in Culicine mosquitoes: in vitro validation of Pol III promoters

ACS Synthetic Biology 9 (3), 678-681


CRISPR-Cas9-based "gene drive" technologies have been proposed as a novel and effective means of controlling human diseases vectored by mosquitoes. However, more complex designs than those demonstrated to date-and an expanded molecular toolbox with which to build them-will be required to overcome the issues of resistance formation/evolution and drive spatial/temporal limitation. Foreseeing this need, we assessed the sgRNA transcriptional activities of 33 phylogenetically diverse insect Polymerase III promoters using three disease-relevant Culicine mosquito cell lines (Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus). We show that U6 promoters work across species with a range of transcriptional activity levels and find 7SK promoters to be especially promising because of their broad phylogenetic activity. We further show that U6 promoters can be substantially truncated without affecting transcriptional levels. These results will be of great utility to researchers involved in developing the next generation of gene drives.

Wood B A, Mioulet V, Henry E, Gray A, Azhar M, Thapa B, Diederich S, Hoffmann B, Beer M, King D P, Eschbaumer M (2020)

Inactivation of foot-and-mouth disease virus A/IRN/8/2015 with commercially available lysis buffers

Journal of Virological Methods 278, 113835


Laboratories working with foot-and-mouth disease virus (FMDV) must maintain a high level of biocontainment. However, if infectious virus is reliably inactivated during sample processing, molecular and serological testing can be performed at a lower level of containment. In this study, three commercial lysis buffers (AL, AVL, and MagMAX CORE) were tested in two laboratories for their ability to inactivate FMDV A/IRN/8/2015 in different sample matrices (cell culture supernatant, epithelial tissue suspension and milk). Residual infectivity after the addition of lysis buffer was evaluated by inoculating susceptible cell cultures. No cytopathic effect was observed for all three lysis buffers, indicating that the buffers are capable of reducing viral infectivity (estimated range 3.1 to >5.1 Log10). These results highlight the capacity of lysis buffers to decrease FMDV infectivity; however, additional validation experiments should be conducted, particularly if different sample matrices and/or lysis buffers are used.


Herpesvirus of turkeys (HVT), used originally as a vaccine against Marek’s disease (MD), has recently been shown to be a highly effective viral vector for generation of recombinant vaccines that deliver protective antigens of other avian pathogens. Until the recent launch of commercial HVT-vectored dual insert vaccines, most of the HVT-vectored vaccines in the market carry a single foreign gene and are usually developed with slow and less efficient conventional recombination methods. There is immense value in developing multivalent HVT-vectored vaccines capable of inducing simultaneous protection against multiple avian pathogens, particularly to overcome the interference between individual recombinant HVT vaccines. Here we demonstrate the use of a previously developed CRISPR/Cas9 gene editing protocol for the insertion of ILTV gD-gI and the H9N2 AIV hemagglutinin expression cassettes into the distinct locations of the recombinant HVT-IBDV VP2 viral genome, to generate the triple insert HVT-VP2-gDgI-HA recombinant vaccine. The insertion, protein expression, and stability of each insert were then evaluated by PCR, immunostaining and Western blot analyses. The successful generation of the first triple insert recombinant HVT vaccine with the potential for the simultaneous protection against three major avian viral diseases in addition to MD is a major innovation in vaccination-based control of major poultry diseases.


In the 21(st) century, the emergence of H7N9 and H1N1/2009 influenza viruses originating from animals and causing severe human infections has prompted investigations into the genetic alterations required for cross-species transmission. We previously found that substitution of the human-origin PA gene segment in avian influenza virus (AIV) could overcome barriers to cross-species transmission. Recently, it was reported that the PA gene segment encodes both the PA protein and a second protein, PA-X. Herein, we investigated the role of PA-X. We found that an H9N2 avian influenza reassortant virus bearing a human-origin H1N1/2009 PA gene was attenuated in mice after the loss of PA-X. Reverse genetics analyses of PA-X substitutions conserved in human influenza viruses indicated that R195K, K206R and P210L substitutions conferred significantly increased replication and pathogenicity on H9N2 virus in mice and ferrets. PA-X R195K was present in all human H7N9 and H1N1/2009 viruses, and predominated in human H5N6 viruses. Compared with PA-X 195R, H7N9 influenza viruses bearing PA-X 195K showed increased replication and transmission in ferrets. We further showed that PA-X 195K enhanced lung inflammatory responses, potentially due to decreased host shutoff function. A competitive transmission study in ferrets indicated that 195K provides a replicative advantage over 195R in H1N1/2009 viruses. By contrast, PA-X 195K did not influence the virulence of H9N2 AIV in chickens, suggesting that the effects of this substitution were mammal-specific. Therefore, future surveillance efforts should scrutinize this region of PA-X because of its potential impact on cross-species transmission of influenza viruses.ImportanceFour influenza pandemics in humans [the Spanish flu of 1918 (H1N1), the Asian flu of 1957 (H2N2), the Hong Kong flu of 1968 (H3N2), and the swine-origin flu of 2009 (H1N1)] are all proposed to have been caused by avian or swine influenza viruses, which acquired virulence factors through adaptive mutation or reassortment with circulating human viruses. Currently, influenza viruses circulating in animals are repeatedly transmitted to humans, posing a significant threat to public health. However, the molecular properties accounting for interspecies transmission of influenza viruses remain unclear. In the present study, we demonstrated that PA-X plays an important role in cross-species transmission of influenza viruses. At least three human-specific amino acid substitutions in PA-X dramatically enhanced the adaptation of animal influenza viruses in mammals. In particular, PA-X 195K might have contributed to cross-species transmission of H7N9, H5N6, and H1N1/2009 viruses from animal reservoirs to humans.


Bcl-2 (B cell lymphoma-2)-related gene Nr-13 plays a major role in the regulation of cell death in developing avian B-cells. With over 65% sequence similarity to the chicken Nr-13, herpesvirus of turkeys (HVT)-encoded HVT079 and HVT096 gene product vNr-13 is the first known alphaherpesvirus-encoded Bcl-2 homolog. HVT-infected cells were reported to be relatively more resistant to serum starvation, suggested that vNr-13 could be involved in protecting the cells. Here we describe CRISPR/Cas9-based editing of exon 1 of HVT079 and HVT096 genes from the HVT genome to generate the mutant HVT-ΔvNr-13 to gain insights into its functional roles. Overall, both wild type HVT and HVT-ΔvNr-13 showed similar growth kinetics; however, at early time points, HVT-ΔvNr-13 showed lower growth of 1.3- to 1.7-fold of cell-associated virus and 3- to 6.2- fold of cell-free virus. In the transfected cells, HVT vNr-13 mainly showed diffuse cytoplasmic distribution with faint nuclear staining. Further, vNr-13 localized to the mitochondria and endoplasmic reticulum (ER), and demonstrated to disrupt mitochondrial network morphology in the transfected cells. In the wildtype HVT-infected cells, vNr-13 expression appeared to be directly involved in the disruption of the mitochondrial network, as the mitochondrial network morphology was substantially restored in the HVT-ΔvNr-13-infected cells. IncuCyte(R) S3 real time apoptosis monitoring demonstrated that vNr-13 unequivocally involved in the apoptosis inhibition, and it is associated with increase of PFU, especially at serum-free conditions of later stages of viral replication cycle. Furthermore, HVT blocks apoptosis in infected cells, but activates apoptosis in the non-infected bystander cells.

B cell lymphoma-2 (Bcl-2)-family proteins play important role in regulating apoptosis during homeostasis, tissue development and infectious diseases. Several viruses encode homologs of cellular Bcl-2-proteins (vBcl-2) to inhibit apoptosis, which enable them to replicate and persist in the infected cells and to evade/modulate the immune response of the host. Herpesvirus of turkeys (HVT) is a non-pathogenic alphaherpesvirus of turkeys and chickens, widely used as live vaccine against Marek's disease and recombinant vaccine viral vectors for protecting against multiple avian diseases. Identical copies of the HVT genes HVT079 and HVT096 encode the Bcl-2 homolog vNr-13 While previous studies have identified the potential ability of vNr-13 in inhibiting apoptosis induced by serum deprivation, detailed investigations on the functions of vNr-13 are unknown. Using CRISPR/Cas9-based ablation of vNr-13 gene, we demonstrated HVT vNr-13 role in early stages of viral replication cycle, mitochondrial morphology disruption, and apoptosis inhibition in later stages of viral replication.


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