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

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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.

Rajko-Nenow P, Golender N, Bumbarov V, Brown H, Frost L, Darpel K, Tennakoon C, Flannery J, Batten C (2020)

Complete coding sequence of a novel bluetongue virus isolated from a commercial sheeppox vaccine

Microbiology Resource Announcements 9 (10), e01539-19


The full genome sequences of two isolates of bluetongue virus (BTV) from a commercial sheeppox vaccine were determined. Strain SPvvvv/02 shows low sequence identity to its closest relative, strain BTV-26 KUW2010/02, indicating the probable detection of a novel BTV genotype, whereas strain SPvvvv/03 shows high sequence identity to strain BTV-28/1537/14.

Pedrera M, Macchi F, McLean R K, Franceschi V, Thakur N, Russo L, Medfai L, Todd S, Tchilian E Z, Audonnet J-C, Chappell K, Isaacs A, Watterson D, Young P R, Marsh G A, Bailey D, Graham S P, Donofrio G (2020)

Bovine herpesvirus-4-vectored delivery of Nipah virus glycoproteins enhances T cell immunogenicity in pigs

Vaccines 8 (1), 115


Nipah virus (NiV) is an emergent pathogen capable of causing acute respiratory illness and fatal encephalitis in pigs and humans. A high fatality rate and broad host tropism makes NiV a serious public and animal health concern. There is therefore an urgent need for a NiV vaccines to protect animals and humans. In this study we investigated the immunogenicity of bovine herpesvirus (BoHV-4) vectors expressing either NiV attachment (G) or fusion (F) glycoproteins, BoHV-4-A-CMV-NiV-GΔTK or BoHV-4-A-CMV-NiV-FΔTK, respectively in pigs. The vaccines were benchmarked against a canarypox (ALVAC) vector expressing NiV G, previously demonstrated to induce protective immunity in pigs. Both BoHV-4 vectors induced robust antigenspecific antibody responses. BoHV-4-A-CMV-NiV-GΔTK stimulated NiV-neutralizing antibody titers comparable to ALVAC NiV G and greater than those induced by BoHV-4-A-CMV-NiV-FΔTK. In contrast, only BoHV-4-A-CMV-NiV-FΔTK immunized pigs had antibodies capable of significantly neutralizing NiV G and F-mediated cell fusion. All three vectored vaccines evoked antigen-specific CD4 and CD8 T cell responses, which were particularly strong in BoHV-4-A-CMVNiV-GΔTK immunized pigs and to a lesser extent BoHV-4-A-CMV-NiV-FΔTK. These findings emphasize the potential of BoHV-4 vectors for inducing antibody and cell-mediated immunity in pigs and provide a solid basis for the further evaluation of these vectored NiV vaccine candidates.


Wild migratory birds are often implicated in the introduction, maintenance, and global dissemination of different pathogens, such as influenza A viruses (IAV) and antimicrobial-resistant (AMR) bacteria. Trapping of migratory birds during their resting periods at the northern coast of Egypt is a common and ancient practice performed mainly for selling in live bird markets (LBM). In the present study, samples were collected from 148 wild birds, representing 14 species, which were being offered for sale in LBM. All birds were tested for the presence of AIV and enterobacteriaceae. Ten samples collected from Northern Shoveler birds (Spatula clypeata) were positive for IAV and PCR sub-typing and pan HA/NA sequencing assays detected H5N8, H9N2, and H6N2 viruses in four, four, and one birds, respectively. Sequencing of the full haemagglutinin (HA) gene revealed a high similarity with currently circulating IAV in Egypt. From all the birds, E.coli was recovered from 37.2% and Salmonella from 20.2%, with 66%–96% and 23%–43% isolates being resistant to at least one of seven selected critically important antimicrobials (CIA), respectively. The presence of enzootic IAV and the wide prevalence of AMR enterobacteriaceae in wild birds highlight the potential role of LBM in the spread of different pathogens from and to wild birds. Continued surveillance of both AIV and antimicrobial-resistant enterobacteriaceae in wild birds’ habitats is urgently needed. 


Passive immunisation with neutralising antibodies can be a potent therapeutic strategy if used pre- or post-exposure to a variety of pathogens. Herein, we investigated whether recombinant monoclonal antibodies (mAbs) could be used to protect chickens against avian influenza. Avian influenza viruses impose a significant economic burden on the poultry industry and pose a zoonotic infection risk for public health worldwide. Traditional control measures including vaccination do not provide rapid protection from disease, highlighting the need for alternative disease mitigation measures. In this study, previously generated neutralizing anti-H9N2 virus monoclonal antibodies were converted to single-chain variable fragment antibodies (scFvs). These recombinant scFv antibodies were produced in insect cell cultures and the preparations retained neutralization capacity against an H9N2 virus in vitro. To evaluate recombinant scFv antibody efficacy in vivo, chickens were passively immunized with scFvs one day before, and for seven days after virus challenge. Groups receiving scFv treatment showed partial virus load reductions measured by plaque assays and decreased disease manifestation. These results indicate that antibody therapy could reduce clinical disease and shedding of avian influenza virus in infected chicken flocks.


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