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

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Modern phylogeography aims at reconstructing the geographic diffusion of organisms based on their genomic sequences and spatial information. Phylogeographic approaches usually ignore the possibility of recombination, which decouples the evolutionary and geographic histories of different parts of the genome. Genomic regions of recombining or reassorting pathogens often originate and evolve at different times and locations, which characterised their unique spatial histories. Measuring the extent of these differences requires new methods to compare geographic information on phylogenetic trees reconstructed from different parts of the genome. Here we develop for the first time a set of measures of phylogeographic incompatibility aimed at detecting differences between geographical histories in terms of distances between phylogeographies. We study the effect of varying demography and recombination on phylogeographic incompatibilities using coalescent simulations. We further apply these measures to the evolutionary history of human and livestock pathogens, either reassorting or recombining, such as the Victoria and Yamagata lineages of influenza B and the O/Ind-2001 foot-and-mouth disease virus strain. Our results reveal diverse geographical paths of diffusion that characterise the origins and evolutionary histories of different viral genes and genomic segments. phylogeography, recombination, viral evolution.


Mycobacterium tuberculosis (M.tb) infection results in approximately 1.3 million human deaths each year. M.tb resides primarily inside macrophages, and maintains persistent infection. In response to infection and inflammation, platelet activating factor C-16 (PAF C-16), a phospholipid compound, is released by various cells including neutophils and monocytes. We have recently shown that PAF C-16 can directly inhibit the growth of two representative non-pathogenic mycobacteria, Mycobacterium bovis BCG and Mycobacterium smegmatis (M. smegmatis), by damaging the bacterial cell membrane. Here, we have examined the effect of PAF C-16 on M. smegmatis residing within macrophages, and identified mechanisms involved in their growth inhibitory function. Our results demonstrated that exogenous PAF C-16 inhibited the growth of M. smegmatis inside phagocytic cells of monocytic cell line, THP-1; this effect was partially blocked by PAF receptor antagonists, suggesting the involvement of PAF receptor-mediated signaling pathways. Arachidonic acid, a downstream metabolite of PAF C-16 signaling pathway, directly inhibited the growth of M. smegmatis in vitro. Moreover, the inhibition of phospholipase C and phospholipase A2 activities, involved in PAF C-16 signaling pathway, increased survival of intracellular M. smegmatis. Interestingly, we also observed that inhibition of inducible nitric oxide synthase (iNOS) enzyme and antibody-mediated neutralization of TNF-α partially mitigated the intracellular growth inhibitory effect of PAF C-16. Use of a number of PAF C-16 structural analogs, including Lyso-PAF, 2-O-methyl PAF, PAF C-18 and Hexanolamino PAF, revealed that the presence of acetyl group (CH3CO) at sn-2 position of the glycerol backbone of PAF is important for the intracellular growth inhibition activity against M. smegmatis. Taken together, these results suggest that exogenous PAF C-16 treatment inhibits intracellular M. smegmatis growth, at least partially, in a nitric oxide and TNF-α dependent manner.

King S, Rajko-Nenow P, Ashby M, Frost L, Carpenter S, Batten C (2020)

Outbreak of African horse sickness in Thailand, 2020

Transboundary and Emerging Diseases
Publisher’s version:


African horse sickness was confirmed in horses in Thailand during March 2020. The virus was determined to belong to serotype 1 and is phylogenetically closely related to isolates from South Africa. This is the first incidence of African horse sickness occurring in Southeast Asia and of serotype 1 outside of Africa.


Gammacoronavirus infectious bronchitis virus (IBV) causes an economically important respiratory disease of poultry. Protective immunity is associated with the major structural protein, spike (S) glycoprotein, which induces neutralising antibodies and defines the serotype. Cross-protective immunity between serotypes is limited and can be difficult to predict. In this study, the ability of two recombinant IBV vaccine candidates, BeauR-M41(S) and BeauR-4/91(S), to induce cross-protection against a third serotype, QX, was assessed. Both rIBVs are genetically based on the Beaudette genome with only the S gene derived from either M41 or 4/91, two unrelated serotypes. The use of these rIBVs allowed for the assessment of the potential of M41 and 4/91 S glycoproteins to induce cross-protective immunity against a heterologous QX challenge. The impact of the order of vaccination was also assessed. Homologous primary and secondary vaccination with BeauR-M41(S) or BeauR-4/91(S) resulted in a significant reduction of infectious QX load in the trachea at four days post-challenge, whereas heterologous primary and secondary vaccination with BeauR-M41(S) and BeauR-4/91(S) reduced viral RNA load in the conjunctiva-associated lymphoid tissue (CALT). Both homologous and heterologous vaccination regimes reduced clinical signs and birds recovered more rapidly as compared with an unvaccinated/challenge control group. Despite both rIBV BeauR-M41(S) and BeauR-4/91(S) displaying limited replication in vivo, serum titres in these vaccinated groups were higher as compared with the unvaccinated/challenge control group. This suggests that vaccination with rIBV primed the birds for a boosted humoral response to heterologous QX challenge. Collectively, vaccination with the rIBV elicited limited protection against challenge, with failure to protect against tracheal ciliostasis, clinical manifestations, and viral replication. The use of a less attenuated recombinant vector that replicates throughout the respiratory tract could be required to elicit a stronger and prolonged protective immune response.


Avian influenza viruses of the subtype H6Nx are being detected globally with increasing frequency. Some H6Nx lineages are becoming enzootic in Asian poultry and sporadic incursions into European poultry are occurring more frequently. H6Nx viruses that contain mammalian adaptation motifs pose a zoonotic threat and have caused human cases. Although currently understudied globally, H6Nx avian influenza viruses pose a substantial threat to both poultry and human health. In this review we examine the current state of knowledge of H6Nx viruses including their global distribution, tropism, transmission routes and human health risk.


Background: Reverse genetics is used in many laboratories around the world and enables the creation of tailormade influenza viruses with a desired genotype or phenotype. However, the process is not flawless, and difficulties remain during cloning of influenza gene segments into reverse genetics vectors (pHW2000, pHH21, pCAGGS). Reverse genetics begins with making cDNA copies of influenza gene segments and cloning them into bidirectional (pHW2000) or uni-directional plasmids (pHH21, pCAGGS) followed by transfection of the recombinant plasmid(s) to HEK-293 T or any other suitable cells which are permissive to transfection. However, the presence of internal restriction sites in the gene segments of many field isolates of avian influenza viruses makes the cloning process difficult, if employing conventional methods. Further, the genetic instability of influenza gene-containing plasmids in bacteria (especially Polymerase Basic 2 and Polymerase Basic 1 genes; PB2 and PB1) also leads to erroneous incorporation of bacterial genomic sequences into the influenza gene of interest.

Methods: Herein, we report an easy and efficient ligation and restriction enzyme independent (LREI) cloning method for cloning influenza gene segments into pHW2000 vector. The method involves amplification of megaprimers followed by PCR amplification of megaprimers using a bait plasmid, DpnI digestion and transformation.

Results: Hard-to-clone genes: PB2 of A/chicken/Bangladesh/23527/2014 (H9N2) and PB1 of A/chicken/Bangladesh/23527/2014 (H9N2), A/chicken/Jiangxi/02.05YGYXG023-P/2015 (H5N6) and A/Chicken/Vietnam/H7F-14-BN4–315/2014 (H9N2) were cloned into pHW2000 using our LREI method and recombinant viruses were subsequently rescued.

Conclusion: The LREI cloning procedure represents an alternative strategy for cloning influenza gene segments which have internal restriction sites for the enzymes used in reverse genetics. Further, the problem of genetic instability in bacteria can be alleviated by growing recombinant bacterial cultures at a lower temperature. This technique can be applied to clone any influenza gene segment using universal primers, which would help in rapid generation of influenza viruses and facilitate influenza research and vaccine development.

Graham S P, McLean R K, Spencer A J, Belij-Rammerstorfer S, Wright D, Ulaszewska M, Edwards J C, Hayes J W P, Martini V, Thakur N, Conceicao C, Dietrich I, Shelton H, Waters R, Ludi A, Wilsden G, Browning C, Bialy D, Bhat S, Stevenson-Leggett P, Hollinghurst P, Gilbride C, Pulido D, Moffat K, Sharpe H, Allen E, Mioulet V, Chiu C, Newman J, Asfor A S, Burman A, Crossley S, Huo J, Owens R J, Carroll M, Hammond J A, Tchilian E, Bailey D, Charleston B, Gilbert S C, Tuthill T J, Lambe T (2020)

Evaluation of the immunogenicity of prime-boost vaccination with the replication-deficient viral vectored COVID-19 vaccine candidate ChAdOx1 nCoV-19

npj Vaccines 5 (1), 69


Clinical development of the COVID-19 vaccine candidate ChAdOx1 nCoV-19, a replication-deficient simian adenoviral vector expressing the full-length SARS-CoV-2 spike (S) protein was initiated in April 2020 following non-human primate studies using a single immunisation. Here, we compared the immunogenicity of one or two doses of ChAdOx1 nCoV-19 in both mice and pigs. Whilst a single dose induced antigen-specific antibody and T cells responses, a booster immunisation enhanced antibody responses, particularly in pigs, with a significant increase in SARS-CoV-2 neutralising titres.


African swine fever (ASF) is a devastating disease in pigs, with no vaccines for control. The genetic manipulation of African swine fever virus (ASFV) is often tedious and time consuming. Here, we describe a method to manipulate the virus genome to produce gene deletion viruses in a much-reduced time. This method combines the conventional homologous recombination with fluorescent-activated cells sorting (FACS), to isolate and purify viruses expressing fluorescent reporter genes. With three rounds of single cell isolation via FACS and two rounds of limiting dilution, we deleted two additional genes, EP153R and EP402R, from Benin 97/1 ASFV lacking the DP148R gene. By combining different fluorescent markers, this method has the potential to greatly facilitate studies on understanding ASFV gene functions and develop candidate live-attenuated vaccines.


Using the best animal models to study immune responses against specific pathogens or vaccines can dramatically accelerate our understanding. Veterinary species are well studied, particularly livestock, to reduce their disease burden. They have also proven to be powerful models, especially for zoonotic pathogens and novel vaccination strategies. A prerequisite for any model selection is having the right quality and range of species?specific immunological reagents. To help promote the widest possible use of veterinary species, an open access website ( has been created as a central community annotated hub for veterinary immunological reagents. The website is also the portal into services offered by the UK Immunological Toolbox project that includes antibody generation, sequencing and recombinant expression. The funding for this effort is linked into sustainable sources but ultimate success still relies on community engagement to continually increase the quality and quantity of information. It is hoped that as more users and reagent owners engage it will become an essential resource for researchers, veterinarians and clinicians alike by removing barriers that prevent the use of the most informative animal models.

Flannery J, Frost L, Fay P, Hicks H, Henstock M, Smreczak M, Orłowska A, Rajko-Nenow P, Darpel K, Batten C (2020)

BTV-14 infection in sheep elicits viraemia with mild clinical symptoms

Microorganisms 8 (6), 892


In 2011, Bluetongue virus serotype 14 (BTV-14) was detected in Russia during routine surveillance, and was subsequently found in a number of European countries. The strain had high sequence similarity to a BTV-14 vaccine strain. We aimed to determine the risk of this BTV-14 strain causing disease in a UK sheep breed. Four Poll Dorset sheep were infected with a Polish isolate of BTV-14 and infection kinetics were monitored over 28 days. BTV RNA was detected in EDTA blood by 4 days post-infection (dpi) and remained detectable at 28 days post-infection (dpi). Peak viraemia occurred at 6 and 7 dpi with Ct values ranging between 24.6 and 27.3 in all infected animals. BTV antibodies were detected by 10 dpi using a commercial ELISA and neutralising antibodies were detected from 10 dpi. BTV was isolated between 6 and 12 dpi. All infected sheep developed mild clinical signs such as reddening of conjunctiva and mucosal membranes, with one sheep demonstrating more overt clinical signs. Two uninoculated control animals remained clinically healthy and did not have detectable BTV RNA or antibodies. The overall mild clinical symptoms caused by this BTV-14 in this highly susceptible sheep breed were in accordance with the asymptomatic infections observed in the affected countries.


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