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

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Background: The recent reclassification of formaldehyde as a presumed carcinogen prompted the investigation into the comparative efficacy of hydrogen peroxide as a fumigant in microbiological safety cabinets.

Introduction: The aim of the study was to quantify the biocidal efficacy of formaldehyde fumigation, including variables such as exposure time and concentration, and then to compare this to the biocidal efficacy achieved from a hydrogen peroxide vapor fumigation system. The study also investigated the ability of both fumigants to permeate the microbiological safety cabinet (MBSC), including the workspace, under the work tray, and after the HEPA filters. Furthermore, the effect of organic soiling on efficacy was also assessed. Infectious bronchitis virus (IBV) was used as the biological target to develop this study model.

Methods: A model using IBV was developed to determine the efficacy of formaldehyde and hydrogen peroxide as fumigants. Virus was dried on stainless steel discs, and variables including concentration, time, protein soiling, and location within an MBSC were assessed.

Results: It was demonstrated that formaldehyde fumigation could achieve a 6-log reduction in the titer of the virus throughout the cabinet, and high protein soiling in the presentation did not affect efficacy. Appropriate cycle parameters for the hydrogen peroxide system were developed, and when challenged with IBV, it was shown that vaporized hydrogen peroxide could achieve an equal 6-log titer reduction as formaldehyde within the cabinet workspace and overcome the presence of soiling.

Conclusion: Hydrogen peroxide was demonstrated to be a viable alternative to formaldehyde under most situations tested. However, the hydrogen peroxide system did not achieve an equal titer reduction above the cabinet’s first HEPA filter using the cabinet workspace cycle, and further optimization of the hydrogen peroxide cycle parameters, including pulsing of the cabinet fans, may be required to achieve this.


Zoonotic coronavirus (CoV) infections, such as those responsible for the current severe acute respiratory syndrome-CoV 2 (SARS-CoV-2) pandemic, cause grave international public health concern. In infected cells, the CoV RNA-synthesizing machinery associates with modified endoplasmic reticulum membranes that are transformed into the viral replication organelle (RO). Although double-membrane vesicles (DMVs) appear to be a pan-CoV RO element, studies to date describe an assortment of additional CoV-induced membrane structures. Despite much speculation, it remains unclear which RO element(s) accommodate viral RNA synthesis. Here we provide detailed 2D and 3D analyses of CoV ROs and show that diverse CoVs essentially induce the same membrane modifications, including the small open double-membrane spherules (DMSs) previously thought to be restricted to gamma- and delta-CoV infections and proposed as sites of replication. Metabolic labeling of newly synthesized viral RNA followed by quantitative electron microscopy (EM) autoradiography revealed abundant viral RNA synthesis associated with DMVs in cells infected with the beta-CoVs Middle East respiratory syndrome-CoV (MERS-CoV) and SARS-CoV and the gamma-CoV infectious bronchitis virus. RNA synthesis could not be linked to DMSs or any other cellular or virus-induced structure. Our results provide a unifying model of the CoV RO and clearly establish DMVs as the central hub for viral RNA synthesis and a potential drug target in CoV infection.


Various viruses have evolved a plethora of strategies to manipulate DNA damage machinery and commandeer cells to maximize their own replication. On the contrary, host cells have also evolved to maintain cellular genomic stability and suppress carcinogenesis through multifaceted cellular DNA damage response (DDR). Most studies have focused on the relationship between DNA virus replication and the host DDR pathway, but few have investigated the interface between the DDR pathway and ribonucleic acid (RNA) viruses, such as oncolytic Newcastle disease virus (NDV). In the present study, we report for the first time that F-HN co-expression and NDV infection trigger ataxia telangiectasia-mutated (ATM)-dependent DSB lesions in tumor cells to promote viral replication and syncytium formation, respectively. We found that late NDV infection and membrane fusion activated the ATM-Chk2 axis in tumor cells, which expands prior understanding of the membrane fusion process and NDV infection. Our findings reveal new links among oncolytic NDV replication, membrane fusion, and DDR and deepen insights into NDV replication.


Live attenuated vaccines are considered to be the fastest route to the development of a safe and efficacious African swine fever (ASF) vaccine. Infection with the naturally attenuated OURT88/3 strain induces protection against challenge with virulent isolates from the same or closely related genotypes. However, adverse clinical signs following immunisation have been observed. Here, we attempted to increase the OURT88/3 safety profile by deleting I329L, a gene previously shown to inhibit the host innate immune response. The resulting virus, OURT88/3ΔI329L, was tested in vitro to evaluate the replication and expression of type I interferon (IFN) and in vivo by immunisation and lethal challenge experiments in pigs. No differences were observed regarding replication; however, increased amounts of both IFN-β and IFN-α were observed in macrophages infected with the deletion mutant virus. Unexpectedly, the deletion of I329L markedly reduced protection against challenge with the virulent OURT88/1 isolate. This was associated with a decrease in both antibody levels against VP72 and the number of IFN-γ-producing cells in the blood of non-protected animals. Furthermore, a significant increase in IL-10 levels in serum was observed in pigs immunised with OURT88/3ΔI329L following challenge. Interestingly, the deletion of the I329L gene failed to attenuate the virulent Georgia/2007 isolate.


The main target cells for African swine fever virus (ASFV) replication in pigs are of monocyte macrophage lineage and express markers typical of the intermediate to late stages of differentiation. The lack of a porcine cell line, which accurately represents these target cells, limits research on virus host interactions and the development of live-attenuated vaccine strains. We show here that the continuously growing, growth factor dependent ZMAC-4 porcine macrophage cell line is susceptible to infection with eight different field isolates of ASFV. Replication in ZMAC-4 cells occurred with similar kinetics and to similar high titres as in primary porcine bone marrow cells. In addition we showed that twelve passages of an attenuated strain of ASFV, OURT88/3, in ZMAC-4 cells did not reduce the ability of this virus to induce protection against challenge with virulent virus. Thus, the ZMAC-4 cells provide an alternative to primary cells for ASFV replication.


Receptor recognition and binding is the first step of viral infection and a key determinant of host specificity. The inability of avian influenza viruses to effectively bind human-like sialylated receptors is a major impediment to their efficient transmission in humans and pandemic capacity. Influenza H9N2 viruses are endemic in poultry across Asia and parts of Africa where they occasionally infect humans and are therefore considered viruses with zoonotic potential. We previously described H9N2 viruses, including several isolated from human zoonotic cases, showing a preference for human-like receptors. Here we take a mutagenesis approach, making viruses with single or multiple substitutions in H9 haemagglutinin to determine the genetic basis of preferences for alternative avian receptors and for human-like receptors. We describe amino acid motifs at positions 190, 226 and 227 that play a major role in determining receptor specificity, and several other residues such as 159, 188, 193, 196, 198 and 225 play a smaller role. Furthermore, we show changes at residues 135, 137, 147, 157, 158, 184, 188, and 192 can also modulate virus receptor avidity and that substitutions that increased or decreased the net positive charge around the haemagglutinin receptor-binding site show increases and decreases in avidity, respectively. The motifs we identify as increasing preference for the human-receptor will help guide future H9N2 surveillance efforts and facilitate our understanding of the emergence of influenza viruses with high zoonotic potential.

Mignotte A, Garros C, Gardes L, Balenghien T, Duhayon M, Rakotoarivony I, Tabourin L, Poujol L, Mathieu B, Ibanez-Justicia A, Deniz A, Cvetkovikj A, Purse B V, Ramilo D W, Stougiou D, Werner D, Pudar D, Petric D, Veronesi E, Jacobs F, Kampen H, Pereira da Fonseca I, Lucientes J, Navarro J, de la Puente J M, Stefanovska J, Searle K R, Khallaayoune K, Culverwell C L, Larska M, Bourquia M, Goffredo M, Bisia M, England M, Robin M, Quaglia M, Miranda-Chueca M A, Bodker R, Estrada-Pena R, Carpenter S, Tchakarova S, Boutsini S, Sviland S, Schafer S M, Ozolina Z, Seglina Z, Vatansever Z, Huber K (2020)

The tree that hides the forest: cryptic diversity and phylogenetic relationships in the Palaearctic vector Obsoletus/Scoticus Complex (Diptera: Ceratopogonidae) at the European level

Parasites and Vectors 13 (1), 265


BACKGROUND: Culicoides obsoletus is an abundant and widely distributed Holarctic biting midge species, involved in the transmission of bluetongue virus (BTV) and Schmallenberg virus (SBV) to wild and domestic ruminants. Females of this vector species are often reported jointly with two morphologically very close species, C. scoticus and C. montanus, forming the Obsoletus/Scoticus Complex. Recently, cryptic diversity within C. obsoletus was reported in geographically distant sites. Clear delineation of species and characterization of genetic variability is mandatory to revise their taxonomic status and assess the vector role of each taxonomic entity. Our objectives were to characterize and map the cryptic diversity within the Obsoletus/Scoticus Complex.

METHODS: Portion of the cox1 mitochondrial gene of 3763 individuals belonging to the Obsoletus/Scoticus Complex was sequenced. Populations from 20 countries along a Palaearctic Mediterranean transect covering Scandinavia to Canary islands (North to South) and Canary islands to Turkey (West to East) were included. Genetic diversity based on cox1 barcoding was supported by 16S rDNA mitochondrial gene sequences and a gene coding for ribosomal 28S rDNA. Species delimitation using a multi-marker methodology was used to revise the current taxonomic scheme of the Obsoletus/Scoticus Complex.

RESULTS: Our analysis showed the existence of three phylogenetic clades (C. obsoletus clade O2, C. obsoletus clade dark and one not yet named and identified) within C. obsoletus. These analyses also revealed two intra-specific clades within C. scoticus and raised questions about the taxonomic status of C. montanus.

CONCLUSIONS: To our knowledge, our study provides the first genetic characterization of the Obsoletus/Scoticus Complex on a large geographical scale and allows a revision of the current taxonomic classification for an important group of vector species of livestock viruses in the Palaearctic region.

McCleary S, Strong R, McCarthy R R, Edwards J C, Howes E L, Stevens L M, Sanchez-Cordon P J, Nunez A, Watson S, Mileham A J, Lillico S G, Tait-Burkard C, Proudfoot C, Ballantyne M, Whitelaw C B A, Steinbach F, Crooke H R (2020)

Substitution of warthog NF-κB motifs into RELA of domestic pigs is not sufficient to confer resilience to African swine fever virus

Scientific Reports 10 (1), 8951


African swine fever virus (ASFV) causes a lethal, haemorrhagic disease in domestic swine that threatens pig production across the globe. Unlike domestic pigs, warthogs, which are wildlife hosts of the virus, do not succumb to the lethal effects of infection. There are three amino acid differences between the sequence of the warthog and domestic pig RELA protein; a subunit of the NF-κB transcription factor that plays a key role in regulating the immune response to infections. Domestic pigs with all 3 or 2 of the amino acids from the warthog RELA orthologue have been generated by gene editing. To assess if these variations confer resilience to ASF we established an intranasal challenge model with a moderately virulent ASFV. No difference in clinical, virological or pathological parameters were observed in domestic pigs with the 2 amino acid substitution. Domestic pigs with all 3 amino acids found in warthog RELA were not resilient to ASF but a delay in onset of clinical signs and less viral DNA in blood samples and nasal secretions was observed in some animals. Inclusion of these and additional warthog genetic traits into domestic pigs may be one way to assist in combating the devastating impact of ASFV.

Gorbalenya A E, Krupovic M, Mushegian A, Kropinski A M, Siddell S G, Varsani A, Adams M J, Davison A J, Dutilh B E, Harrach B, Harrison R L, Junglen S, King A M Q, Knowles N J, Lefkowitz E J, Nibert M L, Rubino L, Sabanadzovic S, Sanfaçon H, Simmonds P, Walker P J, Zerbini F M, Kuhn J H (2020)

The new scope of virus taxonomy: partitioning the virosphere into 15 hierarchical ranks

Nature Microbiology 5 (5), 668-674


Virus taxonomy emerged as a discipline in the middle of the twentieth century. Traditionally, classification by virus taxonomists has been focussed on the grouping of relatively closely related viruses. However, during the past few years, the International Committee on Taxonomy of Viruses (ICTV) has recognized that the taxonomy it develops can be usefully extended to include the basal evolutionary relationships among distantly related viruses. Consequently, the ICTV has changed its Code to allow a 15-rank classification hierarchy that closely aligns with the Linnaean taxonomic system and may accommodate the entire spectrum of genetic divergence in the virosphere. The current taxonomies of three human pathogens, Ebola virus, severe acute respiratory syndrome coronavirus and herpes simplex virus 1 are used to illustrate the impact of the expanded rank structure. This new rank hierarchy of virus taxonomy will stimulate further research on virus origins and evolution, and vice versa, and could promote crosstalk with the taxonomies of cellular organisms.

Evans S W, Beal J, Berger K, Bleijs D A, Cagnetti A, Ceroni F, Epstein G L, Garcia-Reyero N, Gillum D R, Harkess G, Hillson N J, Hogervorst P A M, Jordan J L, Lacroix G, Moritz R, OhEigeartaigh S S, Palmer M J, van Passel M W J (2020)

Embrace experimentation in biosecurity governance

Science 368 (6487), 138-140


As biological research and its applications rapidly evolve, new attempts at the governance of biology are emerging, challenging traditional assumptions about how science works and who is responsible for governing. However, these governance approaches often are not evaluated, analyzed, or compared. This hinders the building of a cumulative base of experience and opportunities for learning. Consider “biosecurity governance,” a term with no internationally agreed definition, here defined as the processes that influence behavior to prevent or deter misuse of biological science and technology. Changes in technical, social, and political environments, coupled with the emergence of natural diseases such as coronavirus disease 2019 (COVID-19), are testing existing governance processes. This has led some communities to look beyond existing biosecurity models, policies, and procedures. But without systematic analysis and learning across them, it is hard to know what works. We suggest that activities focused on rethinking biosecurity governance present opportunities to “experiment” with new sets of assumptions about the relationship among biology, security, and society, leading to the development, assessment, and iteration of governance hypotheses.


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