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

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Franzoni G, Graham S P, Sanna G, Angioi P, Fiori M S, Anfossi A, Amadori M, Dei Giudici S, Oggiano A (2018)

Interaction of porcine monocyte-derived dendritic cells with African swine fever viruses of diverse virulence

Veterinary Microbiology 216, 190-197


African swine fever (ASF) is a devastating disease for which there is no vaccine. The ASF virus (ASFV) can infect dendritic cell (DC), but despite the critical role these cells play in induction of adaptive immunity, few studies investigated their response to ASFV infection. We characterized the in vitro interactions of porcine monocyte-derived DCs (moDC) with a virulent (22653/14), a low virulent (NH/P68) and an avirulent (BA71V) ASFV strain. At a high multiplicity of infection (MOI = 1), all three strains infected immature moDC. Maturation of moDC, with IFN-α/TNF-α, increased susceptibility to infection with 22653/14 and other virulent strains, but reduced susceptibility to NH/P68 and BA71V. The reduced moDC susceptibility to BA71V/NH/P68 was IFN-α mediated, whereas increased susceptibility to 22653/14 was induced by TNF-α. Using an MOI of 0.01, we observed that BA71V replicated less efficiently in moDC compared to the other isolates and we detected increased replication of NH/P68 compared to 22653/14. We observed that BA71V and NH/P68, but not 22653/14, downregulated expression of MHC class I on infected cells. All three strains decreased CD16 expression on moDC, whereas ASFV infection resulted in CD80/86 down-regulation and MHC class II DR up-regulation on mature moDC. None of the tested strains induced a strong cytokine response to ASFV and only modest IL-1α was released after BA71V infection. Overall our results revealed differences between strains and suggest that ASFV has evolved mechanisms to replicate covertly in inflammatory DC, which likely impairs the induction of an effective immune response.

Swatek K N, Aumayr M, Pruneda J N, Visser L J, Berryman S, Kueck A F, Geurink P P, Ovaa H, van Kuppeveld F J M, Tuthill T J, Skern T, Komander D (2018)

Irreversible inactivation of ISG15 by a viral leader protease enables alternative infection detection strategies

Proceedings of the National Academy of Sciences of the United States of America 115 (10), 2371-2376


In response to viral infection, cells mount a potent inflammatory response that relies on ISG15 and ubiquitin posttranslational modifications. Many viruses use deubiquitinases and deISGylases that reverse these modifications and antagonize host signaling processes. We here reveal that the leader protease, Lbpro, from foot-and-mouth disease virus (FMDV) targets ISG15 and to a lesser extent, ubiquitin in an unprecedented manner. Unlike canonical deISGylases that hydrolyze the isopeptide linkage after the C-terminal GlyGly motif, Lbpro cleaves the peptide bond preceding the GlyGly motif. Consequently, the GlyGly dipeptide remains attached to the substrate Lys, and cleaved ISG15 is rendered incompetent for reconjugation. A crystal structure of Lbpro bound to an engineered ISG15 suicide probe revealed the molecular basis for ISG15 proteolysis. Importantly, anti-GlyGly antibodies, developed for ubiquitin proteomics, are able to detect Lbpro cleavage products during viral infection. This opens avenues for infection detection of FMDV based on an immutable, host-derived epitope.

Chang P, Yao Y, Tang N, Sadeyen J-R, Sealy J, Clements A, Bhat S, Munir M, Bryant J, Iqbal M (2018)

The application of NHEJ-CRISPR/Cas9 and Cre-Lox system in the generation of bivalent duck enteritis virus vaccine against avian influenza virus

Viruses 10 (2), 81


Duck-targeted vaccines to protect against avian influenza are critically needed to aid in influenza disease control efforts in regions where ducks are endemic for highly pathogenic avian influenza (HPAI). Duck enteritis virus (DEV) is a promising candidate viral vector for development of vaccines targeting ducks, owing to its large genome and narrow host range. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system is a versatile gene-editing tool that has proven beneficial for gene modification and construction of recombinant DNA viral vectored vaccines. Currently, there are two commonly used methods for gene insertion: non-homologous end-joining (NHEJ) and homology-directed repair (HDR). Owing to its advantages in efficiency and independence from molecular requirements of the homologous arms, we utilized NHEJ-dependent CRISPR/Cas9 to insert the influenza hemagglutinin (HA) antigen expression cassette into the DEV genome. The insert was initially tagged with reporter green fluorescence protein (GFP), and a Cre-Lox system was later used to remove the GFP gene insert. Furthermore, a universal donor plasmid system was established by introducing double bait sequences that were independent of the viral genome. In summary, we provide proof of principle for generating recombinant DEV viral vectored vaccines against the influenza virus using an integrated NHEJ-CRISPR/Cas9 and Cre-Lox system.

Jankovich J K, Chapman D, Hansen D T, Robida M D, Loskutov A, Craciunescu F, Borovkov A, Kibler K, Goatley L, King K, Netherton C L, Taylor G, Jacobs B, Sykes K, Dixon L K (2018)

Immunisation of pigs by DNA prime and recombinant vaccinia virus boost to identify and rank African swine fever virus immunogenic and protective proteins

Journal of Virology 92 (8),


African swine fever virus (ASFV) causes an acute haemorrhagic fever in domestic pigs with high socio-economic impact. No vaccine is available limiting options for control. Although live attenuated ASFV can induce up to one hundred percent protection against lethal challenge, little is known of the antigens which induce this protective response. To identify additional ASFV immunogenic and potentially protective antigens we cloned 47 viral genes in individual plasmids for gene vaccination and in recombinant vaccinia viruses. These antigens were selected to include proteins with different functions and timing of expression. Pools of up to 22 antigens were delivered by DNA prime and recombinant vaccinia virus boost to groups of pigs. Responses of immune lymphocytes from pigs to individual recombinant proteins and to ASFV were measured by interferon gamma ELISpot assays to identify a subset of the antigens that consistently induced the highest responses. All 47 antigens were then delivered to pigs by DNA prime and recombinant vaccinia virus boost and pigs were challenged with a lethal dose of Georgia 2007/1 ASFV isolate. Although pigs developed clinical and pathological signs consistent with acute ASFV, viral genome levels were significantly reduced in blood and several lymph tissues in those pigs immunised with vectors expressing ASFV antigens compared with control pigs.ImportanceThe lack of a vaccine limits the options to control African swine fever. Advances have been made in the development of genetically modified live attenuated ASFV that can induce protection against challenge. However there may be safety issues relating to the use of these in the field. There is little information about ASFV antigens that can induce a protective immune response against challenge. We carried out a large screen of 30% of ASFV antigens by delivering individual genes in different pools to pigs by DNA immunization prime and recombinant vaccinia virus boost. The response in immunized pigs to these individual antigens was compared to identify the most immunogenic. Lethal challenge of pigs immunised with a pool of antigens resulted in reduced levels of virus in blood and lymph tissues compared to pigs immunised with control vectors. Novel immunogenic ASFV proteins have been identified to test further as vaccine candidates.

Marotta C R, Dos Santos P N, Cordeiro M D, Matos P C M, Barros J H D S, Madeira M D F, Bell-Sakyi L, Fonseca A H (2018)

Trypanosoma rhipicephalis sp. nov. (Protozoa: Kinetoplastida) isolated from Rhipicephalus microplus (Acari: Ixodidae) ticks in Rio de Janeiro, Brazil

Parasitology Open 4, e2


Parasites of the genus Trypanosoma are unicellular flagellated microorganisms of the Trypanosomatidae. This study describes an isolate of the genus Trypanosoma naturally infecting Rhipicephalus microplus ticks, characterized through molecular, morphological and biological analysis. Trypanosome cultures, designated strain P1RJ, were obtained by isolation from R. microplus haemolymph in cultures of the tick cell line IDE8. After isolation, strain P1RJ grew well axenically in L15B medium at temperatures of 30, 32 and 34 °C. The new trypanosome remained stable in axenic culture over 14 passages in L15B at 30 °C and was successfully cryopreserved and resuscitated. Morphometric analysis was performed on randomly selected developmental forms. 18S rRNA and 24Sα rDNA sequence analyses confirmed that strain P1RJ is a new species of the genus Trypanosoma. The nucleotide sequences described were submitted to Genbank. Pathogenicity, involvement in vertebrate hosts, epidemiology, developmental cycle and transmission mechanisms of strain P1RJ are still unknown. Therefore, more studies will be necessary to determine life cycle aspects of this trypanosome, for which we propose the name Trypanosoma rhipicephalis sp. nov.

Cozens D, Grahame E, Sutherland E, Taylor G, Berry C C, Davies R L (2018)

Development and optimization of a differentiated airway epithelial cell model of the bovine respiratory tract

Scientific Reports 8 (1), 853


Cattle are subject to economically-important respiratory tract infections by various bacterial and viral pathogens and there is an urgent need for the development of more realistic in vitro models of the bovine respiratory tract to improve our knowledge of disease pathogenesis. In the present study, we have optimized the culture conditions in serum-free medium that allow bovine bronchial epithelial cells (BBECs) grown at an air-liquid interface to differentiate into a three-dimensional epithelium that is highly representative of the bovine airway. Epidermal growth factor was required to trigger both proliferation and differentiation of BBECs whilst retinoic acid was also essential for mucociliary differentiation. Triiodothyronine was demonstrated not to be important for the differentiation of BBECs. Oxygen concentration had a minimal effect although optimal ciliation was achieved when BBECs were cultured at 14% oxygen tension. Insert pore-density had a significant effect on the growth and differentiation of BBECs; a high-pore-density was required to trigger optimum differentiation. The established BBEC model will have wide-ranging applications for the study of bacterial and viral infections of the bovine respiratory tract; it will contribute to the development of improved vaccines and therapeutics and will reduce the use of cattle in in vivo experimentation.

Qian K, Cheng X, Zhang D, Shao H, Yao Y, Nair V, Qin A (2018)

Antiviral effect of lithium chloride on replication of avian leukosis virus subgroup J in cell culture

Archives of Virology 163 (4), 987-995


Lithium chloride (LiCl) has been reported to possess antiviral activity against several viruses. In the current study, we assessed the antiviral activity effect of LiCl on ALV-J infection in CEF cells by using real-time PCR, Western blot analysis, IFA and p27 ELISA analysis. Our results showed that both viral RNA copy number and protein level decreased significantly in a dose and time dependent manner. Time-course analysis revealed that the antiviral effect was more pronounced when CEFs were treated at the post infection stage rather than at early absorption or pre-absorption stages. Further experiments demonstrated that LiCl did not affect virus attachment or entry, but rather affected early virus replication. We also found that inhibition of viral replication after LiCl treatment was associated with reduced mRNA levels of pro-inflammatory cytokines. These results demonstrate that LiCl effectively blocked ALV-J replication in CEF cells and may be used as an antiviral agent against ALV-J.

Glidden C K, Beechler B, Buss P E, Charleston B, de Klerk-Lorist L-M, Maree F F, Muller T, Pérez-Martin E, Scott K A, van Schalkwyk O L, Jolles A (2018)

Detection of pathogen exposure in African buffalo using non-specific markers of inflammation

Frontiers in Immunology 8, 1944


Detecting exposure to new or emerging pathogens is a critical challenge to protecting human, domestic animal and wildlife health. Yet current techniques to detect infections typically target known pathogens of humans or economically important animals. In the face of the current surge in infectious disease emergence, non-specific disease surveillance tools are urgently needed. Tracking common host immune responses indicative of recent infection may have potential as a non-specific diagnostic approach for disease surveillance. The challenge to immunologists is to identify the most promising markers, which ideally should be highly conserved across pathogens and host species, become upregulated rapidly and consistently in response to pathogen invasion, and remain elevated beyond clearance of infection. This study combined an infection experiment and a longitudinal observational study to evaluate the utility of non-specific markers of inflammation (NSMI; two acute phase proteins (haptoglobin and serum amyloid A), two pro-inflammatory cytokines (IFNγ and TNF-α)) as indicators of pathogen exposure in a wild mammalian species, African buffalo (Syncerus caffer). Specifically, in the experimental study we asked (1) How quickly do buffalo mount NSMI responses upon challenge with an endemic pathogen, foot-and-mouth disease virus; (2) for how long do NSMI remain elevated after viral clearance and; (3) how pronounced is the difference between peak NSMI concentration and baseline NSMI concentration? In the longitudinal study, we asked (4) Are elevated NSMI associated with recent exposure to a suite of bacterial and viral respiratory pathogens in a wild population? Among the four NSMI that we tested, haptoglobin showed the strongest potential as a surveillance marker in African buffalo: Concentrations quickly and consistently reached high levels in response to experimental infection, remaining elevated for almost a month. Moreover, elevated haptoglobin was indicative of recent exposure to two respiratory pathogens assessed in the longitudinal study. We hope this work motivates studies investigating suites of non-specific markers of inflammation as indicators for pathogen exposure in a broader range of both pathogen and host species, potentially transforming how we track disease burden in natural populations.

Li H, Bradley K C, Long J S, Frise R, Ashcroft J W, Hartgroves L C, Shelton H, Makris S, Johansson C, Cao B, Barclay W S (2018)

Internal genes of a highly pathogenic H5N1 influenza virus determine high viral replication in myeloid cells and severe outcome of infection in mice

PLoS Pathog 14 (1), e1006821


The highly pathogenic avian influenza (HPAI) H5N1 influenza virus has been a public health concern for more than a decade because of its frequent zoonoses and the high case fatality rate associated with human infections. Severe disease following H5N1 influenza infection is often associated with dysregulated host innate immune response also known as cytokine storm but the virological and cellular basis of these responses has not been clearly described. We rescued a series of 6:2 reassortant viruses that combined a PR8 HA/NA pairing with the internal gene segments from human adapted H1N1, H3N2, or avian H5N1 viruses and found that mice infected with the virus with H5N1 internal genes suffered severe weight loss associated with increased lung cytokines but not high viral load. This phenotype did not map to the NS gene segment, and NS1 protein of H5N1 virus functioned as a type I IFN antagonist as efficient as NS1 of H1N1 or H3N2 viruses. Instead we discovered that the internal genes of H5N1 virus supported a much higher level of replication of viral RNAs in myeloid cells in vitro but not in epithelial cells and that this was associated with high induction of type I IFN in myeloid cells. We also found that in vivo during H5N1 recombinant virus infection cells of haematopoetic origin were infected and produced type I IFN and proinflammatory cytokines. Taken together our data infer that human and avian influenza viruses are differently controlled by host factors in alternative cell types; internal gene segments of avian H5N1 virus uniquely drove high viral replication in myeloid cells, which triggered an excessive cytokine production, resulting in severe immunopathology.

Klassmann A, Ferretti L (2018)

The third moments of the site frequency spectrum

Theoretical Population Biology 120, 16-28


The analysis of patterns of segregating (i.e. polymorphic) sites in aligned sequences is routine in population genetics. Quantities of interest include the total number of segregating sites and the number of sites with mutations of different frequencies, the so-called site frequency spectrum. For neutrally evolving sequences, some classical results are available, including the expected value and variance of the spectrum in the Kingman coalescent model without recombination as calculated by Fu (1995). In this work, we use similar techniques to compute the third moments of the frequencies of three linked sites. Based on these results, we derive analytical results for the bias of Tajima’s D and other neutrality tests. As a corollary, we obtain the second moments of the frequencies of two linked mutations conditional on the presence of a third mutation with a certain frequency. These moments can be used for the normalisation of new neutrality tests relying on these spectra.


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