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

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Santhakumar D, Iqbal M, Nair V, Munir M (2017)

Chicken IFN Kappa: a novel cytokine with antiviral activities

Scientific Reports 7 (1), 2719


Interferons (IFNs) are essential components of the host innate immune system and define first-line of defence against pathogens. In mammals, several type I IFNs are identified, however, only limited data is available on the repertoire of IFNs in avian species. Here we report the characterization of chicken IFN-kappa (chIFN-kappa) near the type I IFN locus on the sex-determining Z chromosome. Genetic, evolutionary and syntenic analyses indicate that chIFN-kappa is a type I IFN with conserved genetic features and promoter binding sites. chIFN-kappa regulated the IFN-stimulated response element signalling pathways and activated a panel of IFN-regulated genes, antiviral mediators and transcriptional regulators. Priming of chicken primary fibroblasts and tracheal organ cultures with chIFN-kappa imparted cellular protections against viral infections both in vitro and ex vivo. To determine whether chIFN-kappa defines the antiviral state in developing chicken embryos, we used replication-competent retroviral RCAS vector system to generate transgenic chicken embryos that constitutively and stably expressed chIFN-kappa. We could demonstrate that chIFN-kappa markedly inhibited the replication of avian RNA viruses in ovo. Collectively, these results shed the light on the repertoire of IFNs in avian species and provide functional data on the interaction of the chIFN-kappa with RNA viruses of poultry and public health importance.

Verreck F A W, Tchilian E Z, Vervenne R A W, Sombroek C C, Kondova I, Eissen O A, Sommandas V, van der Werff N M, Verschoor E, Braskamp G, Bakker J, Langermans J A M, Heidt P J, Ottenhoff T H M, van Kralingen K W, Thomas A W, Beverley P C L, Kocken C H M (2017)

Variable BCG efficacy in rhesus populations: Pulmonary BCG provides protection where standard intra-dermal vaccination fails

Tuberculosis 104, 46-57


M.bovis BCG vaccination against tuberculosis (TB) notoriously displays variable protective efficacy in different human populations. In non-human primate studies using rhesus macaques, despite efforts to standardise the model, we have also observed variable efficacy of BCG upon subsequent experimental M. tuberculosis challenge. In the present head-to-head study, we establish that the protective efficacy of standard parenteral BCG immunisation varies among different rhesus cohorts. This provides different dynamic ranges for evaluation of investigational vaccines, opportunities for identifying possible correlates of protective immunity and for determining why parenteral BCG immunisation sometimes fails. We also show that pulmonary mucosal BCG vaccination confers reduced local pathology and improves haematological and immunological parameters post-infection in animals that are not responsive to induction of protection by standard intra-dermal BCG. These results have important implications for pulmonary TB vaccination strategies in the future.

Le T T, Chang P, Benton D J, McCauley J W, Iqbal M, Cass A E G (2017)

Dual recognition element lateral flow assay toward multiplex strain specific influenza virus detection

Analytical Chemistry 89 (12), 6781-6786


Different influenza virus strains have caused a number of recent outbreaks killing scores of people and causing significant losses in animal farming. Simple, rapid, sensitive, and specific detection of particular strains, such as a pandemic strain versus a previous seasonal influenza, plays a crucial role in the monitoring, controlling, and management of outbreaks. In this paper we describe a dual recognition element lateral flow assay (DRELFA) which pairs a nucleic acid aptamer with an antibody for use as a point-of-care platform which can detect particular strains of interest. The combination is used to overcome the individual limitations of antibodies’ cross-reactivity and aptamers’ slow binding kinetics. In the detection of influenza viruses, we show that DRELFA can discriminate a particular virus strain against others of the same subtype or common respiratory diseases while still exhibiting fast binding kinetic of the antibody-based lateral flow assay (LFA). The improvement in specificity that DRELFA exhibits is an advantage over the currently available antibody-based LFA systems for influenza viruses, which offer discrimination between influenza virus types and subtypes. Using quantitative real-time PCR (qRT-PCR), it showed that the DRELFA is very effective in localizing the analyte to the test line (consistently over 90%) and this is crucial for the sensitivity of the device. In addition, color intensities of the test lines showed a good correlation between the DRELFA and the qRT-PCR over a 50-fold concentration range. Finally, lateral flow strips with a streptavidin capture test line and an anti-antibody control line are universally applicable to specific detection of a wide range of different analytes.

Franzoni G, Bonelli P, Graham S P, Anfossi A G, Giudici S D, Pilo G, Pittau M, Nicolussi P, Oggiano A (2017)

Comparative phenotypic and functional analyses of the effects of autologous plasma and recombinant human macrophage-colony stimulating factor (M-CSF) on porcine monocyte to macrophage differentiation

Veterinary Immunology and Immunopathology 187, 80-88


Porcine monocyte-derived macrophages (moM?) have been employed as a model cell in numerous studies of the porcine immune system. However, the lack of a standardized method for moM? differentiation hampers the comparison of results coming from the use of different laboratory protocols. In this study we compared the use of varying concentrations of autologous plasma (10, 20 and 30% v/v) or recombinant human macrophage-colony stimulating factor (hM-CSF; 50, 100, and 200 ng/ml) to differentiate porcine monocytes into macrophages. Changes in cell morphology and surface marker expression were assessed by confocal microscopy and flow cytometry. Macrophage differentiation was evaluated by analysing TNF-? response to LPS stimulation and determining cytokine secretion patterns under both basal conditions and after classical and alternative activation. The effects of the differentiation methods on metabolic activity and susceptibility to infection with the myelotropic African swine fever virus (ASFV) were also evaluated. Monocytes cultured using the different culture conditions tested augmented in dimension and cellular complexity, but increasing porcine plasma concentrations resulted in a dose dependent enhancement in granularity and a marked pleomorphism. As expected, CD163, MHC class II DR and CD203a expression were up-regulated in both hM-CSF (M-CSF-moM?) and autologous plasma cultured macrophages (AP-moM?), although a lower percentage of CD163+ cells were found following differentiation with high percentages of porcine plasma. We observed enhanced number of viable cells using high concentration of hM-CSF compared to porcine plasma, suggesting a proliferative effect. Irrespective of differentiation conditions, monocyte differentiation into macrophages resulted in an increased susceptibility to ASFV and yielded larger amounts of LPS-induced TNF-?. AP-moM? showed a higher basal release of IL-1RA compared to those cultured with hM-CSF and displayed a reduced ability to respond to classical activation, suggesting that the use of high percentages of porcine plasma led to the acquisition of a M2-like phenotype. We conclude that all the protocols tested in this study can be considered as suitable to produce porcine moM?, although the use of hM-CSF provides high responsiveness to M1 polarization. Since a higher phenotypic and functional inter-animal variability was observed in AP-moM?, we propose that the use of low concentration of hM-CSF should be adopted as the method of choice to provide a better reproducibility between experiments.

Baratelli M, Pedersen L E, Trebbien R, Larsen L E, Jungersen G, Blanco E, Nielsen J, Montoya M (2017)

Identification of cross-reacting T-cell epitopes in structural and non-structural proteins of swine and pandemic H1N1 influenza A virus strains in pigs

Journal of General Virology 98 (5), 895-899


Heterologous protection against swine influenza viruses (SwIVs) of different lineages is an important concern for the pig industry. Cross-protection between 'avian-like' H1N1 and 2009 pandemic H1N1 lineages has been observed previously, indicating the involvement of cross-reacting T-cells. Here, reverse vaccinology was applied to identify cross-reacting MHC class I T-cell epitopes from two different SwIV H1 lineages in pigs. In silico prediction followed by in vitro and in vivo testing was used to identify SLA-1*0702 T-cell epitopes in heterologous SwIV-infected pigs. Following viral infection, tetramer specific T-cell populations were identified. The majority of the identified T-cell epitopes were conserved between the examined lineages, suggesting that targeting cross-reactive T-cell epitopes could be used to improve vaccines against SwIV in SLA-1*0702-positive pigs.

Sanz Bernardo B, Goodbourn S, Baron M D (2017)

Control of the induction of type I interferon by peste des petits ruminants virus

PLOS ONE 12 (5), e0177300


Peste des petits ruminants virus (PPRV) is a morbillivirus that produces clinical disease in goats and sheep. We have studied the induction of interferon-β (IFN-β) following infection of cultured cells with wild-type and vaccine strains of PPRV, and the effects of such infection with PPRV on the induction of IFN-β through both MDA-5 and RIG-I mediated pathways. Using both reporter assays and direct measurement of IFN-β mRNA, we have found that PPRV infection induces IFN-β only weakly and transiently, and the virus can actively block the induction of IFN-β. We have also generated mutant PPRV that lack expression of either of the viral accessory proteins (V&C) to characterize the role of these proteins in IFN-β induction during virus infection. Both PPRV_ΔV and PPRV_ΔC were defective in growth in cell culture, although in different ways. While the PPRV V protein bound to MDA-5 and, to a lesser extent, RIG-I, and over-expression of the V protein inhibited both IFN-β induction pathways, PPRV lacking V protein expression can still block IFN-β induction. In contrast, PPRV C bound to neither MDA-5 nor RIG-I, but PPRV lacking C protein expression lost the ability to block both MDA-5 and RIG-I mediated activation of IFN-β. These results shed new light on the inhibition of the induction of IFN-β by PPRV.

Chakraborty P, Vervelde L, Dalziel R G, Wasson P S, Nair V, Dutia B M, Kaiser P (2017)

Marek's disease virus infection of phagocytes: a de novo in vitro infection model

Journal of General Virology 98 (5), 1080-1088


Marek's disease virus (MDV) is an alphaherpesvirus that induces T-cell lymphomas in chickens. Natural infections in vivo are caused by the inhalation of infected poultry house dust and it is presumed that MDV infection is initiated in the macrophages from where the infection is passed to B cells and activated T cells. Virus can be detected in B and T cells and macrophages in vivo, and both B and T cells can be infected in vitro. However, attempts to infect macrophages in vitro have not been successful. The aim of this study was to develop a model for infecting phagocytes [macrophages and dendritic cells (DCs)] with MDV in vitro and to characterize the infected cells. Chicken bone marrow cells were cultured with chicken CSF-1 or chicken IL-4 and chicken CSF-2 for 4 days to produce macrophages and DCs, respectively, and then co-cultured with FACS-sorted chicken embryo fibroblasts (CEFs) infected with recombinant MDV expressing EGFP. Infected phagocytes were identified and sorted by FACS using EGFP expression and phagocyte-specific mAbs. Detection of MDV-specific transcripts of ICP4 (immediate early), pp38 (early), gB (late) and Meq by RT-PCR provided evidence for MDV replication in the infected phagocytes. Time-lapse confocal microscopy was also used to demonstrate MDV spread in these cells. Subsequent co-culture of infected macrophages with CEFs suggests that productive virus infection may occur in these cell types. This is the first report of in vitro infection of phagocytic cells by MDV.

Zhang F, Perez-Martin E, Juleff N, Charleston B, Seago J (2017)

A replication-competent foot-and-mouth disease virus expressing a luciferase reporter

Journal of Virological Methods 247, 38-44


Bioluminescence is a powerful tool in the study of viral infection both in vivo and in vitro. Foot-and-mouth disease virus (FMDV) has a small RNA genome with a limited tolerance to foreign RNA entities. There has been no success in making a reporter FMDV expressing a luciferase in infected cell culture supernatants. We report here for the first time a replication-competent FMDV encoding Nanoluciferase, named as Nano-FMDV. Nano-FMDV is genetically stable during serial passages in cells and exhibits growth kinetics and plaque morphology similar to its parental virus. There are applications for the use of Nano-FMDV such as real-time monitoring of FMDV replication in vitro and in vivo.

Tchilian E, Holzer B (2017)

Harnessing local immunity for an effective universal swine influenza vaccine

Viruses 9 (5),
Publisher’s version:


Influenza A virus infections are a global health threat to humans and are endemic in pigs, contributing to decreased weight gain and suboptimal reproductive performance. Pigs are also a source of new viruses of mixed swine, avian, and human origin, potentially capable of initiating human pandemics. Current inactivated vaccines induce neutralising antibody against the immunising strain but rapid escape occurs through antigenic drift of the surface glycoproteins. However, it is known that prior infection provides a degree of cross-protective immunity mediated by cellular immune mechanisms directed at the more conserved internal viral proteins. Here we review new data that emphasises the importance of local immunity in cross-protection and the role of the recently defined tissue-resident memory T cells, as well as locally-produced, and sometimes cross-reactive, antibody. Optimal induction of local immunity may require aerosol delivery of live vaccines, but it remains unclear how long protective local immunity persists. Nevertheless, a universal vaccine might be extremely useful for disease prevention in the face of a pandemic. As a natural host for influenza A viruses, pigs are both a target for a universal vaccine and an excellent model for developing human influenza vaccines.

Simmonds P, Adams M J, Benko M, Breitbart M, Brister J R, Carstens E B, Davison A J, Delwart E, Gorbalenya A E, Harrach B, Hull R, King A M Q, Koonin E V, Krupovic M, Kuhn J H, Lefkowitz E J, Nibert M L, Orton R, Roossinck M J, Sabanadzovic S, Sullivan M B, Suttle C A, Tesh R B, van der Vlugt R A, Varsani A, Zerbini F M (2017)

Consensus statement: virus taxonomy in the age of metagenomics

Nature Reviews Microbiology 15 (3), 161-168


The number and diversity of viral sequences that are identified in metagenomic data far exceeds that of experimentally characterized virus isolates. In a recent workshop, a panel of experts discussed the proposal that, with appropriate quality control, viruses that are known only from metagenomic data can, and should be, incorporated into the official classification scheme of the International Committee on Taxonomy of Viruses (ICTV). Although a taxonomy that is based on metagenomic sequence data alone represents a substantial departure from the traditional reliance on phenotypic properties, the development of a robust framework for sequence-based virus taxonomy is indispensable for the comprehensive characterization of the global virome. In this Consensus Statement article, we consider the rationale for why metagenomic sequence data should, and how it can, be incorporated into the ICTV taxonomy, and present proposals that have been endorsed by the Executive Committee of the ICTV.


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