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

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Orabi A, Hussein A, Saleh A A, El-Magd M A, Munir M (2017)

Evolutionary insights into the fusion protein of Newcastle disease virus isolated from vaccinated chickens in 2016 in Egypt

Archives of Virology early online,


Newcastle disease virus (NDV) infections are one of the most devastating causes of economic losses in the poultry industry and despite extensive vaccination, outbreaks are being reported around the globe especially from developing and tropical countries. Analysis of NDV field strains from vaccinated flocks would highlight essential areas of consideration not only to design effective immunization strategies but also to devise vaccines that provide sterile immunity. For this purpose, 91 NDV suspected outbreaks were investigated and screened for NDV genetic material. A total of 16 NDV-positive isolates were examined using biological, genetics and bioinformatics analysis to assess the epidemiological association and to identify motifs that are under vaccine-induced immune pressures. In line with the clinical outcomes, all isolates showed the 112RRQKR|F117 cleavage motif and phylogenetic analysis revealed grouping of isolates into the genotype VII, and specifically sub-genotype VIId. Further analysis of the putative fusion protein sequence showed a number of substitutions (n=10) in functionally important domains and based on these differences, the studied isolates could be categorized into four distinct groups (A-D). Importantly, two residues (N30 and K71) were conserved in the commercial live vaccine and Egyptian field strains that are present in class II, genotype II. Collectively, these data enhance our knowledge of the evolution of genotype VIId NDV under the vaccine-induced immune pressures. In addition, our findings suggest that the use of genotype II-type vaccines in Egypt may be implicated in the emergence of new variants rather than providing benefits against NDV infections.

Kennedy D A, Cairns C, Jones M J, Bell A S, Salathé R M, Baigent S J, Nair V K, Dunn P A, Read A F (2017)

Industry-wide surveillance of Marek's disease virus on commercial poultry farms

Avian Diseases 61 (2), 153-164


Marek's disease virus is a herpesvirus of chickens that costs the worldwide poultry industry more than US$1 billion annually. Two generations of Marek's disease vaccines have shown reduced efficacy over the last half century due to evolution of the virus. Understanding where the virus is present may give insight into whether continued reductions in efficacy are likely. We conducted a 3-yr surveillance study to assess the prevalence of Marek's disease virus on commercial poultry farms, determine the effect of various factors on virus prevalence, and document virus dynamics in broiler chicken houses over short (weeks) and long (years) timescales. We extracted DNA from dust samples collected from commercial chicken and egg production facilities in Pennsylvania, USA. Quantitative PCR was used to assess wild-type virus detectability and concentration. Using data from 1018 dust samples with Bayesian generalized linear mixed effects models, we determined the factors that correlated with virus prevalence across farms. Maximum likelihood and autocorrelation function estimation on 3727 additional dust samples were used to document and characterize virus concentrations within houses over time. Overall, wild-type virus was detectable at least once on 36 of 104 farms at rates that varied substantially between farms. Virus was detected in one of three broiler-breeder operations (companies), four of five broiler operations, and three of five egg layer operations. Marek's disease virus detectability differed by production type, bird age, day of the year, operation (company), farm, house, flock, and sample. Operation (company) was the most important factor, accounting for between 12% and 63.4% of the variation in virus detectability. Within individual houses, virus concentration often dropped below detectable levels and reemerged later. These data characterize Marek's disease virus dynamics, which are potentially important to the evolution of the virus.

Tiede C, Bedford R, Heseltine S J, Smith G, Wijetunga I, Ross R, AlQallaf D, Roberts A P, Balls A, Curd A, Hughes R E, Martin H, Needham S R, Zanetti-Domingues L C, Sadigh Y, Peacock T P, Tang A A, Gibson N, Kyle H, Platt G W, Ingram N, Taylor T, Coletta L P, Manfield I, Knowles M, Bell S, Esteves F, Maqbool A, Prasad R K, Drinkhill M, Bon R S, Patel V, Goodchild S A, Martin-Fernandez M, Owens R J, Nettleship J E, Webb M E, Harrison M, Lippiat J D, Ponnambalam S, Peckham M, Smith A, Ferrigno P K, Johnson M, McPherson M J, Tomlinson D C (2017)

Affimer proteins are versatile and renewable affinity reagents

Elife 6, e24903


Molecular recognition reagents are key tools for understanding biological processes and are used universally by scientists to study protein expression, localisation and interactions. Antibodies remain the most widely used of such reagents and many show excellent performance, although some are poorly characterised or have stability or batch variability issues, supporting the use of alternative binding proteins as complementary reagents for many applications. Here we report on the use of Affimer proteins as research reagents. We selected 12 diverse molecular targets for Affimer selection to exemplify their use in common molecular and cellular applications including the (a) selection against various target molecules; (b) modulation of protein function in vitro and in vivo; (c) labelling of tumour antigens in mouse models; and (d) use in affinity fluorescence and super-resolution microscopy. This work shows that Affimer proteins, as is the case for other alternative binding scaffolds, represent complementary affinity reagents to antibodies for various molecular and cell biology applications.

Grant C F, Carr B V, Kotecha A, van den Born E, Stuart D I, Hammond J A, Charleston B (2017)

The B cell response to foot-and-mouth disease virus in cattle following sequential vaccination with multiple serotypes

Journal of Virology 91 (9),


Foot-and-mouth disease virus (FMDV) is a highly contagious viral disease. Antibodies are pivotal in providing protection against FMDV infection. Serological protection against one FMDV serotype does not confer interserotype protection. However, some historical data have shown that interserotype protection can be induced following sequential FMDV challenge with multiple FMDV serotypes. In this study, we have investigated the kinetics of the FMDV-specific antibody-secreting cell (ASC) response following homologous and heterologous inactivated FMDV vaccination regimes. We have demonstrated that the kinetics of the B cell response are similar for all four FMDV serotypes tested following a homologous FMDV vaccination regime. When a heterologous vaccination regime was used with the sequential inoculation of three different inactivated FMDV serotypes (O, A, and Asia1 serotypes) a B cell response to FMDV SAT1 and serotype C was induced. The studies also revealed that the local lymphoid tissue had detectable FMDV-specific ASCs in the absence of circulating FMDV-specific ASCs, indicating the presence of short-lived ASCs, a hallmark of a T-independent 2 (TI-2) antigenic response to inactivated FMDV capsid.IMPORTANCE We have demonstrated the development of intraserotype response following a sequential vaccination regime of four different FMDV serotypes. We have found indication of short-lived ASCs in the local lymphoid tissue, further evidence of a TI-2 response to FMDV.

Eldaghayes I, Dayhum A, Kammon A, Sharif M, Ferrari G, Bartels C, Sumption K, King D P, Grazioli S, Brocchi E (2017)

Exploiting serological data to understand the epidemiology of foot-and-mouth disease virus serotypes circulating in Libya

Open Veterinary Journal 7 (1), 01-Nov


Sporadic outbreaks of foot-and-mouth disease (FMD) have occurred in Libya for almost fifty years. During the spring of 2013, a countrywide serosurvey was undertaken to assess the level of FMD virus circulation and identify FMD virus serotypes in the country. A total of 4221 sera were collected, comprising samples from large ruminants (LR; n=1428 samples from 357 farms) and small ruminants (SR; n=2793 samples from 141 farms). FMD sero-prevalence of NSP antibodies determined by ELISA were 19.0% (271/1428) with 95% CI (16.9 - 21.0) and 13.5% (378/2793) with 95% CI (12.3 - 14.8) for LR and SR samples, respectively. The sero-prevalence of NSP antibodies in LR was 12.3% and 19.8% for age group < 1 year and >/= 1 year, respectively (X2= 4.95, P= 0.026), while in SR was 3.7%, 13.6% and 21.3% for age group < 1 year, 1-2 year and > 2 year, respectively (X2= 118.1, P= 0.000). These observed NSP serologic profiles support the hypothesis of an endemic level of FMD circulation in Libya. All positive sera were tested for SP antibodies for O, A and SAT-2 FMD virus serotypes. Serotype O was the dominant circulating serotype followed by serotype A, while evidence of SAT-2 was not found. These data provide an insight into the wider epidemiology of FMD in Libya, and contribute to field and laboratory investigations that during 2013 serotype O (O/ME-SA/Ind-2001 lineage) was isolated from clinical samples collected from the country.

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.


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