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

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Rodriguez L, Nogales A, Iqbal M, Perez D R, Martinez-Sobrido L (2018)

Identification of the amino acid residues responsible for inhibition of host gene expression by influenza A H9N2 NS1

Frontiers in Microbiology 9, 2546


H9N2 influenza A viruses (IAV) are considered low pathogenic avian influenza viruses (LPAIV). These viruses are endemic in poultry in many countries in Asia, the Middle East and parts of Africa. Several cases of H9N2-associated infections in humans as well as in pigs have led the World Health Organization (WHO) to include these viruses among those with pandemic potential. To date, the processes and mechanisms associated with H9N2 IAV adaptation to mammals are poorly understood. The nonstructural protein 1 (NS1) from IAV is a virulence factor that counteracts the innate immune responses. Here, we evaluated the ability of the NS1 protein from A/quail/Hong Kong/G1/97 (HK/97) H9N2 to inhibit host immune responses. We found that HK/97 NS1 protein counteracted interferon (IFN) responses but was not able to inhibit host gene expression in human or avian cells. In contrast, the NS1 protein from earlier H9N2 IAV strains, including the first H9N2 A/turkey/Wisconsin/1/1966 (WI/66), were able to inhibit both IFN and host gene expression. Using chimeric constructs between WI/66 and HK/97 NS1 proteins, we identified the region and amino acid residues involved in inhibition of host gene expression. Amino acid substitutions L103F, I106M, P114S, G125D and N139D in HK/97 NS1 resulted in binding to the 30-kDa subunit of the cleavage and polyadenylation specificity factor (CPSF30) and, in consequence, inhibition of host gene expression. Notably, changes in the same amino acid residues resulted in the lack of inhibition of host gene expression by WI/66 NS1. Importantly, our results identified a new combination of amino acids required for NS1 binding to CPSF30 and inhibition of host gene expression. These results also confirm previous studies demonstrating strain specific differences in the ability of NS1 proteins to inhibit host gene expression. 

Ferretti L, Weinreich D, Tajima F, Achaz G (2018)

Evolutionary constraints in fitness landscapes

Heredity ,


In the last years, several genotypic fitness landscapes—combinations of a small number of mutations—have been experimentally resolved. To learn about the general properties of “real” fitness landscapes, it is key to characterize these experimental landscapes via simple measures of their structure, related to evolutionary features. Some of the most relevant measures are based on the selectively acessible paths and their properties. In this paper, we present some measures of evolutionary constraints based on (i) the similarity between accessible paths and (ii) the abundance and characteristics of “chains” of obligatory mutations, that are paths going through genotypes with a single fitter neighbor. These measures have a clear evolutionary interpretation. Furthermore, we show that chains are only weakly correlated to classical measures of epistasis. In fact, some of these measures of constraint are non-monotonic in the amount of epistatic interactions, but have instead a maximum for intermediate values. Finally, we show how these measures shed light on evolutionary constraints and predictability in experimentally resolved landscapes.

Ferretti L, Ribeca P, Ramos-Onsins S (2018)

The site frequency/dosage spectrum of autopolyploid populations

Frontiers in Genetics 9,


The Site Frequency Spectrum (SFS) and the heterozygosity of allelic variants are among the most important summary statistics for population genetic analysis of diploid organisms. We discuss the generalisation of these statistics to populations of autopolyploid organisms in terms of the joint Site Frequency/Dosage Spectrum and its expected value for autopolyploid populations that follow the standard neutral model. Based on these results, we present estimators of nucleotide variability from High-Throughput Sequencing (HTS) data of autopolyploids and discuss potential issues related to sequencing errors and variant calling. We use these estimators to generalise Tajima's D and other SFS-based neutrality tests to HTS data from autopolyploid organisms. Finally, we discuss how these approaches fail when the number of individuals is small. In fact, in autopolyploids there are many possible deviations from the Hardy-Weinberg equilibrium, each reflected in a different shape of the individual dosage distribution. The SFS from small samples is often dominated by the shape of these deviations of the dosage distribution from its Hardy-Weinberg expectations.

Ferretti L, Klassmann A, Raineri E, Ramos-Onsins S E, Wiehe T, Achaz G (2018)

The neutral frequency spectrum of linked sites

Theoretical Population Biology 123, 70-79


We introduce the conditional Site Frequency Spectrum (SFS) for a genomic region linked to a focal mutation of known frequency. An exact expression for its expected value is provided for the neutral model without recombination. Its relation with the expected SFS for two sites, 2-SFS, is discussed. These spectra derive from the coalescent approach of Fu (1995) for finite samples, which is reviewed. Remarkably simple expressions are obtained for the linked SFS of a large population, which are also solutions of the multi-allelic Kolmogorov equations. These formulae are the immediate extensions of the well known single site θ/f neutral SFS. Besides the general interest in these spectra, they relate to relevant biological cases, such as structural variants and introgressions. As an application, a recipe to adapt Tajima's D andother SFS-based neutrality tests to a non-recombining region containing a neutral marker is presented.

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

Temporal differentiation of bovine airway epithelial cells grown at an air-liquid interface

Scientific Reports 8, 14893


There is an urgent need to develop improved, physiologically-relevant in vitro models of airway epithelia with which to better understand the pathological processes associated with infection, allergies and toxicological insults of the respiratory tract of both humans and domesticated animals. In the present study, we have characterised the proliferation and differentiation of primary bovine bronchial epithelial cells (BBECs) grown at an air-liquid interface (ALI) at three-day intervals over a period of 42 days from the introduction of the ALI. The differentiated BBEC model was highly representative of the ex vivo epithelium from which the epithelial cells were derived; a columnar, pseudostratified epithelium that was highly reflective of native airway epithelium was formed which comprised ciliated, goblet and basal cells. The hallmark defences of the respiratory tract, namely barrier function and mucociliary clearance, were present, thus demonstrating that the model is an excellent mimic of bovine respiratory epithelium. The epithelium was fully differentiated by day 21 post-ALI and, crucially, remained healthy and stable for a further 21 days. Thus, the differentiated BBEC model has a three-week window which will allow wide-ranging and long-term experiments to be performed in the fields of infection, toxicology or general airway physiology.

Chang P, Sealy J E, Sadeyen J R, Iqbal M (2018)

Amino acid residue 217 in the hemagglutinin 1 glycoprotein is a key mediator of avian influenza H7N9 virus antigenicity

Journal of Virology ,


Avian influenza viruses continue to evolve and acquire mutations that facilitate antigenic drift and virulence change. In 2017, low pathogenicity H7N9 avian influenza viruses evolved to a high pathogenicity phenotype in China. The comparative antigenic analysis between the low and high pathogenicity virus strains showed marked variability. In order to identify residues that may be linked to the antigenic change amongst the H7N9 viruses, we serially passaged the viruses in the presence of homologous ferret antisera. Progeny viruses able to overcome the neutralising capacity of the antisera were sequenced. The analysis showed that the emergent immune escaped viruses contained mutations A125T, A151T and L217Q in the hemagglutinin (HA) glycoprotein as early as passage 5 and these mutations persisted until passage 10. The results revealed that a single mutation L217Q in the HA of H7N9 virus led to 23- and 8-fold reductions in hemagglutination inhibition (HI) titre with ferret and chicken antisera, respectively. Further analysis showed that this change also contributed to antigenic differences between the low and high pathogenicity H7N9 viruses, therein, playing a major role in their antigenic diversification. Therefore, evolutionary changes at amino acid position 217 in the H7N9 viruses can serve as a genetic marker for virus antigenic diversity during vaccine seed matching and selection. The in vitro immune escape mutant selection method used in this study could also aid in the prediction of emerging antigenic variants in the naturally infected or immunised animals

IMPORTANCE Avian influenza H7N9 viruses circulating in poultry and wild birds continue to evolve and acquire important phenotypic changes. Mutations to the virus hemagglutinin (HA) glycoprotein can modulate virus antigenicity and facilitate virus escape from natural or vaccine-induced immunity. The focus of this study was to identify evolutionary markers in the HA of H7N9 that drive escape from antibody-based immunity. To achieve this, we propagated low pathogenicity H7N9 virus in the presence of polyclonal antisera derived from ferrets infected with the same strain of virus (homologous antisera). This selection process was repeated 10 times. The HA gene sequence of viruses recovered after the 5(th) passage showed that the viruses readily acquired mutation at three different amino acid positions (A125T, A151T and L217Q). Further functional analysis of these mutations confirmed that mutation at residue HA 217 was responsible for mediating changes to the immunological properties of the H7N9 virus.

Aksular M, Calvo-Pinilla E, Marin-Lopez A, Ortego J, Chambers A C, King L A, Castillo-Olivares J (2018)

A single dose of African horse sickness virus (AHSV) VP2 based vaccines provides complete clinical protection in a mouse model

Vaccine 36 (46), 7003-7010


African horse sickness is a severe, often fatal, arboviral disease of equids. The control of African horse sickness virus (AHSV) in endemic countries is based currently on the use of live attenuated vaccines despite some biosafety concerns derived from its biological properties. Thus, experimental vaccination platforms have been developed over the years in order to avoid the biosafety concerns associated with the use of attenuated vaccines.

Various studies showed that baculovirus-expressed AHSV-VP2 or modified Vaccinia Ankara virus expressing AHSV-VP2 (MVA-VP2) induced virus neutralising antibodies and protective immunity in small animals and horses.

AHSV is an antigenically diverse pathogen and immunity against AHS is serotype-specific. Therefore, AHS vaccines for use in endemic countries need to induce an immune response capable of protecting against all existing serotypes. For this reason, current live attenuated vaccines are administered as polyvalent preparations comprising combinations of AHSV attenuated strains of different serotypes.

Previous studies have shown that it is possible to induce cross-reactive virus neutralising antibodies against different serotypes of AHSV by using polyvalent vaccines comprising combinations of either different serotype-specific VP2 proteins, or MVA-VP2 viruses. However, these strategies could be difficult to implement if induction of protective immunity is highly dependent on using a two-dose vaccination regime for each serotype the vaccine intends to protect against.

In our study, we have tested the protective capacity of MVA-VP2 and baculovirus-expressed VP2 vaccines when a single dose was used. Groups of interferon alpha receptor knock-out mice were inoculated with either MVA-VP2 or baculovirus-expressed VP2 vaccines using one dose or the standard two-dose vaccination regime. After vaccination, all four vaccinated groups were challenged with AHSV and clinical responses, lethality and viraemia compared between the groups. Our results show that complete clinical protection was achieved after a single vaccination with either MVA-VP2 or baculovirus sub-unit VP2 vaccines.

Rohaim M A, Santhakumar D, Naggar R F E, Iqbal M, Hussein H A, Munir M (2018)

Chickens expressing IFIT5 ameliorate clinical outcome and pathology of highly pathogenic avian influenza and velogenic Newcastle disease viruses

Frontiers in Immunology 9, 2025


Innate antiviral immunity establishes first line of defense against invading pathogens through sensing their molecular structures such as viral RNA. This antiviral potential of innate immunity is mainly attributed to a myriad of IFN-stimulated genes (ISGs). Amongst well-characterized ISGs, we have previously shown that antiviral potential of chicken IFN-induced proteins with tetratricopeptides repeats 5 (chIFIT5) is determined by its interaction potential with 5?ppp containing viral RNA. Here, we generated transgenic chickens using avian sarcoma-leukosis virus (RCAS)-based gene transfer system that constitutively and stably express chIFIT5. The transgenic chickens infected with clinical dose (EID50 104 for HPAIV and 105 EID50 for vNDV) of high pathogenicity avian influenza virus (HPAIV; H5N1) or velogenic strain of Newcastle disease virus (vNDV; Genotype VII) showed marked resistance against infections. While transgenic chickens failed to sustain a lethal dose of these viruses (EID50 105 for HPAIV and 106 EID50 for vNDV), a delayed and lower level of clinical disease and mortality, reduced virus shedding and tissue damage was observed compared to non-transgenic control chickens. These observations suggest that stable expression of chIFIT5 alone is potentially insufficient in providing sterile protection against these highly virulent viruses; however, it is sufficient to ameliorate the clinical outcome of these RNA viruses. These findings propose the potential of innate immune genes in conferring genetic resistance in chickens against highly pathogenic and zoonotic viral pathogens causing sever disease in both animals and humans.

Qian K, Tian X, Shao H, Ye J, Yao Y, Nair V, Qin A (2018)

Identification of novel B-cell epitope in gp85 of subgroup J avian leukosis virus and its application in diagnosis of disease

BMC Veterinary Research 14 (1), 295


The gp85 is the main envelope protein of avian leukosis subgroup J (ALV-J) involved in virus neutralization. Here, we mapped the epitope in ALV-J gp85 by ELISA using synthetic peptides and developed epitope based diagnostic methods for ALV-J infection.

Hodgson S, Moffat K, Hill H, Flannery J T, Graham S P, Baron M D, Darpel K E (2018)

Comparison of the immunogenicity and cross-lineage efficacy of live attenuated peste des petits ruminants virus vaccines PPRV/Nigeria/75/1 and PPRV/Sungri/96

Journal of Virology early view,


Peste des petits ruminants (PPR) is a severe disease of goats and sheep that is widespread in Africa, the Middle East and Asia. Several effective vaccines exist for the disease, based on attenuated strains of the virus (PPRV) that causes PPR. While the efficacy of these vaccines has been established by use in the field, the nature of the protective immune response has not been determined. In addition, while the vaccine derived from PPRV/Nigeria/75/1 (N75) is used in many countries, those developed in India have never been tested for their efficacy outside that country. We have studied the immune response in goats to vaccination with either N75 or the main Indian vaccine, which is based on isolate PPRV/India/Sungri/96 (S96). In addition, we have compared the ability of these two vaccines, in parallel, to protect animals against challenge with pathogenic viruses from the four known genetic lineages of PPRV, representing viruses from different parts of Africa as well as Asia. These studies showed that, while N75 elicited a stronger antibody response than S96, as measured by both ELISA and virus neutralisation, S96 resulted in more pronounced cellular immune responses, as measured by virus antigen-induced proliferation and interferon gamma production. While both vaccines induced comparable numbers of PPRV-specific CD8+ T cells, S96 induced a higher number of CD4+ T cells specifically responding to virus. Despite these quantitative and qualitative differences in the immune responses following vaccination, both vaccines gave complete clinical protection against challenge with all four lineages of PPRV.


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