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

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Lee H J, Lee K Y, Park Y H, Choi H J, Yao Y, Nair V, Han J Y (2017)

Acquisition of resistance to avian leukosis virus subgroup B through mutations on tvb cysteine-rich domains in DF-1 chicken fibroblasts

Veterinary Research 48 (1), 48


Avian leukosis virus (ALV) is a retrovirus that causes tumors in avian species, and its vertical and horizontal transmission in poultry flocks results in enormous economic losses. Despite the discovery of specific host receptors, there have been few reports on the modulation of viral susceptibility via genetic modification. We therefore engineered acquired resistance to ALV subgroup B using CRISPR/Cas9-mediated genome editing technology in DF-1 chicken fibroblasts. Using this method, we efficiently modified the tumor virus locus B (tvb) gene, encoding the TVB receptor, which is essential for ALV subgroup B entry into host cells. By expanding individual DF-1 clones, we established that artificially generated premature stop codons in the cysteine-rich domain (CRD) of TVB receptor confer resistance to ALV subgroup B. Furthermore, we found that a cysteine residue (C80) of CRD2 plays a crucial role in ALV subgroup B entry. These results suggest that CRISPR/Cas9-mediated genome editing can be used to efficiently modify avian cells and establish novel chicken cell lines with resistance to viral infection.

Lee H J, Lee K Y, Jung K M, Park K J, Lee K O, Suh J-Y, Yao Y, Nair V, Han J Y (2017)

Precise gene editing of chicken Na+/H+ exchange type 1 (chNHE1) confers resistance to avian leukosis virus subgroup J (ALV-J)

Developmental and Comparative Immunology 77 (Supp C), 340-349


Avian leukosis virus subgroup J (ALV-J), first isolated in the late 1980s, has caused economic losses to the poultry industry in many countries. As all chicken lines studied to date are susceptible to ALV infection, there is enormous interest in developing resistant chicken lines. The ALV-J receptor, chicken Na+/H+ exchange 1 (chNHE1) and the critical amino acid sequences involved in viral attachment and entry have already been characterized. However, there are no reported attempts to induce resistance to the virus by targeted genome modification of the receptor sequences. In an attempt to induce resistance to ALV-J infection, we used clustered regularly interspaced short palindromic repeats (CRISPR)-associated (CRISPR/Cas9)-based genome editing approaches to modify critical residues of the chNHE1 receptor in chicken cells. The susceptibility of the modified cell lines to ALV-J infection was examined using enhanced green fluorescent protein (EGFP)-expressing marker viruses. We showed that modifying the chNHE1 receptor by artificially generating a premature stop codon induced absolute resistance to viral infection, with mutations of the tryptophan residue at position 38 (Trp38) being very critical. Single-stranded oligodeoxynucleotide (ssODN)-mediated targeted recombination of the Trp38 region revealed that deletions involving the Trp38 residue were most effective in conferring resistance to ALV-J. Moreover, protein structure analysis of the chNHE1 receptor sequence suggested that its intrinsically disordered region undergoes local conformational changes through genetic alteration. Collectively, these results demonstrate that targeted mutations on chNHE1 alter the susceptibility to ALV-J and the technique is expected to contribute to develop disease-resistant chicken lines.

Howson E L A, Kurosaki Y, Yasuda J, Takahashi M, Goto H, Gray A R, Mioulet V, King D P, Fowler V L (2017)

Defining the relative performance of isothermal assays that can be used for rapid and sensitive detection of foot-and-mouth disease virus

Journal of Virological Methods 249, 102-110


This study describes the first multiway comparison of portable isothermal assays for the detection of foot-and-mouth disease virus (FMDV), benchmarked against real-time reverse transcription RT-PCR (rRT-PCR). The selected isothermal chemistries included reverse transcription loop-mediated isothermal amplification (RT-LAMP) and reverse transcription recombinase polymerase amplification (RT-RPA). The analytical sensitivity of RT-LAMP was comparable to rRT-PCR (101 RNA copies), while RT-RPA was one log10 less sensitive (102 RNA copies). Diagnostic performance was evaluated using a panel of 35 samples from FMDV-positive cattle and eight samples from cattle infected with other vesicular viruses. Assay concordance for RT-LAMP and RT-RPA was 86–98% and 67–77%, respectively, when compared to rRT-PCR, with discordant samples consistently having high rRT-PCR cycle threshold values (no false-positives were detected for any assay). In addition, a hierarchy of sample preparation methods, from robotic extraction to simple dilution of samples, for epithelial suspensions, serum and oesophageal-pharyngeal (OP) fluid were evaluated. Results obtained for RT-LAMP confirmed that FMDV RNA can be detected in the absence of RNA extraction. However, simple sample preparation methods were less encouraging for RT-RPA, with accurate results only obtained when using RNA extraction. Although the evaluation of assay performance is specific to the conditions tested in this study, the compatibility of RT-LAMP chemistry with multiple sample types, both in the presence and absence of nucleic acid extraction, provides advantages over alternative isothermal chemistries and alternative pen-side diagnostics such as antigen-detection lateral-flow devices. These characteristics of RT-LAMP enable the assay to be performed over a large diagnostic detection window, providing a realistic means to rapidly confirm positive FMD cases close to the point of sampling.

Harmsen M M, Seago J, Perez E, Charleston B, Eblé P L, Dekker A (2017)

Isolation of single-domain antibody fragments that preferentially detect intact (146s) particles of foot-and-mouth disease virus for use in vaccine quality control

Frontiers in Immunology 8, 960


Intact (146S) foot-and-mouth disease virions (FMDVs) can dissociate into specific (12S) viral capsid degradation products. FMD vaccines normally consist of inactivated virions. Vaccine quality is dependent on 146S virus particles rather than 12S particles. We earlier isolated two llama single-domain antibody fragments (VHHs) that specifically recognize 146S particles of FMDV strain O1 Manisa and shown their potential use in quality control of FMD vaccines during manufacturing. These 146S specific VHHs were specific for particular O serotype strains and did not bind strains from other FMDV serotypes. Here we describe the isolation of 146S specific VHHs against FMDV SAT2 and Asia 1 strains by phage display selection from llama immune libraries. VHHs that bind both 12S and 146S particles were readily isolated but VHHs that bind specifically to 146S particles could only be isolated by phage display selection using prior depletion for 12S particles. We obtained one 146S specific VHH – M332F – that binds to strain Asia 1 Shamir and several VHHs that preferentially bind 146S particles of SAT2 strain SAU/2/00, from which we selected VHH M379F for further characterization. Both M332F and M379F did not bind FMDV strains from other serotypes. In a sandwich ELISA employing unlabeled and biotinylated versions of the same VHH M332F showed high specificity for 146S particles but M379F showed lower 146S-specificity with some cross-reaction with 12S particles. These ELISAs could detect 146S particle concentrations as low as 2.3-4.6 µg/l. They can be used for FMD vaccine quality control and research and development, for example to identify virion stabilizing excipients.

Dixon K L, Sánchez-Cordón J P, Galindo I, Alonso C (2017)

Investigations of pro- and anti-apoptotic factors affecting African swine fever virus replication and pathogenesis

Viruses 9 (9), 241
Publisher’s version:


African swine fever virus (ASFV) is a large DNA virus that replicates predominantly in the cell cytoplasm and is the only member of the Asfarviridae family. The virus causes an acute haemorrhagic fever, African swine fever (ASF), in domestic pigs and wild boar resulting in the death of most infected animals. Apoptosis is induced at an early stage during virus entry or uncoating. However, ASFV encodes anti-apoptotic proteins which facilitate production of progeny virions. These anti-apoptotic proteins include A179L, a Bcl-2 family member; A224L, an inhibitor of apoptosis proteins (IAP) family member; EP153R a C-type lectin; and DP71L. The latter acts by inhibiting activation of the stress activated pro-apoptotic pathways pro-apoptotic pathways. The mechanisms by which these proteins act is summarised. ASF disease is characterised by massive apoptosis of uninfected lymphocytes which reduces the effectiveness of the immune response, contributing to virus pathogenesis. Mechanisms by which this apoptosis is induced are discussed.

Zou H, Su R, Ruan J, Shao H, Qian K, Ye J, Yao Y, Nair V, Qin A (2017)

Double-stranded RNA induces chicken T-cell lymphoma apoptosis by TRIF and NF-κB

Scientific Reports 7 (1), 7547


Toll-like receptor-3 (TLR3), a member of the pathogen recognition receptor family, has been reported to activate immune response and to exhibit pro-apoptotic activity against some tumor cells. However it is unclear whether TLR3 has same function against chicken lymphoma. In this paper we investigated the effect of TLR3 activation on a Marek’s disease lymphoma-derived chicken cell line, MDCC-MSB1. The TLR3 agonist poly (I:C) activated TLR3 pathway and inhibited tumor cells proliferation through caspase-dependent apoptosis. Using pharmacological approaches, we found that an interferon-independent mechanism involving Toll-IL-1-receptor domain-containing adapter-inducing IFN-α (TRIF) and nuclear factor κB (NF-κB) causes the apoptosis of MDCC-MSB1 cells. This is the first report about the function of TLR3 in chicken T-cell lymphoma, especially in signal pathway. The mechanisms underlying TLR3-mediated apoptosis may contribute to the development of new drug to treat lymphomas and oncovirus infections.

Hoa L N M, Tuan N A, My P H, Huong T T K, Chi N T Y, Hau Thu T T, Carrique-Mas J, Duong M T, Tho N D, Hoang N D, Thanh T L, Diep N T, Duong N V, Toan T K, Tung T S, Mai L Q, Iqbal M, Wertheim H, van Doorn H R, Bryant J E, The Vizions C (2017)

Assessing evidence for avian-to-human transmission of influenza A/H9N2 virus in rural farming communities in northern Vietnam

Journal of General Virology early view,


Rural farming communities in northern Vietnam do not routinely practice vaccination for influenza A viruses (IAV) for either humans or poultry, which enables us to study transmission intensity via seroepidemiology. Using samples from a longitudinal cohort of farming households, we determined the number of symptomatic and asymptomatic human infections for seasonal IAV and avian A/H9 over 2 years. As expected, we detected virologically confirmed acute cases of seasonal IAV in humans, as well as large numbers of subclinical seroconversions to A/H1pdm [55/265 (21 %)], A/H3 [95/265 (36 %)] and A/H9 [24/265 (9 %)]. Five of the A/H9 human seroconverters likely represented true infections rather than heterosubtypic immunity, because the individuals seroconverted solely to A/H9. Among co-located poultry, we found significantly higher seroprevalance for A/H5 compared to A/H9 in both chickens and ducks [for northern study sites overall, 337/1105 (30.5 %) seropositive for A/H5 and 123/1105 (11.1 %) seropositive for A/H9].

Bande F, Arshad S S, Omar A R, Hair-Bejo M, Mahmuda A, Nair V (2017)

Global distributions and strain diversity of avian infectious bronchitis virus: a review

Animal Health Research Reviews 18 (1), 70-83


The poultry industry faces challenge amidst global food security crisis. Infectious bronchitis is one of the most important viral infections that cause huge economic loss to the poultry industry worldwide. The causative agent, infectious bronchitis virus (IBV) is an RNA virus with great ability for mutation and recombination; thus, capable of generating new virus strains that are difficult to control. There are many IBV strains found worldwide, including the Massachusetts, 4/91, D274, and QX-like strains that can be grouped under the classic or variant serotypes. Currently, information on the epidemiology, strain diversity, and global distribution of IBV has not been comprehensively reported. This review is an update of current knowledge on the distribution, genetic relationship, and diversity of the IBV strains found worldwide.

Rehman Z U, Meng C, Umar S, Mahrose K M, Ding C, Munir M (2017)

Mast cells and innate immunity: master troupes of the avian immune system

World's Poultry Science Journal 73 (3), 621-632


Mast cells (MCs) are granulated cells of haematopoietic lineage and constitute a major sensory arm of the immune system. MCs dually guard hosts and regulate immune responses against invading pathogens. This property of the MCs is attributed to their adaptability to detect stress signals and pathogens, and the production of signal specific mediators to engage immune cells for clearance of infectious agents. Pathogen-specific signals establish basis for the initiation of adoptive immune responses. These immune regulatory roles of MCs have opened avenues to engage different MCs activators which culminate in effective passive immunisation. The molecular mechanisms and dynamics of functionalities of MCs in host defences have been extensively characterised in mammals and rodents, and research on MCs in avian species is emerging. This review surveys the development, morphology and distribution of MCs in different tissues of the poultry and highlight areas that can be exploited for disease control and prevention.


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