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

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Cozens D, Sutherland E, Lauder M, Taylor G, Berry C C, Davies R L (2019)

Pathogenic Mannheimia haemolytica invades differentiated bovine airway epithelial cells

Infection and Immunity 87 (6),


The Gram-negative bacterium Mannheimia haemolytica is the primary bacterial species associated with bovine respiratory disease (BRD) which is responsible for significant economic losses to the livestock industries worldwide. Healthy cattle are frequently colonised by commensal serotype A2 strains, but disease is usually caused by pathogenic strains of serotype A1. For reasons that are poorly understood, a transition occurs within the respiratory tract and a sudden explosive proliferation of serotype A1 bacteria leads to the onset of pneumonic disease. Very little is known about the interactions of M. haemolytica with airway epithelial cells of the respiratory mucosa which might explain the different abilities of serotype A1 and A2 strains to cause disease. In the present study, host-pathogen interactions in the bovine respiratory tract were mimicked using a novel differentiated bovine bronchial epithelial cell (BBEC) infection model. In this model, differentiated BBECs were inoculated with serotype A1 or A2 strains of M. haemolytica and the course of infection followed over a five-day period by microscopic assessment and measurement of key proinflammatory mediators. We have demonstrated that serotype A1, but not A2, M. haemolytica invades differentiated BBECs by transcytosis and subsequently undergoes rapid intracellular replication before spreading to adjacent cells and causing extensive cellular damage. Our findings suggest that the explosive proliferation of serotype A1 M. haemolytica that occurs within the bovine respiratory tract prior to the onset of pneumonic disease is potentially due to bacterial invasion of, and rapid proliferation within, the mucosal epithelium. The discovery of this previously unrecognised mechanism of pathogenesis is important because it will allow the serotype A1-specific virulence determinants responsible for invasion to be identified and thereby provide opportunities for the development of new strategies for combatting BRD aimed at preventing early colonisation and infection of the bovine respiratory tract.

Oade M S, Keep S, Freimanis G, Orton R, Britton P, Hammond J, Bickerton E (2019)

Attenuation of infectious bronchitis virus in eggs results in different patterns of genomic variation across multiple replicates

Journal of Virology 93 (14),


The gammacoronavirus infectious bronchitis virus (IBV) causes an acute, highly contagious respiratory disease of poultry. Live attenuated vaccines are traditionally generated by serial passage of a virulent strain in embryonated chicken eggs, however the molecular mechanism of attenuation is unknown. The virulent lab adapted strain of IBV, M41-CK, was egg-passaged over one hundred times in four parallel independent replicates. All four final egg-passaged viruses were attenuated in vivo and exhibited similar growth phenotypes in adult chicken kidney cells and ex vivo tracheal organ cultures. The virus populations were sequenced by 454 pyrosequencing at the end of passaging, showing that overall sequence diversity in the IBV population increased but the four replicates only had between 11 and 17 consensus-level single nucleotide polymorphisms (SNPs). Although hotspots of variation were identified in spike and nucleocapsid structural proteins as well as the 3' untranslated region, each attenuated virus possessed a different pattern of genomic variation. Overall, only a small number of consensus-level SNPs were acquired during egg passage, leaving a potentially short route back to virulence. These results highlight the unpredictable nature of attenuation by serial egg passage and the need to develop mechanisms to rationally attenuate IBV for the next generation of effective vaccines.ImportanceInfectious Bronchitis remains a major problem in the global poultry industry, despite the existence of many different vaccines. IBV vaccines are currently developed by serial passage of a virulent strain on embryonated hen's eggs until attenuation, however little is known about the evolution of the viral population during the process of attenuation. High throughput sequencing of four replicates of a serially egg-passaged IBV revealed a different pattern of genomic variation in each attenuated replicate and few consensus-level SNPs. This raises concerns that only a small number of genomic mutations are required to revert to a virulent phenotype, which may result in vaccine breakdown in the field. The observed hotspots of variation in the attenuated viruses has the potential to be used in the rational attenuation of virulent IBV for next generation vaccine design.

Rajko-Nenow P, Brown-Joseph T, Tennakoon C, Flannery J, Oura C A L, Batten C (2019)

Detection of a novel reassortant epizootic hemorrhagic disease virus serotype 6 in cattle in Trinidad, West Indies, containing nine RNA segments derived from exotic EHDV strains with an Australian origin

Infection, Genetics and Evolution 74, 103931


Epizootic hemorrhagic disease virus (EHDV) is a Culicoides-transmitted orbivirus that infects domestic and wild ruminants in many parts of the world. Of the eight proposed serotypes, only EHDV-1, 2 and 6 have been reported to be present in the Americas. Following the identification of a virulent EHDV-6 reasssortant virus in the USA in 2007 (EHDV-6 Indiana), with outer coat protein segments derived from an Australian strain of EHDV and all remaining segments derived from a locally circulating EHDV-2 strain, questions have remained about the origin of the Australian parent strain and how it may have arrived in the USA. When EHDV-6 was identified in asymptomatic cattle imported into the Caribbean island of Trinidad in 2013, full genome sequencing was carried out to further characterise the virus. The EHDV-6 Trinidad strain was a reassortant virus, with 8 of its 10 segments, being derived from the same exotic Australian EHDV-6 strain as the VP2 and VP5 present in the EHDV-6 Indiana strain from the USA. Analyses of the two remaining segments revealed that segment 8 showed the highest nucleotide identity (90.4%) with a USA New Jersey strain of EHDV-1, whereas segment 4 had the highest nucleotide identity (96.5%) with an Australian EHDV-2 strain. This data strongly suggests that the Trinidad EHDV-6 has an Australian origin, receiving its segment 4 from a reassortment event with an EHDV-2 also from Australia. This reassortant virus likely came to the Americas, where it received its segment 8 from a locally-circulating (as yet unknown) EHDV strain. This virus then may have gained entry into the USA, where it further reassorted with a known locally-circulating EHDV-2 strain, the resulting strain being EHDV-6 Indiana. This study therefore identifies, for the first time, the likely minor parent virus of the EHDV-6 currently circulating in the USA.

Iqbal M, Lukosaityte D, Munir M, Nair V (2019)

Meeting report: Global Alliance for Research on Avian Diseases 2018, International Conference, January 17 to 19, 2018, Hanoi, Vietnam

Avian Diseases 63 (1s), 251-257


Poultry production is one of the fastest growing sectors of the livestock industry, growing at a rate of around 5% per year (2015-16) to meet the global demands and food security, as shown by European Union Open Data Portal. One of the major challenges for the sustainable growth of this sector comes from the plethora of diseases, including viral diseases, which have devastating effects on productivity. With a significant growth in poultry production in Asia, South America, and Africa, most of the disease challenges are in these regions. Because of the global nature of these diseases, it is of vital importance to work collaboratively to generate effective mitigation opportunities via innovative strategies. In the spirit of this international collaboration, the second International Conference of the Global Alliance for Research on Avian Diseases (GARAD) was held from January 17 to January 19, 2018, in Hanoi, Vietnam. The conference, attended by over 150 delegates from academia, poultry breeding/farming, and the pharmaceutic industry, discussed the major challenges and research advances related to the control of poultry diseases. The topics reviewed included the continuous threat from avian influenza and its antigenic shifts/drifts, the risks of disease transmission within and from live bird markets, the challenges from antigenic diversity of other avian viruses, innovative approaches for poultry vaccine development, and the potential opportunities to introduce genetic resistance to infectious agents through novel gene editing techniques. In separate interactive sessions, delegates actively debated the challenges, priorities, and opportunities for academia in driving avian disease research, the importance of developing improved disease measures by industry, and the contribution by the farming sector in the low- and middle-income countries.

Sanders C J, Shortall C R, England M, Harrington R, Purse B, Burgin L, Carpenter S, Gubbins S (2019)

Long-term shifts in the seasonal abundance of adult Culicoides biting midges and their impact on potential arbovirus outbreaks

Journal of Applied Ecology 56 (7), 1649-1660


Surveillance of adult Culicoides biting midge flight activity is used as an applied ecological method to guide the management of arbovirus incursions on livestock production in Europe and Australia.
To date the impact of changes in the phenology of adult vector activity on arbovirus transmission has not been defined. We investigated this at two sites in the UK, identifying 150,000 Culicoides biting midges taken from 2867 collections over a nearly 40 year timescale.
Whilst we recorded no change in seasonal activity at one site, shifts in first adult appearance and last adult appearance increased the seasonal activity period of Culicoides species at the other site by 40 days over the time period.
Lengthening of the adult activity season was driven by an increase in abundance of Culicoides and correlated with local increases in temperature and precipitation. This diversity in responses poses significant challenges for predicting future transmission and overwintering risk.
Policy implications. Our analysis not only shows a dramatic and consistent increase in the adult active period of Culicoides biting midges, but also that this varies significantly between sites. This suggests broad‐scale analyses alone are insufficient to understand the potential impacts of changes in climate on arbovirus vector populations. Understanding the impact of climate change on adult Culicoides seasonality and transmission of arboviruses requires the context of changes in a range of other local ecological drivers.

Zhang Y, Luo J, Tang N, Teng M, Reddy V, Moffat K, Shen Z, Nair V, Yao Y (2019)

Targeted editing of the pp38 gene in Marek's disease virus-transformed cell lines using CRISPR/Cas9 system

Viruses 11 (5), 391
Publisher’s version:


Marek's disease virus (MDV), a lymphotropic alpha-herpesvirus associated with T-cell lymphomas in chickens, is an excellent model for herpesvirus biology and virus-induced oncogenesis. Marek's disease (MD) is also one of the cancers against which a vaccine was first used. In the lymphomas and lymphoblastoid cell lines (LCLs) derived from them, MDV establishes latent infection with limited gene expression. Although LCLs are valuable for interrogating viral and host gene functions, molecular determinants associated with the maintenance of MDV latency and lytic switch remain largely unknown, mainly due to the lack of tools for in situ manipulation of the genomes in these cell lines. Here we describe the first application of CRISPR/Cas9 editing approach for precise editing of the viral gene phosphoprotein 38 (pp38), a biomarker for latent/lytic switch in MDV-transformed LCLs MDCC-MSB-1 (Marek's disease cell line MSB-1) and MDCC-HP8. Contradictory to the previous reports suggesting that pp38 is involved in the maintenance of transformation of LCL MSB-1 cells, we show that pp38-deleted cells proliferated at a significant higher rate, suggesting that pp38 is dispensable for the transformed state of these cell lines. Application of CRISPR/Cas9-based gene editing of MDV-transformed cell lines in situ opens up further opportunities towards a better understanding of MDV pathogenesis and virus-host interactions.

Netherton C L, Connell S, Benfield C T O, Dixon L K (2019)

The genetics of life and death: virus-host interactions underpinning resistance to African swine fever, a viral haemorrhagic disease

Frontiers in Genetics 10, 402


Pathogen transmission from wildlife hosts to genetically distinct species is a major driver of disease emergence. African swine fever virus (ASFV) persists in sub-Saharan Africa through a sylvatic cycle between warthogs and soft ticks that infest their burrows. The virus does not cause disease in these animals, however transmission of the virus to domestic pigs or wild boar causes a hemorrhagic fever that is invariably fatal. ASFV transmits readily between domestic pigs and causes economic hardship in areas where it is endemic. The virus is also a significant transboundary pathogen that has become established in Eastern Europe, and has recently appeared in China increasing the risk of an introduction of the disease to other pig producing centers. Although a DNA genome mitigates against rapid adaptation of the virus to new hosts, extended epidemics of African swine fever (ASF) can lead to the emergence of viruses with reduced virulence. Attenuation in the field leads to large deletions of genetic material encoding genes involved in modulating host immune responses. Therefore resistance to disease and tolerance of ASFV replication can be dependent on both virus and host factors. Here we describe the different virus-host interfaces and discuss progress toward understanding the genetic determinants of disease outcome after infection with ASFV.

Mahapatra M, Upadhyaya S, Parida S (2019)

Identification of novel epitopes in serotype O foot-and-mouth disease virus by in vitro immune selection

Journal of General Virology 100 (5), 804-811


Foot-and-mouth disease virus (FMDV) displays various epitopes on the capsid outer surface. In addition to the five neutralizing antigenic sites, there is evidence of the existence of other, yet unidentified, epitopes that are believed to play a role in antibody-mediated protection. Previous attempts to identify these epitopes revealed two additional substitutions at positions VP2-74 and -191 (5M2/5 virus) to be of antigenic significance. However, complete resistance to neutralization was not obtained in the neutralization assay, indicating the existence of other, undisclosed epitopes. Results from this study provides evidence of at least two new neutralizing epitopes involving residues VP3-116 and -195 around the threefold axis that have significant impact on the antigenic nature of the virus. These findings extend our knowledge of the surface features of the FMDV capsid known to elicit neutralizing antibodies, and should help with rational vaccine design.

Gubbins S (2019)

Using the basic reproduction number to assess the risk of transmission of lumpy skin disease virus by biting insects

Transboundary and Emerging Diseases 66 (5), 1873-1883
Publisher’s version:


In recent years, lumpy skin disease virus (LSDV) has emerged as a major threat to cattle outside Africa, where it is endemic. Although evidence suggests that LSDV is transmitted by the bites of blood sucking arthropods, few studies have assessed the risk of transmission posed by particular vector species. Here this risk is assessed by calculating the basic reproduction number (R0) for transmission of LSDV by five species of biting insect: the stable fly, Stomoxys calcitrans, the biting midge, Culicoides nubeculosus, and three mosquito species, Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus. Parameters relating to mechanical transmission of LSDV were estimated using new analyses of previously-published data from transmission experiments, while vector life history parameters were derived from the published literature. Uncertainty and sensitivity analyses were used to compute R0 for each species and to identify those parameters which influence its magnitude. Results suggest that S. calcitrans is likely to be the most efficient at transmitting LSDV, with Ae. aegypti also an efficient vector. By contrast, C. nubeculosus, An. stephensi, and Cx. quinquefasciatus are likely to be inefficient vectors of LSDV. However, there is considerable uncertainty associated with the estimates of R0, reflecting uncertainty in most of the constituent parameters. Sensitivity analysis suggests that future experimental work should focus on estimating the probability of transmission from insect to bovine and on the virus inactivation rate in insects.

Dixon L K, Islam M, Nash R, Reis A L (2019)

African swine fever virus evasion of host defences

Virus Research 266, 25-33


African swine fever virus causes a haemorrhagic fever in domestic pigs and wild boar. The continuing spread in Africa, Europe and Asia threatens the global pig industry. The lack of a vaccine limits disease control. To underpin rational strategies for vaccine development improved knowledge is needed of how the virus interacts with and modulates the host’s responses to infection. The virus long double-stranded DNA genome codes for more than 160 proteins of which many are non-essential for replication in cells but can have important roles in evading the host’s defences. Here we review knowledge of the pathways targeted by ASFV and the mechanisms by which these are inhibited. The impact of deleting single or multiple ASFV genes on virus replication in cells and infection in pigs is summarised providing information on strategies for rational development of modified live vaccines.


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