Publications

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

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Entrican G, Lunney J, Wattegedera S R, Mwangi W, Hope J, Hammond J A (2020)

The Veterinary Immunological Toolbox: Past, Present and Future

Frontiers in Immunology 11, 1651

Abstract

It is well-recognized that research capability in veterinary species is restricted by a lack of immunological reagents relative to the extensive toolboxes for small rodent biomedical model species and humans. This creates a barrier to the strategic development of disease control solutions for livestock, companion animals and wildlife that not only affects animal health but can affect human health by increasing the risk of transmission of zoonotic pathogens. There have been a number of projects aimed at reducing the capability gaps in the veterinary immunological toolbox, the majority of these focusing on livestock species. Various approaches have been taken to veterinary immunological reagent development across the globe and technological advances in molecular biology and protein biochemistry have accelerated toolbox development. While short-term funding initiatives can address specific gaps in capability, they do not account for long-term sustainability of reagents and databases that requires a different funding model. We review the past, present and future of the veterinary immunological toolbox with specific reference to recent developments discussed at the International Union of Immunological Societies (IUIS) Veterinary Immunology Committee (VIC) Immune Toolkit Workshop at the 12th International Veterinary Immunology Symposium (IVIS) in Seattle, USA, 16–19 August 2019. The future availability of these reagents is critical to research for improving animal health, responses to infectious pathogens and vaccine design as well as for important analyses of zoonotic pathogens and the animal /human interface for One Health initiatives.

Abstract

H7N9 avian influenza viruses (AIVs) continue to evolve and remain a huge threat to human health and the poultry industry. Previously, serially passaging the H7N9 A/Anhui/1/2013 virus in the presence of homologous ferret antiserum resulted in immune escape viruses containing amino acid substitutions alanine to threonine at residues 125 (A125T), 151 (A151T) and leucine to glutamine at residue 217 (L217Q) in the hemagglutinin (HA) protein. These HA mutations have also been found in the field isolates in 2019. To investigate the potential threat of the serum escape mutant viruses to humans and poultry, the impact of these HA substitutions, either individually or in combination, on receptor binding, pH of fusion, thermal stability and virus replication were investigated. Our results showed the serum escape mutant formed large plaques in Madin-Darby canine kidney (MDCK) cells and grew robustly in vitro and in ovo. They had a lower pH of fusion and increased thermal stability. Of note, the serum escape mutant completely lost the ability to bind to human-like receptor analogues. Further analysis revealed that N-linked glycosylation, as a result of A125T or A151T substitutions in HA, resulted in reduced receptor binding avidity toward both human and avian-like receptor analogues, and the A125T+A151T mutations completely abolished human-like receptor binding. The L217Q mutation enhanced the H7N9 acid and thermal stability while the A151T mutation dramatically decreased H7N9 HA thermal stability. To conclude, H7N9 AIVs that contain A125T+A151T+L217Q mutations in HA protein might pose a reduced pandemic risk but remain a heightened threat for poultry.

Abstract

Due to the current pandemic, a global shortage of reagents has drawn interest in developing alternatives to increase the number of coronavirus tests. One such alternative is sample pooling. We compared commercial kits that are used in COVID-19 diagnostics in terms of their sensitivity and feasibility for use in pooling. In this preliminary study, we showed that pooling of up to 80 samples did not affect the efficacy of the kits. Additionally, the RNA-dependent RNA polymerase (RdRp) gene is a more suitable target in pooled samples than the envelope (E) gene. This approach could provide an easy method of screening a large number of samples and help adjust different governmental regulations.

Abstract

The Gammacoronavirus infectious bronchitis virus (IBV) is a highly contagious economically important respiratory pathogen of domestic fowl. Reverse genetics allows for the molecular study of pathogenic determinants to enable rational vaccine design. The recombinant IBV (rIBV) Beau-R, a molecular clone of the apathogenic Beaudette strain, has previously been investigated as a vaccine platform. To determine tissues in which Beau-R could effectively deliver antigenic genes, an in vivo study in chickens, the natural host, was used to compare the pattern of viral dissemination of Beau-R to the pathogenic strain M41-CK. Replication of Beau-R was found to be restricted to soft tissue within the beak, whereas M41-CK was detected in beak tissue, trachea and eyelid up to seven days post infection. In vitro assays further identified that, unlike M41-CK, Beau-R could not replicate at 41 °C, the core body temperature of a chicken, but is able to replicate a 37 °C, a temperature relatable to the very upper respiratory tract. Using a panel of rIBVs with defined mutations in the structural and accessory genes, viral replication at permissive and non-permissive temperatures was investigated, identifying that the Beau-R replicase gene was a determinant of temperature sensitivity and that sub-genomic mRNA synthesis had been affected. The identification of temperature sensitive allelic lesions within the Beau-R replicase gene opens up the possibility of using this method of attenuation in other IBV strains for future vaccine development as well as a method to investigate the functions of the IBV replicase proteins.

Conceicao C, Thakur N, Human S, Kelly J T, Logan L, Bialy D, Bhat S, Stevenson-Leggett P, Zagrajek A K, Hollinghurst P, Varga M, Tsirigoti C, Hammond J A, Maier H J, Bickerton E, Shelton H, Dietrich I, Graham S C, Bailey D (2020)

The SARS-CoV-2 Spike protein has a broad tropism 1 for mammalian ACE2 proteins

bioRxiv, 156471

Abstract

SARS-CoV-2 emerged in late 2019, leading to the COVID-19 pandemic that continues to cause significant global mortality in human populations. Given its sequence similarity to SARS-CoV, as well as related coronaviruses circulating in bats, SARS-CoV-2 is thought to have originated in Chiroptera species in China. However, whether the virus spread directly to humans or through an intermediate host is currently unclear, as is the potential for this virus to infect companion animals, livestock and wildlife that could act as viral reservoirs. Using a combination of surrogate entry assays and live virus we demonstrate that, in addition to human ACE2, the Spike glycoprotein of SARS-CoV-2 has a broad host tropism for mammalian ACE2 receptors, despite divergence in the amino acids at the Spike receptor binding site on these proteins. Of the twenty-two different hosts we investigated, ACE2 proteins from dog, cat and rabbit were the most permissive to SARS-CoV-2, while bat and bird ACE2 proteins were the least efficiently used receptors. The absence of a significant tropism for any of the three genetically distinct bat ACE2 proteins we examined indicates that SARS-CoV-2 receptor usage likely shifted during zoonotic transmission from bats into people, possibly in an intermediate reservoir. Interestingly, while SARS-CoV-2 pseudoparticle entry was inefficient in cells bearing the ACE2 receptor from bats or birds the live virus was still able to enter these cells, albeit with markedly lower efficiency. The apparently broad tropism of SARS-CoV-2 at the point of viral entry confirms the potential risk of infection to a wide range of companion animals, livestock and wildlife.

Abstract

The chicken industry of Pakistan is a major livestock sub-sector, playing a pivotal role in economic growth and rural development. This study aimed to characterize and map the structure of broiler and layer production systems, associated value chains, and chicken disease management in Pakistan. Qualitative data were collected in 23 key informant interviews and one focus group discussion on the types of production systems, inputs, outputs, value addition, market dynamics, and disease management. Quantitative data on proportions of commodity flows were also obtained. Value chain maps were generated to illustrate stakeholder groups and their linkages, as well as flows of birds and products. Thematic analysis was conducted to explain the functionality of the processes, governance, and disease management. Major chicken production systems were: (1) Environmentally controlled production (97–98%) and (2) Open-sided house production (2–3%). Broiler management systems were classified as (I) Independent broiler production; (II) Partially integrated broiler production; and (III) Fully integrated broiler production, accounting for 65–75, 15–20, and 10–15% of commercial broiler meat supply, respectively. The management systems for layers were classified as (I) Partially integrated layer production and (II) Independent layer production, accounting for 10 and 80–85% in the egg production, respectively. The share of backyard birds for meat and eggs was 10–15%. Independent, and integrated systems for chicken production could be categorized in terms of value chain management, dominance of actors, type of finished product and target customers involved. Integrated systems predominantly targeted high-income customers and used formal infrastructure. Numerous informal chains were identified in independent and some partially integrated systems, with middlemen playing a key role in the distribution of finished birds and eggs. Structural deficiencies in terms of poor farm management, lack of regulations for ensuring good farming practices and price fixing of products were key themes identified. Both private and public stakeholders were found to have essential roles in passive disease surveillance, strategy development and provision of health consultancies. This study provides a foundation for policy-makers and stakeholders to investigate disease transmission, its impact and control and the structural deficiencies identified could inform interventions to improve performance of the poultry sector in Pakistan.

McNee A, Smith T R F, Holzer B, Clark B, Bessell E, Guibinga G, Brown H, Schultheis K, Fisher P, Ramos S, Nunez A, Bernard M, Graham S, Martini V, Chrun T, Xiao Y, Kash J C, Taubenberger J K, Elliott S, Patel A, Beverley P, Rijal P, Weiner D, Townsend A, Broderick K, Tchilian E (2020)

Establishment of a pig influenza challenge model for evaluation of monoclonal antibody delivery platforms

Journal of Immunology 205, 648-660

Abstract

mAbs are a possible adjunct to vaccination and drugs in treatment of influenza virus infection. However, questions remain whether small animal models accurately predict efficacy in humans. We have established the pig, a large natural host animal for influenza, with many physiological similarities to humans, as a robust model for testing mAbs. We show that a strongly neutralizing mAb (2-12C) against the hemagglutinin head administered prophylactically at 15 mg/kg reduced viral load and lung pathology after pandemic H1N1 influenza challenge. A lower dose of 1 mg/kg of 2-12C or a DNA plasmid-encoded version of 2-12C reduced pathology and viral load in the lungs but not viral shedding in nasal swabs. We propose that the pig influenza model will be useful for testing candidate mAbs and emerging delivery platforms prior to human trials.

Gizaw D, Tesfaye Y, Wood B A, Di Nardo A, Shegu D, Muluneh A, Bilata T, Belayneh R, Fentie A, Asgdome H, Sombo M, Rufael T, Tadesse F, Khan F, Yami M, Gelaye E, Wadsworth J, Knowles N J, King D P (2020)

Molecular characterisation of foot-and-mouth disease viruses circulating in Ethiopia between 2008 and 2019

Transboundary and Emerging Diseases
Publisher’s version: https://doi.org/10.1111/tbed.13675

Abstract

One of the constraints to controlling foot-and-mouth disease (FMD) in East Africa is the incomplete knowledge of the specific FMD virus (FMDV) strains circulating, and the way in which these viruses move across countries in the region. This retrospective study focuses on Ethiopia, which has one of the largest FMD-susceptible livestock populations in Africa. Analyses of FMDV positive samples collected between 2008 and 2019 demonstrate that serotypes O (n=175), A (n=51) and SAT 2 (n=33) were present in the country. Phylogenetic analysis of the VP1 sequences for these viruses showed that there were at least seven different FMD viral clades circulating during this period: O/EA-3, O/EA-4, A/AFRICA/G-I, A/AFRICA/G-IV, A/AFRICA/G-VII, SAT2/VII, and SAT2/XIII. Although these results only represent a snapshot and might not reflect all FMDV lineages that are present, they highlight the importance of serotype O, as well as the complexity and co-existence of FMDV serotypes in Ethiopia and surrounding countries. These sequence data also support the idea that there are two FMDV ecosystems existing in East Africa. Data from retrospective studies, such as these presented here, will be beneficial for vaccine selection and vaccination campaigns to control FMDV within Ethiopia.

Abstract

The positive stranded RNA genomes of picornaviruses comprise a single large open reading frame flanked by 5' and 3' untranslated regions (UTRs). Foot-and-mouth disease virus (FMDV) has an unusually large 5' UTR (1.3 kb) containing five structural domains. These include the internal ribosome entry site (IRES), which facilitates initiation of translation, and the cis-acting replication element (cre). Less well characterised structures are a 5' terminal 360 nucleotide stem-loop, a variable length poly-C-tract of approximately 100-200 nucleotides and a series of two to four tandemly repeated pseudoknots (PKs). We investigated the structures of the PKs by selective 2' hydroxyl acetylation analysed by primer extension (SHAPE) analysis and determined their contribution to genome replication by mutation and deletion experiments. SHAPE and mutation experiments confirmed the importance of the previously predicted PK structures for their function. Deletion experiments showed that although PKs are not essential for replication, they provide genomes with a competitive advantage. However, although replicons and full-length genomes lacking all PKs were replication competent, no infectious virus was rescued from genomes containing less than one PK copy. This is consistent with our earlier report describing the presence of putative packaging signals in the PK region.

van Doremalen N, Haddock E, Feldmann F, Meade-White K, Bushmaker T, Fischer R J, Okumura A, Hanley P W, Saturday G, Edwards N J, Clark M H A, Lambe T, Gilbert S C, Munster V J (2020)

A single dose of ChAdOx1 MERS provides protective immunity in rhesus macaques

Science Advances 6 (24), eaba8399

Abstract

Developing a vaccine to protect against the lethal effects of the many strains of coronavirus is critical given the current global pandemic. For Middle East respiratory syndrome coronavirus (MERS-CoV), we show that rhesus macaques seroconverted rapidly after a single intramuscular vaccination with ChAdOx1 MERS. The vaccine protected against respiratory injury and pneumonia and reduced viral load in lung tissue by several orders of magnitude. MERS-CoV replication in type I and II pneumocytes of ChAdOx1 MERS-vaccinated animals was absent. A prime-boost regimen of ChAdOx1 MERS boosted antibody titers, and viral replication was completely absent from the respiratory tract tissue of these rhesus macaques. We also found that antibodies elicited by ChAdOx1 MERS in rhesus macaques neutralized six different MERS-CoV strains. Transgenic human dipeptidyl peptidase 4 mice vaccinated with ChAdOx1 MERS were completely protected against disease and lethality for all different MERS-CoV strains. The data support further clinical development of ChAdOx1 MERS.

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