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Chinese Hamster cell line (Wg3h), chicken genome RH clones (Laboratoire de génétique cellulaire, Institut National de la Recherche Agronomique, Castanet-Tolosan, France)17, human embryonic kidney (293T) (ATCC), human lung adenocarcinoma epithelial cells (A549) (ATCC) and Madin-Darby canine kidney (MDCK) cells (ATCC) were maintained in cell culture media (Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen) supplemented with 10% fetal calf serum (FCS) (Biosera) and with 1% penicillin-streptomycin (Invitrogen)). Chicken fibroblast (DF-1) (ATCC) cells were maintained in DF-1 cell culture media (DMEM supplemented with 10% FCS, 5% tryptose phosphate broth (Sigma-Aldrich) and 0.1% penicillin-streptomycin (Invitrogen)). Cell lines were maintained at 37 °C in a 5% CO atmosphere. Cell lines were authenticated by RT–PCR and verified negative for mycoplasma. Total RNA was extracted from 1 × 106 cells that were of the same passage as those tested for polymerase, using an RNeasy kit (Qiagen) according to the manufacturer’s instructions. On-column DNA digestion was performed using RNase-free DNase (Qiagen) to remove contaminating genomic DNA. RNA samples were quantified using a Nanodrop Spectrophotometer (Thermo Scientific) and checked for quality using a 2100 Bioanalyzer (Agilent Technologies). All RNA samples had an RNA integrity number (RIN) ≥ 9.8. Array hybridization was performed according to the manufacturer’s instructions (Affymetrix). Labelled samples were hybridized to the Affymetrix Chicken Gene 1.0 ST Arrays in a GeneChip Hybridization Oven for 16 h at 45 °C and 60 rpm in an Affymetrix Hybridization Oven 645. After washing and staining, the arrays were scanned with the Affymetrix GeneChip Scanner 3000 7G. Gene-level expression signal estimates were derived from CEL files generated from raw data using the multi-array analysis (RMA) algorithm implemented from the Affymetrix GeneChip Command Console Software Version 3.0.1. Data Pre-Processing and filtering was done using Partek Software Version 6.6 and included: RMA background correction, quantile normalization across all chips in the experiment, log transformation and median polish summarization. Statistically significant genes were discovered by comparisons between the positive RH clones, parent cells and negative RH clones by two-way ANOVA (variables: clone and passage number) adjusted with the Benjamini–Hochberg multiple-testing correction (false discovery rate (FDR) of P < 0.05). Statistically significant genes were identified and those with fold-change values <|±1.5| were removed. Sequence specific primers were used to amplify targeted cellular transcripts of chicken genes from chromosome 10, including: ANP32A, RAB11A (Ras-related protein Rab-11A), TIPIN (TIMELESS Interacting Protein), RPL4 (Ribosomal Protein L4), PIAS1 (Protein Inhibitor Of Activated STAT, 1), TLE3 (Transducin-Like Enhancer Of Split 3), EIF3J (Eukaryotic Translation Initiation Factor 3, Subunit J) and CTDSPL2 (CTD (Carboxy-Terminal Domain, RNA Polymerase II, Polypeptide A) Small Phosphatase Like 2) from total RNA extracted from RH clone 476, using SuperScript III One-Step RT–PCR System (Invitrogen). Chicken ANP32B, DDX17 (DEAD (Asp-Glu-Ala-Asp) Box Helicase 17), IMPα1, 3 and 7 (Importin α-1,3 and 7) cDNAs were amplified from RNA extracted from DF-1 cells. PCR products were cloned into the pCAGGS expression vector. cDNAs of full length ANP32A isoforms of several species were generated by gene synthesis (GeneArt Strings DNA Fragments) and inserted into the pCAGGS expression vector, based on the following sequences: Chicken ANP32A (chANP32A) (Gallus gallus, XP_413932.3), human ANP32A (huANP32A) (Homo sapiens, NP_006296.1), zebra finch ANP32A (zfANP32A) (Taeniopygia guttata, XP_012424064.1), duck ANP32A (dkANP32A) (Anas platyrhynchos, XP_005023024.1), turkey ANP32A (tyANP32A) (Meleagris gallopavo, XP_010715918.1), ostrich ANP32A (osANP32A) (Struthio camelus australis, XP_009665579.1), pig ANP32A (pgANP32A) (Sus scrofa, XP_003121807.3), mouse ANP32A (muANP32A) (Mus musculus, NP_033802.2), chicken ANP32B (chANP32B) (Gallus gallus, NP_001026105.1) and human ANP32B (huANP32B) (Homo sapiens, NP_006392.1). The sequence of dkANP32A was amended to contain the sequence encoding the intact N terminus by comparison with duck RNA-seq data (from ENA_ERP005909) that had been de novo assembled (using CLC Genomic Workbench 7.5.1), where the appropriate contig was identified by BLASTX against the chicken ANP32A protein sequence. Furthermore, the duck sequence was confirmed by reverse-transcription of ANP32A mRNA derived from duck embryonic fibroblast cells and DNA sequencing. Mutants of these sequences were also generated by gene synthesis, as above, and included: chANP32A 33 (chANP32A with the ‘avian insertion’ deleted (aa176–208)), huANP32A+33 and huANP32B+33 (huANP32A and B with the avian sequence –VLSLVKDRDDKEAPDSDAEGYVEGLDDEEEDED– inserted after aa175 (huANP32A) and aa173 (huANP32B)). All plasmid constructs were verified by DNA sequencing. Primers and sequence information are available upon request. Reverse genetics systems for the following virus strains were used in this study: PR8 (H1N1 A/Puerto Rico/8/1934)30, UDL (H9N2 A/chicken/UDL-01/08)31 (developed in collaboration with M. Iqbal, Pirbright Institute, UK) and Ty05 (H5N1 A/Turkey/Turkey/5/2005) (a kind gift from R. Fouchier, Erasmus University, Netherlands). The PB2(E627K) substitution was made to 50-92 and Ty05 (K627E) by overlapping PCR of the PB2 plasmid as previously described11. Ty05:PR8 6:2 recombinant virus was generated with the HA and NA derived from PR8 and the internal genes of Ty05; virus rescue was performed by co-transfection of the 12-plasmid system: 8 polI plasmids as described above and 4 helper expression plasmids encoding A/Victoria/3/75 (VIC) polymerase components and NP expressed by the pCAGGS vector, as previously described32, 33. UDL virus rescue was performed by co-transfection of a 9 plasmid system including: 7 bidirectional pHW2000 plasmids34 encoding PB2, PA, HA, NA, NP, NS and M genes, together with a polI plasmid encoding PB1 and a pCAGGS expression plasmid of the UDL-PB1 gene. Ty05 virus stocks were propagated in 9-day-old embryonated chicken eggs incubated at 37 °C. UDL virus stocks were generated in MDCK cells with infection media (serum free DMEM supplemented with 1% penicillin-streptomycin and 1 μg ml−1 TPCK-treated trypsin (Lorne Labs)) and incubated at 37 °C. Clinical isolate A/England/691/2010 (H3N2) (Public Health England) was propagated in MDCK cells with infection media. Aliquots of infectious virus were stored at −80 °C. Infectious titres were determined by plaque assay on MDCK cells. 293T cells were transfected with ANP32A (500 ng, 24-well) using Lipofectamine 3000 (Invitrogen) and after 20 h infected with virus diluted in serum free DMEM for 1 h at 33 or 37 °C (MOI as indicated in the relevant figure legends) and replaced with cell culture medium (for qRT–PCR analysis) or DMEM supplemented with 0.1% FCS and 1 μg ml−1 TPCK trypsin (Worthington-Biochemical) (for infectious virus titres). shRNA A549 cells were infected with virus as for 293T cells, except infection media lacked FCS. Infected cell lysates and cell supernatants were harvested at 12 and/or 24 h post-infection. Infectious titres were determined by plaque assay on MDCK cells. All work with infectious agents was conducted in biosafety level 2 facilities, approved by the Health and Safety Executive of the UK and in accordance with local rules, at Imperial College London, UK. Influenza polymerase activity was measured by use of a minigenome reporter which contains the firefly luciferase gene flanked by the non-coding regions of the influenza NS gene segment, transcribed from a species-specific polI plasmid with a mouse terminator sequence35. The human and chicken polI minigenomes (pHOM1-Firefly and pCOM1-Firefly) are described previously36; pMouse-PolI-Firefly was generated by substituting in the mouse polI promoter sequence37. pCAGGS expression plasmids encoding each polymerase component and NP for 50–92 (H5N1 A/Turkey/England/50–92/91), Ty05, VIC and BAV (A/Duck/Bavaria/1/77) are described previously11, 36, 38. UDL PB1, PB2, PA and NP genes were sub-cloned into the pCAGGS plasmid. Mutagenesis of PB2 genes to encode PB2 627K or 627E was performed by overlapping PCR as described previously11, 36. All plasmid constructs were verified by DNA sequencing. Primers and sequence information are available upon request. To measure influenza polymerase activity, 293T cells were transfected in 48-well plates with pCAGGS plasmids encoding the PB1 (20 ng), PB2 (20 ng), PA (10 ng) and NP (40 ng) proteins, together with 20 ng species-specific minigenome reporter and 10 ng Renilla luciferase expression plasmid (pCAGGS-Renilla)39 as an internal control, using Lipofectamine 3000 transfection reagent (Invitrogen) according to manufacturers’ instructions. Wg3h, RH clones and DF-1 cells were transfected as 293T cells but with twice the concentration of DNA and using Lipofectamine 2000 (Invitrogen). Cells were incubated at 37 °C. 20 h after transfection, cells were lysed with 50 μl of passive lysis buffer (Promega), and firefly and Renilla luciferase bioluminescence was measured using a Dual-luciferase system (Promega) with a FLUOstar Omega plate reader (BMG Labtech). The effect of cellular factors on influenza polymerase was examined by polymerase assay after expression of constructs (250 ng) for 24 h. Silencing was achieved by lentivirus delivery of shRNA encoding transgenes. Lentiviral vectors were generated using the TRC1.5-pLKO.1-puro plasmid (MISSION Sigma-Aldrich) containing the shRNA sequence and puromycin selection gene. shRNA sequences for target genes were as follows: huANP32A (TRCN0000006905, 5′-CCGGCCTGAAGATGAGGGAGAAGATCTCGAGATCTTCTCCCTCATCTTCAGGTTTTT-3′ (target sequence, 5′-CCTGAAGATGAGGGAGAAGAT-3′)), huANP32B (TRCN0000077928, 5′-CCGGCCACCCAAAGAGCCAAAGAATCTCGAGATTCTTTGGCTCTTTGGGTGGTTTTTG-3′ (target sequence, 5′-CCACCCAAAGAGCCAAAGAAT-3′)), chANP32A (TRCN0000006902, 5′-CCGGCCTATTGTGATTTGACTGTTTCTCGAGAAACAGTCAAATCACAATAGGTTTTT-3′ (target sequence, 5′-CCTATTGTGATTTGACTGTTT-3′)) and Negative (SHC002, Non-Mammalian shRNA Control) (MISSION Sigma-Aldrich). Lentiviruses were generated by co-transfection in 293T cells with pCMV-delta8.240, pCAGG-VSV G41 and TRC1.5-pLKO.1-puro at a ratio of 1:0.25:1 using Lipofectamine 3000 (Invitrogen), cell culture media was replaced after 16hrs at a reduced volume and supernatant harvested at 36 h post-transfection before being filtered (0.45 μm) and aliquots frozen at −80 °C. 293T, A549 or DF-1 cells were transduced with lentiviral vectors for 16 h before media was replaced. After 72 h incubation, cells were split and cell culture media was replaced containing 0.5 μg ml−1 puromycin (Invivogen). Cells were incubated a further 72 h after selection before analysis. DF-1, 293T or RH clone 476 cells were transfected with 100 nM of siRNA using HiPerFect transfection reagent in 48-well plates, according to manufacturer’s instructions (Qiagen). 48 h later cells were transfected with polymerase and minigenome constructs and harvested after a further 20 h, for luciferase quantification and knockdown analysis. Total RNA was extracted as described previously but with 100 μl of cell lysate added to AVL buffer before continuing with the RNeasy mini kit (Qiagen). siRNAs for target genes were as follows:, AllStars Negative Control, huANP32A (SI02655212 FlexiTube), huANP32B (SI02655380 FlexiTube) (Qiagen), 50-92 NP (5′-AAGGAUCUUAUUUCUUCGGAG-3′), chANP32A (5′-GAGCTGGAATTCTTGAGTACA-3′) (custom RNA oligos, Sigma-Aldrich). Total RNA from RH clones and DF-1 cells were extracted using an RNeasy mini kit (Qiagen), following manufacturer’s instructions. During extraction of RNA, RNeasy columns were treated with RNase-Free DNase (Qiagen). RNA samples were quantified using a Nanodrop Spectrophotometer (Thermo Scientific). Equal concentrations of RNA were subject to first strand synthesis using QuantiTect Reverse Transcription Kit (Qiagen) with primers specific for chANP32A (5′-CAACTGTAGGTCATACGAAGGC-3′) and chANP32B (5′-GGTGGCCTTGAAGTTCTAGC-3′). This product was then quantified with Mesa Green quantitative PCR (qPCR) MasterMix Plus for SYBR Assay I dTTP (Eurogentec) using the primers for first strand synthesis together with chANP32A (5′-GTTTGCAACTGAGGCTAAGC-3′) and chANP32B (5′-ATGAGCATCGTCACCTCGC-3′). Real-time quantitative PCR analysis was performed (Applied Biosystems ViiA 7 Real-Time PCR System) and absolute copy numbers of either chANP32A or B calculated using a standard curve of known concentrations of the corresponding cDNA expression plasmid. Primers were designed to be specific to their target transcripts by using BLASTX against both the hamster and chicken genomes. Purified total RNA (1000 ng) was subject to first-strand cDNA synthesis with gene specific primers, oligo(dT)20 or random hexamers (to amplify mRNA) using SuperScript III (Invitrogen) followed by RNase H treatment (Invitrogen). Primer design was based on Obayashi et al. (2008)42 for quantification of RNA species of the luciferase minigenome driven by reconstituted polymerase, and UDL PB2 RNA species were quantified using a tagged-primer system adapted from Kawakami et al. (2011)43. First strand primers included: luciferase vRNA (5′- TATGAACATTTCGCAGCCTACCGTAGTGTT-3′), luciferase cRNA (5′-AGTAGAAACAAGGGTG-3′), luciferase mRNA (Oligo(dT)20), UDL PB2 vRNA (5′-GGCCGTCATGGTGGCGAAT GATGCGTGATGTATTGGGAAC-3′), UDL PB2 cRNA (5′-GCTAGCTTCAGCTAGGCATC AGTAGAAACAAGGTCGTT-3′), UDL PB2 mRNA (Oligo(dT)20) and 18S ribosomal RNA (Random Hexamers (Invitrogen)). After first strand synthesis, 1 μl of cDNA was subject to real-time quantitative PCR analysis with a gene specific primer pair using SYBR green PCR mix (Applied Biosystems) and analysed on the Applied Biosystems ViiA 7 Real-Time PCR System. Gene specific primer pairs were as follows: Luciferase gene (5′-CCGGAATGATTTGATTGCCA-3′ and 5′-TATGAACATTTCGCAGCCTACCGTAGTGTT-3′), UDL PB2 vRNA (5′-GGCCGTCATGGTGGCGAAT -3′ and 5′-CCTCTCAACACTGCAGATTCC-3′), UDL PB2 cRNA (5′-GCTAGCTTCAGCTAGGCATC -3′ and 5′-GGAATCTGCAGTGTTGAGAGG-3′), UDL PB2 mRNA (5′-GATGCGTGATGTATTGGGAAC-3′ and 5′-CCTCTCAACACTGCAGATTCC-3′) and 18S ribosomal RNA (5′-GCAAATTACCCACTCCCG-3′ and 5′-CTGCAGCAACTTTAATATACGC-3′). Fold change RNA to PB2 627E with Empty vector was calculated by ΔΔC including normalization to C values of 18S ribosomal RNA. Cells were lysed in Passive Lysis buffer (Promega) or NP40 lysis buffer (for cellular fractionation) and prepared in Laemmli 2× buffer (Sigma-Aldrich). Cell proteins were resolved by SDS–PAGE using Mini-PROTEAN TGX Precast Gels (Bio-Rad). Immunoblotting was carried out using the following primary antibodies: anti-chANP32A rabbit polyclonal (LS-B10851, LifeSpan BioSciences, Inc.), anti-huANP32A rabbit polyclonal (AB51013, Abcam), anti-huANP32B rabbit monoclonal (AB184565, Abcam), α-vinculin rabbit monoclonal (AB129002, Abcam), anti-Flag M2 mouse monoclonal (F1804 or F3165, Sigma-Aldrich), anti-PB2 rabbit polyclonal (2N580, a kind gift from Paul Digard, Roslin Institute), and followed with secondary horseradish peroxidase-conjugated (HRP) antibodies: anti-mouse IgG (H/L):HRP goat polyclonal (STAR117P, AbD Serotec) and anti-rabbit IgG:HRP sheep polyclonal (STAR54, AbD Serotec). For quantification of cellular fractions, the following secondary antibodies were used: anti-rabbit IgG (H/L):DyLight 800 (5151P, Cell signalling) and anti-mouse IgG (H/L):DyLight 680 (5470P, Cell signalling). Protein bands were visualized by chemiluminescence (ECL+ western blotting substrate, Pierce) using a FUSION-FX imaging system (Vilber Lourmat). 293T cells were transfected with empty vector or ANP32 plasmid together with the polymerase complex and NP of 50–92 (PB2 627E) and pHOM1-firefly minigenome reporter. After 24 h, cells monolayers were washed in ice-cold PBS and lysed in 0.1% NP40 buffer (50 mM Tris pH 7.5, 150 mM NaCl, 0.1% NP40 and protease inhibitors (EDTA-free COMPLETE tablet (Roche)). Total lysates were centrifuged at 228 g for 5 min at 4 °C. Supernatant was removed (Cytoplasmic fraction) and the nuclear pellet was resuspended in 1% NP40 (as above) and subject to syringing with a 25G needle. Fractions were analysed by immunoblotting. Analysis of Microarray data was performed as previously mentioned, using Affymetrix GeneChip Command Console Software Version 3.0.1. and Partek Software Version 6.6. Statistical analysis of biological replicates was performed by One-way ANOVA with Dunnett’s multiple comparison analysis or Two-way ANOVA with Šidák multiple comparison analysis, using GraphPad Prism 6. Sequence alignments were performed using Geneious R6 software. Quantification of immunoblots was performed using Image Studio Lite V5.2. No statistical methods were used to predetermine sample size. The experiments were not randomized, and the investigators were not blinded to allocation during experiments and outcome assessment.

News Article | November 11, 2015

Two months ago, the US Department of Defense froze operations at nine biodefence laboratories where work is done on dangerous pathogens. Inspectors had discovered live anthrax outside a containment area at the US Army's Dugway Proving Ground — a facility in Utah that tests defence systems against biological and chemical weapons. The discovery at Dugway is the latest of several concerning finds. In June 2014, workers at a US Centers for Disease Control and Prevention (CDC) biosafety-level-3 laboratory in Atlanta, Georgia, sent anthrax samples to three other laboratories on the same campus. The samples were meant to have been sterilized but several factors meant that 41 people were potentially exposed to live bacteria1. Then in May this year, an investigation revealed that for several years, staff at Dugway had been improperly sterilizing anthrax samples, and that live spores may have been sent to 52 laboratories in the United States, Canada, Australia and South Korea. These mishaps — which are by no means unique to anthrax — are worrying on two levels. First, the handling of dangerous pathogens within a controlled environment is one of the easier biological risks to contain. Much harder is ensuring that basic biological research that is known to be potentially dangerous, or that turns out to be so, is carried out safely. Second, it is only going to get harder to ensure the safe and secure use of organisms and their products — whether in basic research or in detecting and preventing the development of biological weapons. Relatively inexpensive and easy-to-use tools and approaches are greatly expanding the possibilities for genetic engineering, including for would-be terrorists. Among them are the gene-editing technique CRISPR/Cas9, and the use of gene drives — where the biased inheritance of particular genes alters entire populations. Meanwhile, myriad developments are undermining existing approaches to non-proliferation. These include: the sale of equipment and materials over the Internet; the accessibility of computing power; and the rise of the open-science movement. What are the prospects for managing the more intractable risks globally if measures to ensure the safe handling of dangerous pathogens are failing at the best-equipped facilities in the country with the most advanced biotechnology in the world? The anthrax incidents occurred despite the use of extensive legislation, protocols and procedures. The problem with the CDC, the US Department of Defense, and the many labs around the world who follow their lead, is not a lack of knowledge or training, or even a lack of engineering resources. It is the lack of a safety culture. Most laboratories handling potentially dangerous biological materials are stuck in compliance mode. To prevent human and environmental catastrophes, and the shutdown of important research, that mindset must be transformed. I never thought I'd write this, but I believe that it is time for experts who advise on biosafety and biosecurity to learn from specialists in nuclear security. I define biosafety and biosecurity as the prevention of the accidental release of potentially harmful organisms or their products and the prevention of the deliberate release of such agents for nefarious purposes. Leaders in these areas include the CDC, the World Health Organization (WHO), and Public Health England in the United Kingdom. The reluctance of those of us in biosecurity and biosafety to learn from the nuclear industry stems from the fact that many of the practices in nuclear security and safety are not transferable to biology. For instance, monitoring the amount of materials entering and leaving a complex makes little sense when a tiny sample can contain militarily significant amounts of a hazardous substance. And expensive security measures — guns, gates, guards and cameras — make sense at nuclear power plants, of which there are only a few hundred worldwide. They are not feasible at the much greater number of labs and hospitals dealing with hazardous biological agents. Moreover, hospital accident-and-emergency buildings and procedures are designed to get patients inside as quickly as possible, not keep them out. And progress within public health and research depends on transparency and open collaboration. What those working with biologicals can learn from practitioners in the nuclear industry — as well as from those in the US Navy, offshore oil drilling, airlines and utilities — is a culture of safety. In all these areas, best practice focuses on preventing failure rather than on maximizing output. The result is what is called a 'high-reliability organization' (HRO). HROs feature the following five characteristics2. First, everyone within the organization constantly asks, 'What can go wrong and how do we prevent it?' Second, workers are sensitive to any deviation from the norm, such as an unexpected change in the temperature of the reactor core in the case of a nuclear power plant, and learn to ascertain which variances can snowball into catastrophic failure. Third, systems are designed to be resilient so that if they do fail, they do so with minimal damage and recovery can be quick. Fourth, workers recognize that the operating environment is complex and changeable, and that mindlessly following standard procedures without paying attention to what else is going on in the environment can be dangerous. Lastly, expertise is valued over seniority, with the recognition that it may be the newest or most junior member of a team who spots a problem or who knows best how to fix it. In HROs, safety is not 'for them' but 'for each and every one of us', and is seen as an investment rather than a short-term cost. Workers are encouraged to hold each other accountable and to report red flags, such as a change in behaviour that might make a colleague more prone to mistakes. Mishaps and near misses too are reported without fear of blame, and mistakes are analysed to learn how to prevent them from recurring. Finally, the process is one of continual improvement: attention to safety does not stop just because certain targets have been met. In biosecurity and biosafety, the CDC is widely seen as the global gold standard. The CDC's handbook Biosafety in Microbiological and Biomedical Laboratories has become the reference for laboratories worldwide. Other resources that it provides (posters, training videos, data and information) along with documents from the WHO, are used as core reference materials, even in the most remote labs. Yet the world's exemplars in the handling of the most dangerous pathogens, and therefore the multitude of public and private organizations who follow them, are stuck in a very different culture from that of HROs. From the CDC to diagnostics and basic-research laboratories worldwide, the emphasis is on ticking boxes and on following rules set by outside authorities, such as the Department of Health and Human Services in the United States or the relevant agencies in the European Union. Safety is generally seen as an inconvenience that detracts from the main task at hand. It is delegated to biosafety officers, and after-the-fact indicators of problems such as the number of accidents, are the predominant metric, with the implicit aim being to ensure that spills, infections and so on are kept below targets with minimal effort. A recent illustration of problems caused by the rote following of rules is the handling of an Ebola patient by staff at Texas Health Presbyterian Hospital in Dallas in 2014, where two nurses contracted the disease. Having never dealt with a suspected Ebola case before, staff checked the CDC website for information on the correct personal protective equipment (PPE) to wear. Unfortunately, that website described PPE more suitable to handling Ebola samples in a laboratory. The PPE the hospital workers initially used left areas of their face and necks exposed. Failure to consider context and all the links in the chain can similarly undermine the value of spending millions of dollars on building and operating containment labs throughout the world. A recent inspection at a major diagnostic lab for animal diseases in Afghanistan, for instance, revealed that standard operating procedures (SOPs) copied from Western labs, for 'safe' operation, were being followed to the letter, including one for the handling of biological waste. The waste was getting bagged up pending incineration. But because there was no budget for petrol for the incinerator, the bags were simply being stored, undermining many of the prior biosafety procedures. Organizations that have successfully implemented a culture of safety have often treated the introduction of a new way of doing things as a business project, akin to a move to a new software platform. Experts in the offshore oil industry have likened the process to moving from directing one play to another3. One must deal with a new script (the vision), a new stage set and scenery (the facilities, equipment and technology used in operations), new stage directions (processes and procedures), new roles (job descriptions), new contracts (hires), and new rehearsals (training and commissioning). For example, the metals manufacturer Alcoa, based in New York, launched a safety drive starting in the late 1980s using such techniques and saw the average rate of lost workdays (due to work-related injuries) drop over a ten-year period from 1.86 to just 0.18 per 100 work years4. As well as this willingness to start afresh, three other steps are crucial. Provide leadership, funds, time and commitment. The process starts with senior management laying out what safety means for their particular organization. All layers of the organization are then involved in identifying what facilities, equipment and practices need to be changed. Lastly, a master plan is drawn up to realize the vision. In some cases, considerable sums will be needed initially. Yet such investments can quickly pay off. Alcoa, for instance, jump-started its safety programme by spending US$3 million over two months to fix unlit passageways in its plants. But based on the US Department of Labor's Accident Cost Calculator, the reduction in time lost due to accidents saved Alcoa around $51.5 million annually. Make safety matter to everyone. People will care about safety at their organization if their immediate bosses and those at the top frequently talk about it and back their talk with actions. If other achievements, such as efficiency or the output of journal papers, are rewarded ahead of safety — as is the case in most basic-research labs — people will pay less heed to it. Those who ignore new safety rules must face significant sanctions. People who refuse to adapt should lose their positions. Various tools can aid managers on this front. For instance, workers can be required to obtain certification before being allowed to perform potentially hazardous tasks. Exploit peer accountability. Most managers of staff who have employment protection, such as tenured professors or civil servants, cannot hire and fire, or give or withhold bonuses. Fortunately, cash does not seem to be a key motivator when it comes to safety. A 2010 study of 1,600 safety professionals across different industries found that people's expectations of their peers seems to be the most important influence on workplace behaviour — ahead even of management's expectations5. And recognition of a job well done can be more motivating than a bonus. For instance, the Gallup Organization, based in Washington DC, has surveyed more than 4 million workers worldwide, and found that employees who are recognized for their achievements have better safety records and fewer accidents on the job. In 2011, the Joint Commission, a non-profit organization that controls hospital certification in the United States, started promoting HRO approaches in hospitals throughout the country6 (see 'Follow the leader'). This followed several serious medical errors, such as surgeons operating on the wrong side of the brain in three patients in one year at the Rhode Island Hospital in Providence. In the case of the hospitals, the actual procedural changes — anything from more-stringent processes for infection control to improved systems for record checking — vary from place to place, but the aim is always to minimize the chances of something going wrong. The CDC is the obvious candidate to pick up the torch and prove that the HRO approach also works in laboratory settings. It has the resources. And where the CDC leads, others follow; if they do not, they risk not being able to acquire funding, collaborate with those in other laboratories or obtain contracts from corporations who demand compliance with best practice. Changing the culture of such a large entity will be difficult. But proof of concept could be achieved first in one unit, such as the Bioterrorism Rapid Response and Advanced Technology Laboratory (BRRAT). Because BRRAT is on the CDC's main campus in Atlanta, top managers from across the organization could more easily be engaged in the process of organizational change. Approaches used at BRRAT could then be rolled out to the entire organization. The HRO approach will be especially valuable for those facing uncertainty. In experimenting with gene drives and CRISPR/Cas9, there are no SOPs to follow. Asking 'What could go wrong?' or 'How could this science be misused?' and 'How can we prevent that from happening?' will embed biosafety and biosecurity considerations into study programmes from the outset. Although research will always have an element of the unknown, under the HRO model, workers are encouraged to constantly monitor outcomes against expectations and to make adjustments on the basis of new evidence. In other words, an HRO approach means reappraising biosafety and biosecurity plans as understanding increases. The biological-research community is capable of taking this road: people working on gene drives, for instance, are actively debating potential safety and security issues7. But HRO principles need to be adopted much more widely. Failure to so could greatly harm society, agriculture and the environment. Take, for instance, the 2007 release of foot-and-mouth disease virus from the Pirbright Institute, an animal-health research centre in Woking, UK. Inadequate sterilization of biological waste, broken waste pipes and unsealed and overflowing manhole covers led to more than 2,000 sheep and cows being slaughtered, at a cost of $200 million. Moreover, failure to be seen to be conducting biology in a safe, responsible and ethical manner undermines the public's support for promising technologies and approaches. That government and public anxieties can quickly block research has been demonstrated repeatedly. Take the nearly eight years of restrictions on human embryonic stem-cell research in the United States, instituted by President George W. Bush in 2001. Or the year-long voluntary moratorium called in 2012 on 'gain-of-function' experiments involving the highly pathogenic avian H5N1 influenza virus8. Here, researchers used genetic engineering to enhance the transmissibility of such flu viruses in mammals in the course of investigating changes that might increase their transmissibility between humans. The research is aimed at predicting which strains we shall need vaccines against in the near future. Biology must move forward on safety and security. Let's not reinvent the wheel, but learn from those doing safety better.

News Article | March 4, 2016

Only a fool ignores well informed advice. And only a very foolish government demands not to receive it in the first place. But that is what the British government is in danger of doing. Last month the Cabinet Office — the ministry that supports the Prime Minister in running the government — introduced a new condition attached to government grants. A new rule warns that money from any grant, either issued direct from departments or through third parties, cannot be used to “support activity intended to influence or attempt to influence Parliament, government or political parties … or attempting to influence legislative or regulatory action”. Despite increasing concern from academics, the Department for Business, Innovation and Skills, responsible for billions of pounds of research funding, could not say as Nature went to press whether the rule would apply to science grants and university funding. The research councils and the Higher Education Funding Council for England — which actually parcel up the money for institutions and academics — are equally in the dark. All of which leaves scientists fearing that they are about to be muzzled. This situation is not as shocking as in Canada, where a previous government deliberately set out to gag its researchers. Instead, the UK case seems to be more cock-up than conspiracy. No official seems to have thought through what it might mean to stop anyone who receives government money saying anything of substance to government. The Cabinet Office says that the clause was introduced to stop bodies that rely on government funds from lobbying government for more funding. What could be lost if this clause is implemented fully is unclear. The specifics of how it will work have not been set out in great detail. But it could cover some of what government-funded scientists already do. A group of cross-party politicians in the House of Lords, for example, is conducting an inquiry into what impact the result of the United Kingdom’s pending referendum, on whether to stay in or quit the European Union, would have on science. Among those giving evidence to the Lords are seven research institutions that are either government-owned or receive substantial government grants, including the Met Office weather agency, animal-health centre the Pirbright Institute in Surrey and the plant experts at the John Innes Centre in Norwich. Then there are nine universities or university centres, plus individual professors. All the evidence culled from this wealth of expertise could be jeopardized by a heavy-handed implementation of this clause — for what is the point of evidence that has no influence? Even if these groups did still give evidence, some of it would have to be watered down or heavily qualified. Academic input has enlightened discussions of climate change, pollinator declines, biomedical ethics and many other issues of crucial importance to the future of the United Kingdom and the wider world. The clause does not even limit itself to activity that tries to influence the United Kingdom; it merely forbids “attempting to influence legislative or regulatory action”. Will some of the world’s leading climate scientists be prevented from contributing to the Intergovernmental Panel on Climate Change’s Summary for Policymakers because they are dependent on government money? Should British wildlife experts not give policy advice to foreign nations attempting to save biodiversity? Surely not, but this is one possible reading of the clause. Scientists in the United Kingdom could be forgiven for feeling baffled by the development, given that the government-funded research councils have spent recent years promoting the ‘impact agenda’. This encourages scientists to make sure that their work has reach outside their own academic disciplines, including influencing policy and legislation. Officials have indicated that the problem can be fixed. Ministers have the power to remove the rule entirely from grants, or add in ‘qualifications’ that could permit some limited additional uses for the money. All researchers supported by government — regardless of what organizational auspice they operate under — should be in no doubt that they have not only a right but a duty to speak out about the implications of their work. There must be a complete exemption of any research from this clause, not just for those who work in academia, but for those who work directly for government.

Knight-Jones T.J.D.,Pirbright Institute | Knight-Jones T.J.D.,Royal Veterinary College VEEPH | Rushton J.,Royal Veterinary College VEEPH
Preventive Veterinary Medicine | Year: 2013

Although a disease of low mortality, the global impact of foot and mouth disease (FMD) is colossal due to the huge numbers of animals affected. This impact can be separated into two components: (1) direct losses due to reduced production and changes in herd structure; and (2) indirect losses caused by costs of FMD control, poor access to markets and limited use of improved production technologies. This paper estimates that annual impact of FMD in terms of visible production losses and vaccination in endemic regions alone amount to between US$6.5 and 21 billion. In addition, outbreaks in FMD free countries and zones cause losses of >US$1.5 billion a year.FMD impacts are not the same throughout the world:. 1.FMD production losses have a big impact on the world's poorest where more people are directly dependent on livestock. FMD reduces herd fertility leading to less efficient herd structures and discourages the use of FMD susceptible, high productivity breeds. Overall the direct losses limit livestock productivity affecting food security.2.In countries with ongoing control programmes, FMD control and management creates large costs. These control programmes are often difficult to discontinue due to risks of new FMD incursion.3.The presence, or even threat, of FMD prevents access to lucrative international markets.4.In FMD free countries outbreaks occur periodically and the costs involved in regaining free status have been enormous.FMD is highly contagious and the actions of one farmer affect the risk of FMD occurring on other holdings; thus sizeable externalities are generated. Control therefore requires coordination within and between countries. These externalities imply that FMD control produces a significant amount of public goods, justifying the need for national and international public investment.Equipping poor countries with the tools needed to control FMD will involve the long term development of state veterinary services that in turn will deliver wider benefits to a nation including the control of other livestock diseases. © 2013 Elsevier B.V.

Ascough S.,Pirbright Institute | Altmann D.M.,Imperial College London
Expert Review of Anti-Infective Therapy | Year: 2015

The emergence of a previously unrecognized route of Bacillus anthracis infection over the last few years has led to concern: sporadic anthrax outbreaks among heroin users in northern Europe have demonstrated the severe pathology associated with the newly described 'injectional anthrax'. With a high case fatality rate and non-specific early symptoms, this is a novel clinical manifestation of an old disease. Lack of awareness of this syndrome among emergency room clinicians can lead to a delayed diagnosis among heroin users; indeed, for many health workers in developed countries, where infection by B. anthracis is rare, this may be the first time they have encountered anthrax infections. As the putative route of contamination of the heroin supply is potentially ongoing, it is important that clinicians and public health workers remain vigilant for early signs of injectional anthrax. © 2015 Informa UK, Ltd.

Reid E.,Pirbright Institute | Charleston B.,Pirbright Institute
Journal of Interferon and Cytokine Research | Year: 2014

The biology of RNA viruses is closely linked to the type I and type III interferon (IFN) response of the host. These viruses display a range of molecular patterns that may be detected by host cells resulting in the induction of IFNs. Consequently, there are many examples of mechanisms employed by RNA viruses to block or delay IFN induction and reduce the expression of IFN-stimulated genes (ISGs), a necessary step in the virus lifecycle because of the capacity of IFNs to block virus replication. Efficient transmission of viruses depends, in part, on maintaining a balance between virus replication and host survival; specialized host cells, such as plasmacytoid dendritic cells, can sense viral molecular patterns and produce IFNs to help maintain this balance. There are now many examples of RNA viruses inducing type I and type III IFNs, and although these IFNs act through different receptors, in many systems studied, they induce a similar spectrum of genes. However, there may be a difference in the temporal expression pattern, with more prolonged expression of ISGs in response to type III IFN compared with type I IFN. There are also examples of synergy between type I and type III IFNs to induce antiviral responses. Clearly, it is important to understand the different roles of these IFNs in the antiviral response in vivo. One of the most striking differences between these 2 IFN systems is the distribution of the receptors: type I IFN receptors are expressed on most cells, yet type III receptor expression is restricted primarily to epithelial cells but has also been demonstrated on other cells, including dendritic cells. There is increasing evidence that type III IFNs are a key control mechanism against RNA viruses that infect respiratory and enteric epithelia. Copyright © 2014, Mary Ann Liebert, Inc.

Nairobi sheep disease virus (NSDV) of the genus Nairovirus causes a haemorrhagic gastroenteritis in sheep and goats with mortality up to 90%; the virus is found in East and Central Africa, and in India, where the virus is called Ganjam virus. NSDV is closely related to the human pathogen Crimean-Congo haemorrhagic fever virus, which also causes a haemorrhagic disease. As with other nairoviruses, replication of NSDV takes place in the cytoplasm and the new virus particles bud into the Golgi apparatus; however, the effect of viral replication on cellular compartments has not been studied extensively. We have found that the overall structure of the endoplasmic reticulum (ER), the ER-Golgi intermediate compartment and the Golgi were unaffected by infection with NSDV. However, we observed that NSDV infection led to the loss of protein disulphide isomerase (PDI), an oxidoreductase present in the lumen of the endoplasmic reticulum (ER) and which assists during protein folding, from the ER. Further investigation showed that NSDV-infected cells have high levels of PDI at their surface, and PDI is also secreted into the culture medium of infected cells. Another chaperone from the PDI family, ERp57, was found to be similarly affected. Analysis of infected cells and expression of individual viral glycoproteins indicated that the NSDV PreGn glycoprotein is involved in redistribution of these soluble ER oxidoreductases. It has been suggested that extracellular PDI can activate integrins and tissue factor, which are involved respectively in pro-inflammatory responses and disseminated intravascular coagulation, both of which manifest in many viral haemorrhagic fevers. The discovery of enhanced PDI secretion from NSDV-infected cells may be an important finding for understanding the mechanisms underlying the pathogenicity of haemorrhagic nairoviruses. © 2014 Lasecka, Baron.

Taylor G.,Pirbright Institute
Current Topics in Microbiology and Immunology | Year: 2013

Bovine respiratory syncytial virus (BRSV), which is an important cause of respiratory disease in young calves, is genetically and antigenically closely related to human (H)RSV. The epidemiology and pathogenesis of infection with these viruses are similar. The viruses are host-specific and infection produces a spectrum of disease ranging from subclinical to severe bronchiolitis and pneumonia, with the peak incidence of severe disease in individuals less than 6 months of age. BRSV infection in calves reproduces many of the clinical signs associated with HRSV in infants, including fever, rhinorrhoea, coughing, harsh breath sounds and rapid breathing. Although BRSV vaccines have been commercially available for decades, there is a need for greater efficacy. The development of effective BRSV and HRSV vaccines face similar challenges, such as the need to vaccinate at an early age in the presence of maternal antibodies, the failure of natural infection to prevent reinfection, and a history of vaccine-augmented disease. Neutralising monoclonal antibodies (mAbs) to the fusion (F) protein of HRSV, which can protect infants from severe HRSV disease, recognise the F protein of BRSV, and vice versa. Furthermore, bovine and human CD8+ T-cells, which are known to be important in recovery from RSV infection, recognise similar proteins that are conserved between HRSV and BRSV. Therefore, not only can the bovine model of RSV be used to evaluate vaccine concepts, it can also be used as part of the preclinical assessment of certain HRSV candidate vaccines. © Springer-Verlag Berlin Heidelberg 2013.

Nair V.,Pirbright Institute
Avian Diseases | Year: 2013

Despite the remarkable progress in our understanding of Marek's disease (MD) and the causative Marek's disease virus (MDV) biology, a number of major features of this complex viral disease remain unknown. Significant information on critical aspects of virus latency in lymphoid cells, and the virus-host interaction in MDV-induced lymphoma, remains to be identified. Moreover, the nature of the unique milieu of the feather follicle epithelial cell that allows cytolytic infection to continue, despite maintaining the latent infection in the lymphoid cells, is not fully understood. Although there has been significant progress in our understanding of the functions of a number of viral genes in the pathogenesis of the disease, the characteristics of the latent infection, how it differs from tumor phase, and whether latency is a prerequisite for the tumor phase are all important questions still to be answered. Reticuloendotheliosis virus-transformed cell lines have been shown to support MDV latency in a manner almost identical to that seen in MDV-transformed cell lines. There are increasing data on the role of epigenetic regulation, including DNA methylation and histone modifications, in maintaining viral latency. Onset of MD tumor is relatively rapid, and recent studies based on chromosomal integration and T-cell repertoire analysis demonstrated the clonal nature of MD lymphomas. Among the viral determinants of oncogenicity, the basic leucine zipper protein Meq is considered to be the most important and the most extensively studied. Deleting the Meq proteins or abolishing some of the important interactions does affect the oncogenicity of the virus. In addition, the noncoding sequences in the viral genome, such as the viral telomerase RNA and the virus-encoded microRNAs, also have significant influence on MDV-encoded oncogenesis. © American Association of Avian Pathologists.

Pirbright Institute | Date: 2014-03-25

The present invention relates to the stabilisation of foot-and-mouth disease virus (FMDV) capsids, by specific substitution of amino acids in a specific region of FMDV VP2. The invention provides stabilised FMDV capsids and vaccines against FMD.

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