Publications by authors named "Veronica L Fowler"

17 Publications

  • Page 1 of 1

Performance of the Innova SARS-CoV-2 antigen rapid lateral flow test in the Liverpool asymptomatic testing pilot: population based cohort study.

BMJ 2021 07 6;374:n1637. Epub 2021 Jul 6.

Institute of Population Health Sciences, University of Liverpool, Liverpool, UK.

Objective: To assess the performance of the SARS-CoV-2 antigen rapid lateral flow test (LFT) versus polymerase chain reaction testing in the asymptomatic general population attending testing centres.

Design: Observational cohort study.

Setting: Community LFT pilot at covid-19 testing sites in Liverpool, UK.

Participants: 5869 asymptomatic adults (≥18 years) voluntarily attending one of 48 testing sites during 6-29 November 2020.

Interventions: Participants were tested using both an Innova LFT and a quantitative reverse-transcriptase polymerase chain reaction (RT-qPCR) test based on supervised self-administered swabbing at testing sites.

Main Outcome Measures: Sensitivity, specificity, and predictive values of LFT compared with RT-qPCR in an epidemic steady state of covid-19 among adults with no classic symptoms of the disease.

Results: Of 5869 test results, 22 (0.4%) LFT results and 343 (5.8%) RT-qPCR results were void (that is, when the control line fails to appear within 30 minutes). Excluding the void results, the LFT versus RT-qPCR showed a sensitivity of 40.0% (95% confidence interval 28.5% to 52.4%; 28/70), specificity of 99.9% (99.8% to 99.99%; 5431/5434), positive predictive value of 90.3% (74.2% to 98.0%; 28/31), and negative predictive value of 99.2% (99.0% to 99.4%; 5431/5473). When the void samples were assumed to be negative, a sensitivity was observed for LFT of 37.8% (26.8% to 49.9%; 28/74), specificity of 99.6% (99.4% to 99.8%; 5431/5452), positive predictive value of 84.8% (68.1% to 94.9%; 28/33), and negative predictive value of 93.4% (92.7% to 94.0%; 5431/5814). The sensitivity in participants with an RT-qPCR cycle threshold (Ct) of <18.3 (approximate viral loads >10 RNA copies/mL) was 90.9% (58.7% to 99.8%; 10/11), a Ct of <24.4 (>10 RNA copies/mL) was 69.4% (51.9% to 83.7%; 25/36), and a Ct of >24.4 (<10 RNA copies/mL) was 9.7% (1.9% to 23.7%; 3/34). LFT is likely to detect at least three fifths and at most 998 in every 1000 people with a positive RT-qPCR test result with high viral load.

Conclusions: The Innova LFT can be useful for identifying infections among adults who report no symptoms of covid-19, particularly those with high viral load who are more likely to infect others. The number of asymptomatic adults with lower Ct (indicating higher viral load) missed by LFT, although small, should be considered when using single LFT in high consequence settings. Clear and accurate communication with the public about how to interpret test results is important, given the chance of missing some cases, even at high viral loads. Further research is needed to understand how infectiousness is reflected in the viral antigen shedding detected by LFT versus the viral loads approximated by RT-qPCR.
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http://dx.doi.org/10.1136/bmj.n1637DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8259455PMC
July 2021

Preliminary optimisation of a simplified sample preparation method to permit direct detection of SARS-CoV-2 within saliva samples using reverse-transcription loop-mediated isothermal amplification (RT-LAMP).

J Virol Methods 2021 03 20;289:114048. Epub 2020 Dec 20.

Hampshire Hospitals NHS Foundation Trust, Department of Microbiology, Basingstoke and North Hants Hospital, Basingstoke, UK.

We describe the optimisation of a simplified sample preparation method which permits rapid and direct detection of SARS-CoV-2 RNA within saliva, using reverse-transcription loop-mediated isothermal amplification (RT-LAMP). Treatment of saliva samples prior to RT-LAMP by dilution 1:1 in Mucolyse™, followed by dilution in 10 % (w/v) Chelex© 100 Resin and a 98 °C heat step for 2 min enabled detection of SARS-CoV-2 RNA in positive saliva samples. Using RT-LAMP, SARS-CoV-2 RNA was detected in as little as 05:43 min, with no amplification detected in 3097 real-time reverse transcription PCR (rRT-PCR) negative saliva samples from staff tested within a service evaluation study, or for other respiratory pathogens tested (n = 22). Saliva samples can be collected non-invasively, without the need for skilled staff and can be obtained from both healthcare and home settings. Critically, this approach overcomes the requirement for, and validation of, different swabs and the global bottleneck in obtaining access to extraction robots and reagents to enable molecular testing by rRT-PCR. Such testing opens the possibility of public health approaches for effective intervention during the COVID-19 pandemic through regular SARS-CoV-2 testing at a population scale, combined with isolation and contact tracing.
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http://dx.doi.org/10.1016/j.jviromet.2020.114048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7750029PMC
March 2021

A highly effective reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay for the rapid detection of SARS-CoV-2 infection.

J Infect 2021 01 30;82(1):117-125. Epub 2020 Nov 30.

Hampshire Hospitals NHS Foundation Trust, Basingstoke & North Hampshire Hospital, Department of Microbiology, Basingstoke, UK. Electronic address:

The COVID-19 pandemic has illustrated the importance of simple, rapid and accurate diagnostic testing. This study describes the validation of a new rapid SARS-CoV-2 RT-LAMP assay for use on extracted RNA or directly from swab offering an alternative diagnostic pathway that does not rely on traditional reagents that are often in short supply during a pandemic. Analytical specificity (ASp) of this new RT-LAMP assay was 100% and analytical sensitivity (ASe) was between 1 × 10 and 1 × 10 copies per reaction when using a synthetic DNA target. The overall diagnostic sensitivity (DSe) and specificity (DSp) of RNA RT-LAMP was 97% and 99% respectively, relative to the standard of care rRT-PCR. When a C cut-off of 33 was employed, above which increasingly evidence suggests there is a low risk of patients shedding infectious virus, the diagnostic sensitivity was 100%. The DSe and DSp of Direct RT-LAMP (that does not require RNA extraction) was 67% and 97%, respectively. When setting C cut-offs of ≤33 and ≤25, the DSe increased to 75% and 100%, respectively, time from swab-to-result, C < 25, was < 15 min. We propose that RNA RT-LAMP could replace rRT-PCR where there is a need for increased sample throughput and Direct RT-LAMP as a near-patient screening tool to rapidly identify highly contagious individuals within emergency departments and care homes during times of increased disease prevalence ensuring negative results still get laboratory confirmation.
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http://dx.doi.org/10.1016/j.jinf.2020.10.039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7703389PMC
January 2021

GoPrime: Development of an In Silico Framework to Predict the Performance of Real-Time PCR Primers and Probes Using Foot-and-Mouth Disease Virus as a Model.

Pathogens 2020 Apr 20;9(4). Epub 2020 Apr 20.

The Pirbright Institute, Ash Road, Pirbright, Surrey GU24 0NF, UK.

Real-time PCR (rPCR) is a widely accepted diagnostic tool for the detection and quantification of nucleic acid targets. In order for these assays to achieve high sensitivity and specificity, primer and probe-template complementarity is essential; however, mismatches are often unavoidable and can result in false-negative results and errors in quantifying target sequences. Primer and probe sequences therefore require continual evaluation to ensure they remain fit for purpose. This paper describes the development of a linear model and associated computational tool (GoPrime) designed to predict the performance of rPCR primers and probes across multiple sequence data. Empirical data were generated using DNA oligonucleotides (n = 90) that systematically introduced variation in the primer and probe target regions of a diagnostic assay routinely used to detect foot-and-mouth disease virus (FMDV); an animal virus that exhibits a high degree of sequence variability. These assays revealed consistent impacts of patterns of substitutions in primer and probe-sites on rPCR cycle threshold (C) and limit of detection (LOD). These data were used to populate GoPrime, which was subsequently used to predict rPCR results for DNA templates (n = 7) representing the natural sequence variability within FMDV. GoPrime was also applicable to other areas of the FMDV genome, with predictions for the likely targets of a FMDV-typing assay consistent with published experimental data. Although further work is required to improve these tools, including assessing the impact of primer-template mismatches in the reverse transcription step and the broader impact of mismatches for other assays, these data support the use of mathematical models for rapidly predicting the performance of rPCR primers and probes in silico.
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http://dx.doi.org/10.3390/pathogens9040303DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7238122PMC
April 2020

Opportunities for enhanced surveillance of foot-and-mouth disease in endemic settings using milk samples.

Transbound Emerg Dis 2019 May 27;66(3):1405-1410. Epub 2019 Feb 27.

Boyd Orr Centre for Population and Ecosystem Health, Institute of Biodiversity, Animal Health and Comparative Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK.

Under-reporting of foot-and-mouth disease (FMD) masks the true prevalence in parts of the world where the disease is endemic. Laboratory testing for the detection of FMD virus (FMDV) is usually reliant upon the collection of vesicular epithelium and fluid samples that can only be collected from acutely infected animals, and therefore animals with sub-clinical infection may not be identified. Milk is a non-invasive sample type routinely collected from dairy farms that has been utilized for surveillance of a number of other diseases. The aim of this study was to examine the application of milk as an alternative sample type for FMDV detection and typing, and to evaluate milk as a novel approach for targeted surveillance of FMD in East Africa. FMDV RNA was detected in 73/190 (38%) individual milk samples collected from naturally infected cattle in northern Tanzania. Furthermore, typing information by lineage-specific rRT-PCR assays was obtained for 58% of positive samples, and corresponded with the virus types identified during outbreak investigations in the study area. The VP1-coding sequence data obtained from milk samples corresponded with the sequence data generated from paired epithelial samples collected from the same animal. This study demonstrates that milk represents a potentially valuable sample type for FMDV surveillance and might be used to overcome some of the existing biases of traditional surveillance methods. However, it is recommended that care is taken during sample collection and testing to minimize the likelihood of cross-contamination. Such approaches could strengthen FMDV surveillance capabilities in East Africa, both at the individual animal and herd level.
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http://dx.doi.org/10.1111/tbed.13146DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6563157PMC
May 2019

The development of two field-ready reverse transcription loop-mediated isothermal amplification assays for the rapid detection of Seneca Valley virus 1.

Transbound Emerg Dis 2019 Jan 15;66(1):497-504. Epub 2018 Nov 15.

OptiGene Limited, Horsham, West Sussex, UK.

Seneca Valley virus 1 (SVV-1) has been associated with vesicular disease in swine, with clinical signs indistinguishable from those of other notifiable vesicular diseases such as foot-and-mouth disease. Rapid and accurate detection of SVV-1 is central to confirm the disease causing agent, and to initiate the implementation of control processes. The development of rapid, cost-effective diagnostic assays that can be used at the point of sample collection has been identified as a gap in preparedness for the control of SVV-1. This study describes the development and bench validation of two reverse transcription loop-mediated amplification (RT-LAMP) assays targeting the 5'-untranslated region (5'-UTR) and the VP3-1 region for the detection of SVV-1 that may be performed at the point of sample collection. Both assays were able to demonstrate amplification of all neat samples diluted 1/100 in negative pig epithelium tissue suspension within 8 min, when RNA was extracted prior to the RT-LAMP assay, and no amplification was observed for the other viruses tested. Simple sample preparation methods using lyophilized reagents were investigated, to negate the requirement for RNA extraction. Only a small delay in the time to amplification was observed for these lyophilized reagents, with a time from sample receipt to amplification achieved within 12 min. Although diagnostic validation is recommended, these RT-LAMP assays are highly sensitive and specific, with the potential to be a useful tool in the rapid diagnosis of SVV-1 in the field.
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http://dx.doi.org/10.1111/tbed.13051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6434928PMC
January 2019

Waves of endemic foot-and-mouth disease in eastern Africa suggest feasibility of proactive vaccination approaches.

Nat Ecol Evol 2018 09 6;2(9):1449-1457. Epub 2018 Aug 6.

Boyd Orr Centre for Population and Ecosystem Health, Institute of Biodiversity, Animal Health and Comparative Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK.

Livestock production in Africa is key to national economies, food security and rural livelihoods, and > 85% of livestock keepers live in extreme poverty. With poverty elimination central to the Sustainable Development Goals, livestock keepers are therefore critically important. Foot-and-mouth disease is a highly contagious livestock disease widespread in Africa that contributes to this poverty. Despite its US$2.3 billion impact, control of the disease is not prioritized: standard vaccination regimens are too costly, its impact on the poorest is underestimated, and its epidemiology is too weakly understood. Our integrated analysis in Tanzania shows that the disease is of high concern, reduces household budgets for human health, and has major impacts on milk production and draft power for crop production. Critically, foot-and-mouth disease outbreaks in cattle are driven by livestock-related factors with a pattern of changing serotype dominance over time. Contrary to findings in southern Africa, we find no evidence of frequent infection from wildlife, with outbreaks in cattle sweeping slowly across the region through a sequence of dominant serotypes. This regularity suggests that timely identification of the epidemic serotype could allow proactive vaccination ahead of the wave of infection, mitigating impacts, and our preliminary matching work has identified potential vaccine candidates. This strategy is more realistic than wildlife-livestock separation or conventional foot-and-mouth disease vaccination approaches. Overall, we provide strong evidence for the feasibility of coordinated foot-and-mouth disease control as part of livestock development policies in eastern Africa, and our integrated socioeconomic, epidemiological, laboratory and modelling approach provides a framework for the study of other disease systems.
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http://dx.doi.org/10.1038/s41559-018-0636-xDOI Listing
September 2018

Efficacy of a high-potency multivalent foot-and-mouth disease virus vaccine in cattle against heterologous challenge with a field virus from the emerging A/ASIA/G-VII lineage.

Vaccine 2018 03 2;36(14):1901-1907. Epub 2018 Mar 2.

The Pirbright Institute, Ash Road, Pirbright, Woking, Surrey GU24 0NF, United Kingdom.

In 2015, outbreaks of foot-and-mouth disease (FMD) in the Middle East were discovered to be caused by a viral lineage (A/ASIA/G-VII), which has recently emerged from the Indian sub-continent. In vitro vaccine matching data generated by the World Reference Laboratory (WRLFMD) indicated that A/ASIA/G-VII field viruses were poorly matched with vaccines (A-SAU-95, A22 IRQ and A-IRN-05) that are already used in the region. In order to assess the likely performance of one of these commercially available FMD vaccines, sixteen cattle were vaccinated with a polyvalent vaccine which contained two serotype A components (A-SAU-95 and A-IRN-05) with a homologous potency of at least 6PD, and two cattle were left unvaccinated as controls. Twenty-one days later, all 18 cattle were challenged by tongue inoculation with an FMDV field isolate A/IRN/22/2015 from the A/ASIA/G-VII lineage, in line with the European Pharmacopeia PPG test conditions. The two control animals developed generalised FMD, and 7/16 vaccinated animals developed at least one foot lesion, thus only 56.3% were defined as protected. For the vaccine components, there was a significant increase in the probability of protection with increasing serological titres for A-SAU-95 (p = 0.03), but not for A-IRN-05 (p = 0.42). Analysis of FMDV in blood and nasal swabs suggested that vaccination reduced shedding and potential onward spread of FMD virus even if the animal developed foot lesions. In summary, the results from this study suggest that whilst this vaccine would not be appropriate for use in an emergency situation (in previously FMD-free countries), it may be partially effective in the field in endemic countries where repeat prophylactic vaccination is practiced. For emergency reactive vaccination, the findings from this study support the idea that a new vaccine strain should be developed that is tailored to the A/ASIA/G-VII lineage.
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http://dx.doi.org/10.1016/j.vaccine.2018.02.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5864508PMC
March 2018

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

J Virol Methods 2017 11 31;249:102-110. Epub 2017 Aug 31.

The Pirbright Institute, Ash Road, Pirbright, Surrey, GU24 0NF, UK. Electronic address:

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 (10 RNA copies), while RT-RPA was one log less sensitive (10 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.
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http://dx.doi.org/10.1016/j.jviromet.2017.08.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5630204PMC
November 2017

Development of a novel real-time RT-PCR assay to detect Seneca Valley virus-1 associated with emerging cases of vesicular disease in pigs.

J Virol Methods 2017 01 29;239:34-37. Epub 2016 Oct 29.

Kansas State Veterinary Diagnostic Laboratory, Kansas State University, 1800 Denison Avenue, Manhattan, KS 66506, United States; Department of Diagnostic Medicine/Pathobiology, Kansas State University, 1800 Denison Avenue, Manhattan, KS 66506, United States. Electronic address:

Seneca Valley virus 1 (SVV-1) can cause vesicular disease that is clinically indistinguishable from foot-and-mouth disease, vesicular stomatitis and swine vesicular disease. SVV-1-associated disease has been identified in pigs in several countries, namely USA, Canada, Brazil and China. Diagnostic tests are required to reliably detect this emerging virus, and this report describes the development and evaluation of a novel real-time (r) reverse-transcription (RT) PCR assay (rRT-PCR), targeting the viral polymerase gene (3D) of SVV-1. This new assay detected all historical and contemporary SVV-1 isolates examined (n=8), while no cross-reactivity was observed with nucleic acid templates prepared from other vesicular disease viruses or common swine pathogens. The analytical sensitivity of the rRT-PCR was 0.79 TCID/ml and the limit of detection was equivalent using two different rRT-PCR master-mixes. The performance of the test was further evaluated using pig nasal (n=25) and rectal swab samples (n=25), where concordant results compared to virus sequencing were generated for 43/50 samples. The availability of this assay, will enable laboratories to rapidly detect SVV-1 in cases of vesicular disease in pigs, negated for notifiable diseases, and could enable existing knowledge gaps to be investigated surrounding the natural epidemiology of SVV-1.
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http://dx.doi.org/10.1016/j.jviromet.2016.10.012DOI Listing
January 2017

Monty Roberts' Public Demonstrations: Preliminary Report on the Heart Rate and Heart Rate Variability of Horses Undergoing Training during Live Audience Events.

Animals (Basel) 2016 Sep 9;6(9). Epub 2016 Sep 9.

The Pirbright Institute, Ash Road, Pirbright, Surrey GU24 0NF, UK.

Effective training of horses relies on the trainer's awareness of learning theory and equine ethology, and should be undertaken with skill and time. Some trainers, such as Monty Roberts, share their methods through the medium of public demonstrations. This paper describes the opportunistic analysis of beat-to-beat (RR) intervals and heart rate variability (HRV) of ten horses being used in Monty Roberts' public demonstrations within the United Kingdom. RR and HRV was measured in the stable before training and during training. The HRV variables standard deviation of the RR interval (SDRR), root mean square of successive RR differences (RMSSD), geometric means standard deviation 1 (SD1) and 2 (SD2), along with the low and high frequency ratio (LF/HF ratio) were calculated. The minimum, average and maximum RR intervals were significantly lower in training (indicative of an increase in heart rate as measured in beats-per-minute) than in the stable ( p = 0.0006; p = 0.01; p = 0.03). SDRR, RMSSD, SD1, SD2 and the LF/HF ratio were all significantly lower in training than in the stable ( p = 0.001; p = 0.049; p = 0.049; p = 0.001; p = 0.01). When comparing the HR and HRV of horses during Join-up (®) to overall training, there were no significant differences in any variable with the exception of maximum RR which was significantly lower ( p = 0.007) during Join-up (®) , indicative of short increases in physical exertion (canter) associated with this training exercise. In conclusion, training of horses during public demonstrations is a low-moderate physiological, rather than psychological stressor for horses. The physiological stress responses observed within this study were comparable or less to those previously reported in the literature for horses being trained outside of public audience events. Furthermore, there is no evidence that the use of Join-up (®) alters HR and HRV in a way to suggest that this training method negatively affects the psychological welfare of horses.
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http://dx.doi.org/10.3390/ani6090055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5035950PMC
September 2016

Development of a reverse transcription loop-mediated isothermal amplification assay for the detection of vesicular stomatitis New Jersey virus: Use of rapid molecular assays to differentiate between vesicular disease viruses.

J Virol Methods 2016 08 23;234:123-31. Epub 2016 Apr 23.

The Pirbright Institute, Ash Road, Pirbright, Surrey, GU24 0NF, United Kingdom.

Vesicular stomatitis (VS) is endemic in Central America and northern regions of South America, where sporadic outbreaks in cattle and pigs can cause clinical signs that are similar to foot-and-mouth disease (FMD). There is therefore a pressing need for rapid, sensitive and specific differential diagnostic assays that are suitable for decision making in the field. RT-LAMP assays have been developed for vesicular diseases such as FMD and swine vesicular disease (SVD) but there is currently no RT-LAMP assay that can detect VS virus (VSV), nor are there any multiplex RT-LAMP assays which permit rapid discrimination between these 'look-a-like' diseases in situ. This study describes the development of a novel RT-LAMP assay for the detection of VSV focusing on the New Jersey (VSNJ) serotype, which has caused most of the recent VS cases in the Americas. This RT-LAMP assay was combined in a multiplex format combining molecular lateral-flow devices for the discrimination between FMD and VS. This assay was able to detect representative VSNJV's and the limit of detection of the singleplex and multiplex VSNJV RT-LAMP assays were equivalent to laboratory based real-time RT-PCR assays. A similar multiplex RT-LAMP assay was developed to discriminate between FMDV and SVDV, showing that FMDV, SVDV and VSNJV could be reliably detected within epithelial suspensions without the need for prior RNA extraction, providing an approach that could be used as the basis for a rapid and low cost assay for differentiation of FMD from other vesicular diseases in the field.
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http://dx.doi.org/10.1016/j.jviromet.2016.04.012DOI Listing
August 2016

Genome Sequences of Nine Vesicular Stomatitis Virus Isolates from South America.

Genome Announc 2016 Apr 14;4(2). Epub 2016 Apr 14.

The Pirbright Institute, Pirbright, Woking, Surrey, United Kingdom.

We report nine full-genome sequences of vesicular stomatitis virus obtained by Illumina next-generation sequencing of RNA, isolated from either cattle epithelial suspensions or cell culture supernatants. Seven of these viral genomes belonged to the New Jersey serotype/species (clade III), while two isolates belonged to the Indiana serotype/species.
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http://dx.doi.org/10.1128/genomeA.00249-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4832157PMC
April 2016

Identification of a novel cell culture adaptation site on the capsid of foot-and-mouth disease virus.

J Gen Virol 2015 Sep 3;96(9):2684-2692. Epub 2015 Jul 3.

The Pirbright Institute, Ash Road, Pirbright, Woking, Surrey GU24 0NF, UK.

Vaccination remains the most effective tool for control of foot-and-mouth disease both in endemic countries and as an emergency preparedness for new outbreaks. Foot-and-mouth disease vaccines are chemically inactivated virus preparations and the production of new vaccines is critically dependent upon cell culture adaptation of field viruses, which can prove problematic. A major driver of cell culture adaptation is receptor availability. Field isolates of foot-and-mouth disease virus (FMDV) use RGD-dependent integrins as receptors, whereas cell culture adaptation often selects for variants with altered receptor preferences. Previously, two independent sites on the capsid have been identified where mutations are associated with improved cell culture growth. One is a shallow depression formed by the three major structural proteins (VP1-VP3) where mutations create a heparan sulphate (HS)-binding site (the canonical HS-binding site). The other involves residues of VP1 and is located at the fivefold symmetry axis. For some viruses, changes at this site result in HS binding; for others, the receptors are unknown. Here, we report the identification of a novel site on VP2 where mutations resulted in an expanded cell tropism of a vaccine variant of A/IRN/87 (called A - ). Furthermore, we show that introducing the same mutations into a different type A field virus (A/TUR/2/2006) resulted in the same expanded cell culture tropism as the A/IRN/87 A -  vaccine variant. These observations add to the evidence for multiple cell attachment mechanisms for FMDV and may be useful for vaccine manufacture when cell culture adaptation proves difficult.
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http://dx.doi.org/10.1099/jgv.0.000222DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4635497PMC
September 2015

Recovery of viral RNA and infectious foot-and-mouth disease virus from positive lateral-flow devices.

PLoS One 2014 14;9(10):e109322. Epub 2014 Oct 14.

Vesicular Disease Reference Laboratory, The Pirbright Institute, Pirbright, Surrey, United Kingdom.

Foot-and-mouth disease Virus (FMDV) is an economically important, highly contagious picornavirus that affects both wild and domesticated cloven hooved animals. In developing countries, the effective laboratory diagnosis of foot-and-mouth disease (FMD) is often hindered by inadequate sample preservation due to difficulties in the transportation and storage of clinical material. These factors can compromise the ability to detect and characterise FMD virus in countries where the disease is endemic. Furthermore, the high cost of sending infectious virus material and the biosecurity risk it presents emphasises the need for a thermo-stable, non-infectious mode of transporting diagnostic samples. This paper investigates the potential of using FMDV lateral-flow devices (LFDs) for dry transportation of clinical samples for subsequent nucleic acid amplification, sequencing and recovery of infectious virus by electroporation. FMDV positive samples (epithelial suspensions and cell culture isolates) representing four FMDV serotypes were applied to antigen LFDs: after which it was possible to recover viral RNA that could be detected using real-time RT-PCR. Using this nucleic acid, it was also possible to recover VP1 sequences and also successfully utilise protocols for amplification of complete FMD virus genomes. It was not possible to recover infectious FMDV directly from the LFDs, however following electroporation into BHK-21 cells and subsequent cell passage, infectious virus could be recovered. Therefore, these results support the use of the antigen LFD for the dry, non-hazardous transportation of samples from FMD endemic countries to international reference laboratories.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0109322PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4196899PMC
June 2015

Preliminary validation of direct detection of foot-and-mouth disease virus within clinical samples using reverse transcription loop-mediated isothermal amplification coupled with a simple lateral flow device for detection.

PLoS One 2014 28;9(8):e105630. Epub 2014 Aug 28.

Livestock Viral Disease Program, The Pirbright Institute, Pirbright, Guildford, Surrey, United Kingdom.

Rapid, field-based diagnostic assays are desirable tools for the control of foot-and-mouth disease (FMD). Current approaches involve either; 1) Detection of FMD virus (FMDV) with immuochromatographic antigen lateral flow devices (LFD), which have relatively low analytical sensitivity, or 2) portable RT-qPCR that has high analytical sensitivity but is expensive. Loop-mediated isothermal amplification (LAMP) may provide a platform upon which to develop field based assays without these drawbacks. The objective of this study was to modify an FMDV-specific reverse transcription-LAMP (RT-LAMP) assay to enable detection of dual-labelled LAMP products with an LFD, and to evaluate simple sample processing protocols without nucleic acid extraction. The limit of detection of this assay was demonstrated to be equivalent to that of a laboratory based real-time RT-qPCR assay and to have a 10,000 fold higher analytical sensitivity than the FMDV-specific antigen LFD currently used in the field. Importantly, this study demonstrated that FMDV RNA could be detected from epithelial suspensions without the need for prior RNA extraction, utilising a rudimentary heat source for amplification. Once optimised, this RT-LAMP-LFD protocol was able to detect multiple serotypes from field epithelial samples, in addition to detecting FMDV in the air surrounding infected cattle, pigs and sheep, including pre-clinical detection. This study describes the development and evaluation of an assay format, which may be used as a future basis for rapid and low cost detection of FMDV. In addition it provides providing "proof of concept" for the future use of LAMP assays to tackle other challenging diagnostic scenarios encompassing veterinary and human health.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0105630PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4148330PMC
October 2015

Progress in the development of DNA vaccines against foot-and-mouth disease.

Expert Rev Vaccines 2012 Apr;11(4):481-93

Institute for Animal Health, Pirbright Laboratory, Surrey GU24 0NF, UK.

DNA vaccines are, in principle, the simplest yet most versatile methods of inducing protective humoral and cellular immune responses. Research involving this type of vaccine against veterinary diseases began in the early 1990s and has since seen the evaluation of more than 30 important viral pathogens, including the economically important foot-and-mouth disease. With the demonstration that DNA vaccines protect against foot-and-mouth disease in sheep and pigs, and the advantages these DNA vaccines have over the conventional formulations, this approach may provide a better solution to the control of this disease. In this review, we provide a comprehensive overview of DNA vaccination strategies for foot-and-mouth disease reported in the literature, in which we highlight the studies that have reported protection in the key target species.
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http://dx.doi.org/10.1586/erv.11.198DOI Listing
April 2012
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