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The Layers of Protection Analysis (LOPA) method is a semi-quantitative risk assessment tool that is used to determine the ability of safeguards to protect against unplanned hazardous scenarios. One possible outcome of a LOPA is that existing and proposed safeguards are deemed sufficient to reduce the risk associated with the hazardous scenario to a level that can be deemed as acceptable. Alternatively, the LOPA may also show that the safeguards are insufficient and therefore additional Safety Instrumented Function(s) (SIF) would be required to reduce risk to an acceptable level. In the latter case, the LOPA method will inform the end user as to the reliability requirements of the safety function in question. The LOPA method has been used extensively in the process industries (e.g., oil and gas) as a useful tool to manage and understand risk and to demonstrate if the facility is ‘safe’ to operate, but much less so in the biosafety sector. This paper describes the LOPA method and provides some practical examples of how it may be applied in microbiological high Containment Level (CL) facilities.

There is need for effective and affordable vaccines against SARS-CoV-2 to tackle the ongoing pandemic. In this study, we describe a protein nanoparticle vaccine against SARS-CoV-2. The vaccine is based on the display of coronavirus spike glycoprotein receptor-binding domain (RBD) on a synthetic virus-like particle (VLP) platform, SpyCatcher003-mi3, using SpyTag/SpyCatcher technology. Low doses of RBD-SpyVLP in a prime-boost regimen induce a strong neutralising antibody response in mice and pigs that is superior to convalescent human sera. We evaluate antibody quality using ACE2 blocking and neutralisation of cell infection by pseudovirus or wild-type SARS-CoV-2. Using competition assays with a monoclonal antibody panel, we show that RBD-SpyVLP induces a polyclonal antibody response that recognises key epitopes on the RBD, reducing the likelihood of selecting neutralisation-escape mutants. Moreover, RBD-SpyVLP is thermostable and can be lyophilised without losing immunogenicity, to facilitate global distribution and reduce cold-chain dependence. The data suggests that RBD-SpyVLP provides strong potential to address clinical and logistic challenges of the COVID-19 pandemic. Vaccines for SARS-COV-2 are needed in the ongoing pandemic. Here the authors characterize a vaccine candidate that presents the receptor-binding domain (RBD) of SARS-CoV-2 spike protein on a synthetic VLP platform using SpyTag/SpyCatcher technology and show immunogenicity of a prime-boost regimen in mice and pigs.

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.

Background Foot-and-mouth disease virus (FMDV) is capable of causing explosive outbreaks among domestic and wild cloven-hoofed animals. Genomic characterisation of FMDV is a crucial component of disease control enabling accurate tracing of disease outbreaks to be undertaken. Nanopore sequencing is an affordable and accessible form of high-throughput sequencing (HTS) technology. However, most published methods for FMDV only sequence genomic fragments or focus upon specific lineages. In this study, a universal FMDV sequencing protocol was developed alongside a bespoke analytical pipeline to sequence any FMDV genome in the absence of prior knowledge regarding the identity of the serotype or lineage. Methods Universal multiplex RT-PCRs were used to amplify overlapping tiles encompassing the entire FMDV genome. The PCR products were pooled and subjected to nanopore sequencing using the portable MinION sequencing device. A bioinformatics pipeline was used to assemble genomes based upon blastn and reference assembly. Results Iterative changes in primer design and pooling resulted in two panels of primers; one set amplifying twenty short fragments (S_scheme), and another set amplifying six longer fragments (L_scheme). Both approaches were shown to be capable of generating FMDV genomes, however the L_scheme was simpler, more reliable and more cost-effective at generating complete genomes. The final L_scheme protocol was assessed using 30 FMDV isolates representing all the currently circulating lineages of FMDV. As part of the development, we successfully trialled the use of this technology in Uganda, a country endemic for FMD. Conclusions The amplification, sequencing and bioinformatics strategy developed here has been assessed using a diverse array of FMDV lineages. Using two multiplex PCR reactions, this approach can successfully generate complete genomes of FMDV in a lineage agnostic fashion. Therefore, the primer sets and approaches described here represent a useful tool for expanding the capacity of laboratories to characterise FMDV at the genomic level.

The Animal Welfare Assessment Grid (AWAG) is a method for assessing quality of life, originally designed for experimental primates. This study adapts the AWAG for use in cattle and pigs, by adapting the factors included for these species and including data which had been collected previously as the standard approach to monitoring these species in research. The intention is that the results presented here will allow the future data collected for experimental cattle and pigs to be optimised for inclusion in an AWAG. Data were collected from two vaccine assessment studies at the Pirbright Institute. Factors were scored for every recorded event using retrospective data and CCTV clips. There was a lack of behavioural data recorded in both studies, which limited the accuracy of assessing each animal’s welfare. This paper emphasises the importance of including behavioural information when assessing welfare and not simply relying on assessment of physical condition. Scores peaked following an exponential rise as animals reached set humane end points. This demonstrated the potential of using the AWAG to aid the decision-making of when euthanasia should be performed. Our study shows the AWAG to be a useful tool for assessing welfare, which can be used in harm:benefit assessment.

Restoring connectivity via assisted migration is a useful but currently underused approach for maintaining genetic diversity and preventing extirpations of threatened species. The use of assisted migration as a conservation strategy may be limited by the difficulty of balancing the benefits of reconnecting populations (including reduced inbreeding depression and increased adaptive capacity) with the perceived risk of outbreeding depression, which requires comprehensive knowledge of the landscape of adaptive, neutral, deleterious, and structural variation across a species' range. Using a combination of reduced‐representation and whole‐genome sequencing, we characterized genomic diversity and differentiation for the Arkansas Darter (Etheostoma cragini) across its range in the Midwestern US. We found strong population structure and large differences in genetic diversity and effective population sizes across drainages. The strength of genetic isolation by river distance differed among drainages, with landscape type surrounding streams and impoundments also contributing to genetic isolation. Despite low effective population sizes in some populations, there was surprisingly little evidence for recent inbreeding (based on the absence of long runs of homozygosity) or for elevated levels of deleterious variation in smaller populations. Considering neutral, adaptive, deleterious, and structural variation allowed us to identify several potential recipient populations that may benefit from translocations and potential donor sites throughout the range. Planning translocation strategies intended for restored connectivity and possible genetic rescue at earlier stages in species decline will likely increase the probability of retaining genetic diversity and population persistence over the long term while minimizing risks associated with translocation.

Foot-and-mouth disease virus (FMDV) is a highly contagious virus that causes one of the most devastating diseases in cloven-hoofed animals. Disease symptoms develop within 2 to 3 days of exposure and include fever and vesicular lesions on the tongue and hooves. Dendritic cells (DC) play an essential role in protective immune responses against pathogens. Therefore, investigating their role during FMDV infection would lead to a better understanding of host-pathogen interactions. In this study, following infection of cattle with FMDV, we investigated the frequency and function of conventional (cDC) and plasmacytoid DC (pDC) in blood by using multi-color flow cytometry. We show that the frequency of cDC and pDC increased following FMDV infection and peaked 3 to 4 days post-infection. During peak viremia, the cattle became lymphopenic, the expression of MHC class II molecules on cDC and pDC was dramatically down-regulated, the processing of exogenous antigen by cDC and pDC was impaired, and there was an increase in IL-10 production by DC and monocytes. Notably, after clearance of FMDV from the blood, MHC class II expression returned to pre-infection levels. Altogether, our study demonstrates that in cattle, FMDV inhibits the function of DC, thereby retarding the initiation of adaptive immune responses, potentially enhancing virus shedding during the acute phase of infection.

In the light of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, we have developed a porcine respiratory coronavirus (PRCV) model for in depth mechanistic evaluation of the pathogenesis, virology and immune responses of this important family of viruses. Pigs are a large animal with similar physiology and immunology to humans and are a natural host for PRCV. Four PRCV strains were investigated and shown to induce different degrees of lung pathology. Importantly, although all four strains replicated equally well in porcine cell lines in vitro and in the upper respiratory tract in vivo , PRCV strains causing more severe lung pathology were also able to replicate in ex vivo tracheal organ cultures as well as in vivo in the trachea and lung. The time course of infection of PRCV 135, which caused the most severe pulmonary pathology, was investigated. Virus was shed from the upper respiratory tract until day 10 post infection, with infection of the respiratory mucosa, as well as olfactory and sustentacular cells, providing an excellent model to study upper respiratory tract disease in addition to the commonly known lower respiratory tract disease from PRCV. Infected animals made antibody and T cell responses that cross reacted with the four PRCV strains and Transmissible Gastroenteritis Virus. The antibody response was reproduced in vitro in organ cultures. Comparison of mechanisms of infection and immune control in pigs infected with PRCVs of differing pathogenicity with human data from SARS-CoV-2 infection and from our in vitro organ cultures, will enable key events in coronavirus infection and disease pathogenesis to be identified.

Nairobi sheep disease virus (NSDV; also called Ganjam virus in India) is a bunyavirus of the genus Nairovirus. It causes a haemorrhagic gastroenteritis in sheep and goats with mortality up to 90%. The virus is closely related to the human pathogen Crimean-Congo haemorrhagic fever virus (CCHFV). Little is currently known about the biology of NSDV. We have generated specific antibodies against the virus nucleocapsid protein (N) and polymerase (L) and used these to characterise NSDV in infected cells and to study its distribution during infection in a natural host. Due to its large size and the presence of a papain-like protease (the OTU-like domain) it has been suggested that the L protein of nairoviruses undergoes an autoproteolytic cleavage into polymerase and one or more accessory proteins. Specific antibodies which recognise either the N-terminus or the C-terminus of the NSDV L protein showed no evidence of L protein cleavage in NSDV-infected cells. Using the specific anti-N and anti-L antibodies, it was found that these viral proteins do not fully colocalise in infected cells; the N protein accumulated near the Golgi at early stages of infection while the L protein was distributed throughout the cytoplasm, further supporting the multifunctional nature of the L protein. These antibodies also allowed us to gain information about the organs and cell types targeted by the virus in vivo. We could detect NSDV in cryosections prepared from various tissues collected post-mortem from experimentally inoculated animals; the virus was found in the mucosal lining of the small and large intestine, in the lungs, and in mesenteric lymph nodes (MLN), where NSDV appeared to target monocytes and/or macrophages.

T cell responses directed against highly conserved viral proteins contribute to the clearance of the influenza virus and confer broadly cross-reactive and protective immune responses against a range of influenza viruses in mice and ferrets. We examined the protective efficacy of mucosal delivery of adenoviral vectors expressing hemagglutinin (HA) and nucleoprotein (NP) from the H1N1 virus against heterologous H3N2 challenge in pigs. We also evaluated the effect of mucosal co-delivery of IL-1β, which significantly increased antibody and T cell responses in inbred Babraham pigs. Another group of outbred pigs was first exposed to pH1N1 as an alternative means of inducing heterosubtypic immunity and were subsequently challenged with H3N2. Although both prior infection and adenoviral vector immunization induced strong T-cell responses against the conserved NP protein, none of the treatment groups demonstrated increased protection against the heterologous H3N2 challenge. Ad-HA/NP+Ad-IL-1β immunization increased lung pathology, although viral load was unchanged. These data indicate that heterotypic immunity may be difficult to achieve in pigs and the immunological mechanisms may differ from those in small animal models. Caution should be applied in extrapolating from a single model to humans.