Identification of a small molecule inhibitor of Ebolavirus genome replication and transcription using in silico screening.

Ebola virus (EBOV) causes a severe haemorrhagic fever in humans and has a mortality rate over 50%. With no licensed drug treatments available, EBOV poses a significant threat. Investigations into possible therapeutics have been severely hampered by the classification of EBOV as a BSL4 pathogen. Here, we describe a drug discovery pathway combining in silico screening of compounds predicted to bind to a hydrophobic pocket on the nucleoprotein (NP); with a robust and rapid EBOV minigenome assay for inhibitor validation at BSL2. One compound (MCCB4) was efficacious (EC50 4.8 μM), exhibited low cytotoxicity (CC50 > 100 μM) and was specific, with no effect on either a T7 RNA polymerase driven firefly luciferase or a Bunyamwera virus minigenome. Further investigations revealed that this small molecule inhibitor was able to outcompete established replication complexes, an essential aspect for a potential EBOV treatment

Heat Shock Protein 70 family members interact with Crimean-Congo hemorrhagic fever virus and Hazara virus nucleocapsid proteins and perform a functional role in the nairovirus replication cycle

The Nairovirus genus of the Bunyaviridae family contains serious human and animal pathogens classified within multiple serogroups and species. Of these serogroups, the Crimean-Congo hemorrhagic fever virus (CCHFV) serogroup comprises sole members CCHFV and Hazara virus (HAZV). CCHFV is an emerging zoonotic virus that causes often-fatal hemorrhagic fever in infected humans for which preventative or therapeutic strategies are not available. In contrast HAZV is non-pathogenic to humans, and thus represents an excellent model to study aspects of CCHFV biology under more accessible biological containment. The three RNA segments that form the nairovirus genome are encapsidated by the viral nucleocapsid protein (N) to form ribonucleoprotein (RNP) complexes that are substrates for RNA synthesis and packaging into virus particles. We used quantitative proteomics to identify cellular interaction partners of CCHFV N, and identified robust interactions with cellular chaperones. These interactions were validated using immunological methods, and the specific interaction between native CCHFV N and cellular chaperones of the HSP70 family was confirmed during live CCHFV infection. Using infectious HAZV we showed for the first time that the nairovirus N-HSP70 association was maintained within both infected cells and virus particles, where N is assembled as RNPs. Reduction of active HSP70 levels in cells using small molecule inhibitors significantly reduced HAZV titres, and a model for chaperone function in the context of high genetic variability is proposed. These results suggest chaperones of the HSP70 family are required for nairovirus replication and thus represent a genetically stable cellular therapeutic target for preventing nairovirus-mediated disease

Development of a multiplex assay for antibody detection in serum against pathogens affecting ruminants

Numerous infectious diseases impacting livestock impose an important economic burden and in some cases also represent a threat to humans and are classified as zoonoses. Some zoonotic diseases are transmitted by vectors and, due to complex environmental and socio‐economic factors, the distribution of many of these pathogens is changing, with increasing numbers being found in previously unaffected countries. Here, we developed a multiplex assay, based on a suspension microarray, able to detect specific antibodies to five important pathogens of livestock (three of them zoonotic) that are currently emerging in new geographical locations: Rift Valley fever virus (RVFV), Crimean‐Congo haemorrhagic fever virus (CCHFV), Schmallenberg virus (SBV), Bluetongue virus (BTV) and the bacteria complex Mycobacterium tuberculosis. Using the Luminex platform, polystyrene microspheres were coated with recombinant proteins from each of the five pathogens. The mix of microspheres was used for the simultaneous detection of antibodies against the five corresponding diseases affecting ruminants. The following panel of sera was included in the study: 50 sera from sheep experimentally infected with RVFV, 74 sera from calves and lambs vaccinated with SBV, 26 sera from cattle vaccinated with Mycobacterium bovis, 30 field sera from different species of ruminants infected with CCHFV and 88 calf sera infected with BTV. Finally, to determine its diagnostic specificity 220 field sera from Spanish farms free of the five diseases were assessed. All the sera were classified using commercial ELISAs specific for each disease, used in this study as the reference technique. The results showed the multiplex assay exhibited good performance characteristics with values of sensitivity ranging from 93% to 100% and of specificity ranging from 96% to 99% depending on the pathogen. This new tool allows the simultaneous detection of antibodies against five important pathogens, reducing the volume of sample needed and the time of analysis where these pathogens are usually tested individually

Tula orthohantavirus nucleocapsid protein is cleaved in infected cells and may sequester activated caspase-3 during persistent infection to suppress apoptosis

The family Hantaviridae mostly comprises rodent-borne segmented negative-sense RNA viruses, many of which are capable of causing devastating disease in humans. In contrast, hantavirus infection of rodent hosts results in a persistent and inapparent infection through their ability to evade immune detection and inhibit apoptosis. In this study, we used Tula hantavirus (TULV) to investigate the interplay between viral and host apoptotic responses during early, peak and persistent phases of virus infection in cell culture. Examination of early-phase TULV infection revealed that infected cells were refractory to apoptosis, as evidenced by the complete lack of cleaved caspase-3 (casp-3C) staining, whereas in non-infected bystander cells casp-3C was highly abundant. Interestingly, at later time points, casp-3C was abundant in infected cells, but the cells remained viable and able to continue shedding infectious virus, and together these observations were suggestive of a TULV-associated apoptotic block. To investigate this block, we viewed TULV-infected cells using laser scanning confocal and wide-field deconvolution microscopy, which revealed that TULV nucleocapsid protein (NP) colocalized with, and sequestered, casp-3C within cytoplasmic ultrastructures. Consistent with casp-3C colocalization, we showed for the first time that TULV NP was cleaved in cells and that TULV NP and casp-3C could be co-immunoprecipitated, suggesting that this interaction was stable and thus unlikely to be solely confined to NP binding as a substrate to the casp-3C active site. To account for these findings, we propose a novel mechanism by which TULV NP inhibits apoptosis by spatially sequestering casp-3C from its downstream apoptotic targets within the cytosol

Evidence-informed health policy 3 – Interviews with the directors of organizations that support the use of research evidence

Background: Previous surveys of organizations that support the development of evidence-informed health policies have focused on organizations that produce clinical practice guidelines (CPGs) or undertake health technology assessments (HTAs). Only rarely have surveys focused at least in part on units that directly support the use of research evidence in developing health policy on an international, national, and state or provincial level (i.e., government support units, or GSUs) that are in some way successful or innovative or that support the use of research evidence in low- and middle-income countries (LMICs). Methods: We drew on many people and organizations around the world, including our project reference group, to generate a list of organizations to survey. We modified a questionnaire that had been developed originally by the Appraisal of Guidelines, Research and Evaluation in Europe (AGREE) collaboration and adapted one version of the questionnaire for organizations producing CPGs and HTAs, and another for GSUs. We sent the questionnaire by email to 176 organizations and followed up periodically with non-responders by email and telephone. Results: We received completed questionnaires from 152 (86%) organizations. More than one-half of the organizations (and particularly HTA agencies) reported that examples from other countries were helpful in establishing their organization. A higher proportion of GSUs than CPG-or HTA-producing organizations involved target users in the selection of topics or the services undertaken. Most organizations have few (five or fewer) full-time equivalent (FTE) staff. More than four-fifths of organizations reported providing panels with or using systematic reviews. GSUs tended to use a wide variety of explicit valuation processes for the research evidence, but none with the frequency that organizations producing CPGs, HTAs, or both prioritized evidence by its quality. Between one-half and two-thirds of organizations do not collect data systematically about uptake, and roughly the same proportions do not systematically evaluate their usefulness or impact in other ways. Conclusion: The findings from our survey, the most broadly based of its kind, both extend or clarify the applicability of the messages arising from previous surveys and related documentary analyses, such as how the 'principles of evidence-based medicine dominate current guideline programs' and the importance of collaborating with other organizations. The survey also provides a description of the history, structure, processes, outputs, and perceived strengths and weaknesses of existing organizations from which those establishing or leading similar organizations can draw

Characterization and applications of a Crimean-Congo hemorrhagic fever virus nucleoprotein-specific Affimer: Inhibitory effects in viral replication and development of colorimetric diagnostic tests

Crimean-Congo hemorrhagic fever orthonairovirus (CCHFV) is one of the most widespread medically important arboviruses, causing human infections that result in mortality rates of up to 60%. We describe the selection of a high-affinity small protein (Affimer-NP) that binds specifically to the nucleoprotein (NP) of CCHFV. We demonstrate the interference of Affimer-NP in the RNA-binding function of CCHFV NP using fluorescence anisotropy, and its inhibitory effects on CCHFV gene expression in mammalian cells using a mini-genome system. Solution of the crystallographic structure of the complex formed by these two molecules at 2.84 Å resolution revealed the structural basis for this interference, with the Affimer-NP binding site positioned at the critical NP oligomerization interface. Finally, we validate the in vitro application of Affimer-NP for the development of enzyme-linked immunosorbent and lateral flow assays, presenting the first published point-of-care format test able to detect recombinant CCHFV NP in spiked human and animal sera

Human skeletal muscle plasmalemma alters its structure to change its Ca2+-handling following heavy-load resistance exercise

High-force eccentric exercise results in sustained increases in cytoplasmic Ca2+ levels ([Ca2+]cyto), which can cause damage to the muscle. Here we report that a heavy-load strength training bout greatly alters the structure of the membrane network inside the fibres, the tubular (t-) system, causing the loss of its predominantly transverse organization and an increase in vacuolation of its longitudinal tubules across adjacent sarcomeres. The transverse tubules and vacuoles displayed distinct Ca2+-handling properties. Both t-system components could take up Ca2+ from the cytoplasm but only transverse tubules supported store-operated Ca2+ entry. The retention of significant amounts of Ca2+ within vacuoles provides an effective mechanism to reduce the total content of Ca2+ within the fibre cytoplasm. We propose this ability can reduce or limit resistance exercise-induced, Ca2+-dependent damage to the fibre by the reduction of [Ca2+]cyto to help maintain fibre viability during the period associated with delayed onset muscle soreness

Chems4EU: chemsex use and its impacts across four European countries in HIV-positive men who have sex with men attending HIV services

Introduction: Chemsex in a European context is the use of any of the following drugs to facilitate sex: crystal methamphetamine, mephedrone and gamma-hydroxybutyrate (GHB)/gamma-butyrolactone (GBL) and, to a lesser extent, cocaine and ketamine. This study describes the prevalence of self-reported recreational drug use and chemsex in HIV-positive men who have sex with men (MSM) accessing HIV services in four countries. It also examines the problematic impacts and harms of chemsex and access to chemsex-related services. Methods: This is a cross-sectional multi-centre questionnaire study of HIV-positive MSM accessing nine HIV services in the UK, Spain, Greece and Italy. Results: In all, 1589 HIV-positive MSM attending HIV services in four countries completed the questionnaire. The median age of participants was 38 years (interquartile range: 32–46 years) and 1525 (96.0%) were taking antiretroviral therapy (ART). In the previous 12 months, 709 (44.6%) had used recreational drugs, 382 (24.0%) reported chemsex and 104 (6.5%) reported injection of chemsex-associated drugs (‘slamsex’). Of the 382 engaging in chemsex, 155 (40.6%) reported unwanted side effects as a result of chemsex and 81 (21.2%) as a result of withdrawal from chemsex. The reported negative impacts from chemsex were on work (25.1%, 96), friends/family (24.3%, 93) and relationships (28.3%, 108). Fifty-seven (14.9%) accessed chemsex-related services in the past year, 38 of whom (67%) felt the service met their needs. Discussion: A quarter of participants self-reported chemsex in the past 12 months. There were high rates of harms from chemsex across all countries, including negative impacts on work, friends/family and relationships. Although a minority of those engaging in chemsex accessed support, most found this useful

Single electron emission in two-phase xenon with application to the detection of coherent neutrino-nucleus scattering

We present an experimental study of single electron emission in ZEPLIN-III, a two-phase xenon experiment built to search for dark matter WIMPs, and discuss applications enabled by the excellent signal-to-noise ratio achieved in detecting this signature. Firstly, we demonstrate a practical method for precise measurement of the free electron lifetime in liquid xenon during normal operation of these detectors. Then, using a realistic detector response model and backgrounds, we assess the feasibility of deploying such an instrument for measuring coherent neutrino-nucleus elastic scattering using the ionisation channel in the few-electron regime. We conclude that it should be possible to measure this elusive neutrino signature above an ionisation threshold of $\sim$3 electrons both at a stopped pion source and at a nuclear reactor. Detectable signal rates are larger in the reactor case, but the triggered measurement and harder recoil energy spectrum afforded by the accelerator source enable lower overall background and fiducialisation of the active volume