Genetic reconstitution of the human Adenovirus type 2 temperature-sensitive 1 mutant defective in endosomal escape

Human Adenoviruses infect the upper and lower respiratory tracts, the urinary and digestive tracts, lymphoid systems and heart, and give rise to epidemic conjunctivitis. More than 51 human serotypes have been identified to-date, and classified into 6 species A-F. The species C Adenoviruses Ad2 and Ad5 (Ad2/5) cause upper and lower respiratory disease, but how viral structure relates to the selection of particular infectious uptake pathways is not known. An adenovirus mutant, Ad2-ts1 had been isolated upon chemical mutagenesis in the past, and shown to have unprocessed capsid proteins. Ad2-ts1 fails to package the viral protease L3/p23, and Ad2-ts1 virions do not efficiently escape from endosomes. It had been suggested that the C22187T point mutation leading to the substitution of the conserved proline 137 to leucine (P137L) in the L3/p23 protease was at least in part responsible for this phenotype. To clarify if the C22187T mutation is necessary and sufficient for the Ad2-ts1 phenotype, we sequenced the genes encoding the structural proteins of Ad2-ts1, and confirmed that the Ad2-ts1 DNA carries the point mutation C22187T. Introduction of C22187T to the wild-type Ad2 genome in a bacterial artificial chromosome (Ad2-BAC) gave Ad2-BAC46 virions with the full Ad2-ts1 phenotype. Reversion of Ad2-BAC46 gave wild-type Ad2 particles indicating that P137L is necessary and sufficient for the Ad2-ts1 phenotype. The kinetics of Ad2-ts1 uptake into cells were comparable to Ad2 suggesting similar endocytic uptake mechanisms. Surprisingly, infectious Ad2 or Ad5 but not Ad2-ts1 uptake required CALM (clathrin assembly lymphoid myeloid protein), which controls clathrin-mediated endocytosis and membrane transport between endosomes and the trans-Golgi-network. The data show that no other mutations than P137L in the viral protease are necessary to give rise to particles that are defective in capsid processing and endosomal escape. This provides a basis for genetic analyses of distinct host requirements for Ad endocytosis and escape from endosomes

A standardised bioassay method using a bench‐top spray tower to evaluate entomopathogenic fungi for control of the greenhouse whitefly, Trialeurodes vaporariorum

BACKGROUND: Bioassays evaluating entomopathogenic fungi (EPF) isolates for effective microbial control of whitefly are a fundamental part of the screening process for bioprotectants, but development of repeatable, robust bioassays is not straightforward. Currently, there is no readily available standardised method to test the efficacy of EPF on whitefly. Here, we describe the calibration and use of a spray tower to deliver a standardised protocol to assess EPF activity; the method was validated using 18 EPF from four genera in tests against greenhouse whitefly, Trialeurodes vaporariorum (Westwood). RESULTS: At 138 kPa, the sprayer delivered 0.062 mL mm−2 (620 L ha−1) and an even deposition of spray across the central 1590 mm2 of the spray area. Average conidial deposition for all EPF was 252 conidia mm−2 and equivalent to 2.5 × 1012 conidia ha−1 at an application concentration of 1 × 107 conidia mL−1. Conidial deposition of a test Beauveria bassiana suspension increased with increasing application concentration. Egg laying by T. vaporariorum adults was restricted to 177 mm2 using clip cages specifically designed to ensure that third‐instar T. vaporariorum received a uniform spray coverage. Nymphs occupied 373 ± 5 mm2 of the leaf after migrating during the first instar. Average T. vaporariorum mortality totaled 8–89% 14 days after application of 1 × 107 conidia mL−1 of each EPF isolate. CONCLUSION: Combining the calibrated sprayer and bioassay method provides a reliable, standardised approach to test the virulence of EPF against whitefly nymphs. This laboratory‐based assay is affordable, replicable and allows the user to alter the dose of conidia applied to the target

TLR7-mediated skin inflammation remotely triggers chemokine expression and leukocyte accumulation in the brain

Background: The relationship between the brain and the immune system has become increasingly topical as, although it is immune-specialised, the CNS is not free from the influences of the immune system. Recent data indicate that peripheral immune stimulation can significantly affect the CNS. But the mechanisms underpinning this relationship remain unclear. The standard approach to understanding this relationship has relied on systemic immune activation using bacterial components, finding that immune mediators, such as cytokines, can have a significant effect on brain function and behaviour. More rarely have studies used disease models that are representative of human disorders. Methods: Here we use a well-characterised animal model of psoriasis-like skin inflammation—imiquimod—to investigate the effects of tissue-specific peripheral inflammation on the brain. We used full genome array, flow cytometry analysis of immune cell infiltration, doublecortin staining for neural precursor cells and a behavioural read-out exploiting natural burrowing behaviour. Results: We found that a number of genes are upregulated in the brain following treatment, amongst which is a subset of inflammatory chemokines (CCL3, CCL5, CCL9, CXCL10, CXCL13, CXCL16 and CCR5). Strikingly, this model induced the infiltration of a number of immune cell subsets into the brain parenchyma, including T cells, NK cells and myeloid cells, along with a reduction in neurogenesis and a suppression of burrowing activity. Conclusions: These findings demonstrate that cutaneous, peripheral immune stimulation is associated with significant leukocyte infiltration into the brain and suggest that chemokines may be amongst the key mediators driving this response

Interoception and inflammation in psychiatric disorders

Despite a historical focus on neurally-mediated interoceptive signaling mechanisms, humoral (and even cellular) signals also play an important role in communicating bodily physiological state to the brain. These signaling pathways can perturb neuronal structure, chemistry and function leading to discrete changes in behavior. They are also increasingly implicated in the pathophysiology of psychiatric disorders. The importance of these humoral signaling pathways is perhaps most powerfully illustrated in the context of infection and inflammation. Here we provide an overview of how immune activation of neural and humoral interoceptive mechanisms interact to mediate discrete changes in brain and behavior and highlight how activation of these pathways at specific points in neural development may predispose to psychiatric disorder. As our mechanistic understanding of these interoceptive pathways continues to emerge it is revealing novel therapeutic targets, potentially heralding an exciting new era of immunotherapies in psychiatry

Contributions and complexities from the use of in-vivo animal models to improve understanding of human neuroimaging signals.

Many of the major advances in our understanding of how functional brain imaging signals relate to neuronal activity over the previous two decades have arisen from physiological research studies involving experimental animal models. This approach has been successful partly because it provides opportunities to measure both the hemodynamic changes that underpin many human functional brain imaging techniques and the neuronal activity about which we wish to make inferences. Although research into the coupling of neuronal and hemodynamic responses using animal models has provided a general validation of the correspondence of neuroimaging signals to specific types of neuronal activity, it is also highlighting the key complexities and uncertainties in estimating neural signals from hemodynamic markers. This review will detail how research in animal models is contributing to our rapidly evolving understanding of what human neuroimaging techniques tell us about neuronal activity. It will highlight emerging issues in the interpretation of neuroimaging data that arise from in-vivo research studies, for example spatial and temporal constraints to neuroimaging signal interpretation, or the effects of disease and modulatory neurotransmitters upon neurovascular coupling. We will also give critical consideration to the limitations and possible complexities of translating data acquired in the typical animals models used in this area to the arena of human fMRI. These include the commonplace use of anaesthesia in animal research studies and the fact that many neuropsychological questions that are being actively explored in humans have limited homologues within current animal models for neuroimaging research. Finally we will highlighting approaches, both in experimental animals models (e.g. imaging in conscious, behaving animals) and human studies (e.g. combined fMRI-EEG), that mitigate against these challenges