A fluorescent reporter system enables spatiotemporal analysis of host cell modification during herpes simplex virus-1 replication.

Herpesviruses are large and complex viruses that have a long history of coevolution with their host species. One important factor in the virus-host interaction is the alteration of intracellular morphology during viral replication with critical implications for viral assembly. However, the details of this remodeling event are not well understood, in part because insufficient tools are available to deconstruct this highly heterogeneous process. To provide an accurate and reliable method of investigating the spatiotemporal dynamics of virus-induced changes to cellular architecture, we constructed a dual-fluorescent reporter virus that enabled us to classify four distinct stages in the infection cycle of herpes simplex virus-1 at the single cell level. This timestamping method can accurately track the infection cycle across a wide range of multiplicities of infection. We used high-resolution fluorescence microscopy analysis of cellular structures in live and fixed cells in concert with our reporter virus to generate a detailed and chronological overview of the spatial and temporal reorganization during viral replication. The highly orchestrated and striking relocation of many organelles around the compartments of secondary envelopment during transition from early to late gene expression suggests that the reshaping of these compartments is essential for virus assembly. We furthermore find that accumulation of HSV-1 capsids in the cytoplasm is accompanied by fragmentation of the Golgi apparatus with potential impact on the late steps of viral assembly. We anticipate that in the future similar tools can be systematically applied for the systems-level analysis of intracellular morphology during replication of other viruses

Intermittent bulk release of human cytomegalovirus

Human Cytomegalovirus (HCMV) can infect a variety of cell types by using virions of varying glycoprotein compositions. It is still unclear how this diversity is generated, but spatio-temporally separated envelopment and egress pathways might play a role. So far, one egress pathway has been described in which HCMV particles are individually enveloped into small vesicles and are subsequently exocytosed continuously. However, some studies have also found enveloped virus particles inside multivesicular structures but could not link them to productive egress or degradation pathways. We used a novel 3D-CLEM workflow allowing us to investigate these structures in HCMV morphogenesis and egress at high spatio-temporal resolution. We found that multiple envelopment events occurred at individual vesicles leading to multiviral bodies (MViBs), which subsequently traversed the cytoplasm to release virions as intermittent bulk pulses at the plasma membrane to form extracellular virus accumulations (EVAs). Our data support the existence of a novel bona fide HCMV egress pathway, which opens the gate to evaluate divergent egress pathways in generating virion diversity

Impact of opioid-free analgesia on pain severity and patient satisfaction after discharge from surgery: multispecialty, prospective cohort study in 25 countries

Background: Balancing opioid stewardship and the need for adequate analgesia following discharge after surgery is challenging. This study aimed to compare the outcomes for patients discharged with opioid versus opioid-free analgesia after common surgical procedures.Methods: This international, multicentre, prospective cohort study collected data from patients undergoing common acute and elective general surgical, urological, gynaecological, and orthopaedic procedures. The primary outcomes were patient-reported time in severe pain measured on a numerical analogue scale from 0 to 100% and patient-reported satisfaction with pain relief during the first week following discharge. Data were collected by in-hospital chart review and patient telephone interview 1 week after discharge.Results: The study recruited 4273 patients from 144 centres in 25 countries; 1311 patients (30.7%) were prescribed opioid analgesia at discharge. Patients reported being in severe pain for 10 (i.q.r. 1-30)% of the first week after discharge and rated satisfaction with analgesia as 90 (i.q.r. 80-100) of 100. After adjustment for confounders, opioid analgesia on discharge was independently associated with increased pain severity (risk ratio 1.52, 95% c.i. 1.31 to 1.76; P < 0.001) and re-presentation to healthcare providers owing to side-effects of medication (OR 2.38, 95% c.i. 1.36 to 4.17; P = 0.004), but not with satisfaction with analgesia (beta coefficient 0.92, 95% c.i. -1.52 to 3.36; P = 0.468) compared with opioid-free analgesia. Although opioid prescribing varied greatly between high-income and low- and middle-income countries, patient-reported outcomes did not.Conclusion: Opioid analgesia prescription on surgical discharge is associated with a higher risk of re-presentation owing to side-effects of medication and increased patient-reported pain, but not with changes in patient-reported satisfaction. Opioid-free discharge analgesia should be adopted routinely

25th annual computational neuroscience meeting: CNS-2016

The same neuron may play different functional roles in the neural circuits to which it belongs. For example, neurons in the Tritonia pedal ganglia may participate in variable phases of the swim motor rhythms [1]. While such neuronal functional variability is likely to play a major role the delivery of the functionality of neural systems, it is difficult to study it in most nervous systems. We work on the pyloric rhythm network of the crustacean stomatogastric ganglion (STG) [2]. Typically network models of the STG treat neurons of the same functional type as a single model neuron (e.g. PD neurons), assuming the same conductance parameters for these neurons and implying their synchronous firing [3, 4]. However, simultaneous recording of PD neurons shows differences between the timings of spikes of these neurons. This may indicate functional variability of these neurons. Here we modelled separately the two PD neurons of the STG in a multi-neuron model of the pyloric network. Our neuron models comply with known correlations between conductance parameters of ionic currents. Our results reproduce the experimental finding of increasing spike time distance between spikes originating from the two model PD neurons during their synchronised burst phase. The PD neuron with the larger calcium conductance generates its spikes before the other PD neuron. Larger potassium conductance values in the follower neuron imply longer delays between spikes, see Fig. 17.Neuromodulators change the conductance parameters of neurons and maintain the ratios of these parameters [5]. Our results show that such changes may shift the individual contribution of two PD neurons to the PD-phase of the pyloric rhythm altering their functionality within this rhythm. Our work paves the way towards an accessible experimental and computational framework for the analysis of the mechanisms and impact of functional variability of neurons within the neural circuits to which they belong

Preferential Budding of Vesicular Stomatitis Virus from the Basolateral Surface of Polarized Epithelial Cells Is Not Solely Directed by Matrix Protein or Glycoprotein

Vesicular stomatitis virus has been shown to bud basolaterally, and the matrix protein, but not glycoprotein, was proposed to mediate this asymmetry. Using polarized T84 monolayers, we demonstrate that no single viral protein is sufficient for polarized budding. Particles are released from the apical and basolateral surfaces and are indistinguishable, indicating that there is no apical assembly defect. We propose that aspects of host cell polarity create a more efficient budding process at the basolateral surface

Moral injury, spiritual care and the role of chaplains: an exploratory scoping review of literature and resources

This scoping review considered the role of chaplains with regard to ‘moral injury’. Moral injury is gaining increasing notoriety. This is due to greater recognition that trauma (in its various forms) can cause much deeper inflictions and afflictions than just physiological or psychological harm, for there may also be wounds affecting the ‘soul’ that are far more difficult to heal—if at all. As part of a larger research program exploring moral injury, a scoping review of literature and other resources was implemented utilising Arksey and O’Malley’s scoping method (Int J Soc Res Methodol 8(1):19–32, 2005) to focus upon moral injury, spirituality (including religion) and chaplaincy. Of the total number of articles and/or resources noting the term ‘moral injury’ in relation to spiritual/religious issues (n\ua0=\ua0482), the results revealed 60 resources that specifically noted moral injury and chaplains (or other similar bestowed title). The majority of these resources were clearly positive about the role (or the potential role) of chaplains with regard to mental health issues and/or moral injury. The World Health Organization International Classification of Diseases: Australian Modification of Health Interventions to the International Statistical Classification of Diseases and related Health problems (10th revision, vol 3—WHO ICD-10-AM, Geneva, 2002), was utilised as a coding framework to classify and identify distinct chaplaincy roles and interventions with regard to assisting people with moral injury. Several recommendations are made concerning moral injury and chaplaincy, most particularly the need for greater research to be conducted

The brain parenchyma has a type I interferon response that can limit virus spread

The brain has a tightly regulated environment that protects neurons and limits inflammation, designated “immune privilege.” However, there is not an absolute lack of an immune response. We tested the ability of the brain to initiate an innate immune response to a virus, which was directly injected into the brain parenchyma, and to determine whether this response could limit viral spread. We injected vesicular stomatitis virus (VSV), a transsynaptic tracer, or naturally occurring VSV-derived defective interfering particles (DIPs), into the caudate–putamen (CP) and scored for an innate immune response and inhibition of virus spread. We found that the brain parenchyma has a functional type I interferon (IFN) response that can limit VSV spread at both the inoculation site and among synaptically connected neurons. Furthermore, we characterized the response of microglia to VSV infection and found that infected microglia produced type I IFN and uninfected microglia induced an innate immune response following virus injection

Temporal Proteomic Analysis of Herpes Simplex Virus 1 Infection Reveals Cell-Surface Remodeling via pUL56-Mediated GOPC Degradation.

Herpesviruses are ubiquitous in the human population and they extensively remodel the cellular environment during infection. Multiplexed quantitative proteomic analysis over the time course of herpes simplex virus 1 (HSV-1) infection was used to characterize changes in the host-cell proteome and the kinetics of viral protein production. Several host-cell proteins are targeted for rapid degradation by HSV-1, including the cellular trafficking factor Golgi-associated PDZ and coiled-coil motif-containing protein (GOPC). We show that the poorly characterized HSV-1 pUL56 directly binds GOPC, stimulating its ubiquitination and proteasomal degradation. Plasma membrane profiling reveals that pUL56 mediates specific changes to the cell-surface proteome of infected cells, including loss of interleukin-18 (IL18) receptor and Toll-like receptor 2 (TLR2), and that cell-surface expression of TLR2 is GOPC dependent. Our study provides significant resources for future investigation of HSV-host interactions and highlights an efficient mechanism whereby a single virus protein targets a cellular trafficking factor to modify the surface of infected cells