Structural and functional characterisation of feline calicivirus entry

The Caliciviridae are a group of small, non-enveloped viruses with a positive sense, single stranded RNA genome. Caliciviruses include the noroviruses, responsible for winter vomiting disease, as well as several important veterinary pathogens. Feline calicivirus (FCV) is an excellent model for studying calicivirus entry, having a known protein receptor and being readily propagated in cell culture. Here we explore calicivirus entry, using FCV. Virus entry is the critical first step of infection and is therefore an important area of study. Both alpha 2-6 linked sialic acid and feline junctional adhesion molecule A (fJAM-A) have been identified as receptors for FCV. The attachment of FCV to fJAM-A, is followed by uptake via clathrin mediated endocytosis. Little is known, however, on the viral escape mechanism leading to delivery of the viral RNA into the cytoplasm. We set out to explore the nature of FCV attachment and uncoating using structural, biochemical and biophysical analyses. By cryogenic electron microscopy we have characterized the virus-receptor interaction at high-resolution. Using electron microscopy and an RNA release assay, we have investigated virion uncoating. Finally, we have explored the importance of receptor glycosylation, and oligomerisation. Our analysis has allowed us to construct an atomic model of the major capsid protein VP1. Upon binding to fJAM-A, FCV undergoes a conformational change (rotation and tilting of the capsomeres). Flexibility in the receptor decorated virion has prevented high-resolution structure analysis of the conformational change or the virus-receptor interaction. We have, however, seen that the structural changes are limited to the capsid spikes. We hypothesised that the conformational change may be a priming step that would prepare the virus for uncoating upon internalisation. We found that upon lowering the pH below 5, receptor decorated virions disassembled, supporting this hypothesis. Disassembly of the virus-receptor complex at low pH presented a tool for estimating the quantity of receptor needed to prime the capsid for uncoating. Cryo-EM studies reveal that FCV bound fJAM-A is monomeric although the receptor was found to be dimeric in solution as previously described for the human and murine homologues. Furthermore, it is hypothesised that this is the form found at tight junctions between cells. We propose that disruption of fJAM-A homodimers may be the mechanism by which induction of viral uptake by endocytosis is triggered. Finally, we have confirmed the presence of an N-linked glycosylation on fJAM-A and show that the removal of this carbohydrate moiety does not affect viral binding in vitro

Consultation patterns and clinical correlates of consultation in a tertiary care setting

<p>Abstract</p> <p>Background</p> <p>Consultation in hospital is an essential tool for acquiring subspecialty support when managing patients. There is limited knowledge on the utilization of subspecialty consultation from hospital based general internists. Consultation patterns to medical subspecialists and the patient factors that may influence consultation are reported for general medical services.</p> <p>Methods and findings</p> <p>Hospital discharge data were obtained for patients from medical services over a 2-year period. Consultations requested to medicine subspecialties were identified, and then reported by type and frequency. Information on demographic factors, clinical diagnoses, length of stay (LOS), time in critical care units, and disposition were compared for patients with and without consultation.</p> <p>3979 patients were hospitalized during the study and 2885 consultations occurred. Almost half of the patients received at least one consultation (48.3%). Gastroenterology (26.3%), infectious diseases (14.6%) and respirology (13.6%) were the most frequently consulted services. Patients with consultation had a greater number of total diagnoses (7.3 vs. 5.5, P < 0.001), a greater mean LOS (15.9 vs. 6.8 days), were more likely to spend time in the ICU (11.5% vs. 3.5%) and CCU (4.3% vs. 1.2%), and to expire in hospital (10.7% vs. 4.9%).</p> <p>Conclusion</p> <p>Consultation occurs frequently and its presence is an indicator of patient complexity and high use of health system resources. Analysis of consultation patterns for specific patient populations could assist in optimizing efficiency in health care delivery. Targeting quality improvement strategies toward optimizing consultation processes, engaging heavily utilized subspecialties in educational roles and assisting with resource planning are areas for future consideration.</p

Experimental Investigations of Micro Rotor-Tip Vortices

The focus of this project is to run a micro-rotor at varying RPMs, analyzing the flow underneath the blades and above the ground based on flow visualization. This study is related to the understanding of the factors affecting the stability and performance of a rotorcraft. A variety of factors are manipulated, which includes the RPM, blade angle of attack, and ground effect presence. The investigation involves using Planar Laser Imaging where a laser is directed into a test chamber (in our case, the test chamber is filled with smoke) and is matched with a high-speed camera to capture flow visualizations in a non-intrusive, relatively simple manner. Our data primarily consists of flow field velocity measurements, force measurements, and flow field visualization. The flow visualization and velocity data were obtained via the high-speed camera and an accompanying laser system that passed through a diverging lens to illuminate the smoke. Particle Image Velocimetry (PIV) has been performed on the rotor wake, and the preliminary flow field analysis is also presented. Current activities include torque measurements and PIV at varying angles

Vesivirus 2117 capsids more closely resemble sapovirus and lagovirus particles than other known vesivirus structures

Vesivirus 2117 is an adventitious agent that in 2009, was identified as a contaminant of CHO cells propagated in bioreactors at a pharmaceutical manufacturing plant belonging to Genzyme. The consequent interruption in supply of Fabrazyme and Cerezyme (drugs used to treat Fabry and Gaucher disease respectively), caused significant economic losses. Vesivirus 2117 is a member of the Caliciviridae; a family of small icosahedral viruses encoding a positive sense RNA genome. We have used cryo-electron microscopy and three dimensional image reconstruction to calculate a structure of vesivirus 2117 virus like particles as well as feline calicivirus and a chimeric sapovirus. We present a structural comparison of several members of the Caliciviridae, showing that the distal P domain of vesivirus 2117 is morphologically distinct from that seen in other known vesivirus structures. Furthermore, at intermediate resolutions we found a high level of structural similarity between vesivirus 2117 and Caliciviridae from other genera, such as sapovirus and rabbit haemorrhagic disease virus. Phylogenetic analysis confirms vesivirus 2117 as a vesivirus closely related to canine vesiviruses. We postulate that morphological differences in virion structure seen between vesivirus clades may reflect differences in receptor usage

Calicivirus VP2 forms a portal-like assembly following receptor engagement

To initiate infection, many viruses enter their host cells by triggering endocytosis following receptor engagement. However, the mechanisms by which non-enveloped viruses escape the endosome are poorly understood. Here we present near-atomic-resolution cryo-electron microscopy structures for feline calicivirus both undecorated and labelled with a soluble fragment of its cellular receptor, feline junctional adhesion molecule A. We show that VP2, a minor capsid protein encoded by all caliciviruses1,2, forms a large portal-like assembly at a unique three-fold axis of symmetry, following receptor engagement. This assembly—which was not detected in undecorated virions—is formed of twelve copies of VP2, arranged with their hydrophobic N termini pointing away from the virion surface. Local rearrangement at the portal site leads to the opening of a pore in the capsid shell. We hypothesize that the portal-like assembly functions as a channel for the delivery of the calicivirus genome, through the endosomal membrane, into the cytoplasm of a host cell, thereby initiating infection. VP2 was previously known to be critical for the production of infectious virus3; our findings provide insights into its structure and function that advance our understanding of the Caliciviridae

Microwave hyperthermia represses human papillomavirus oncoprotein activity and induces cell death due to cell stress in 3D tissue models of anogenital precancers and cancers

Background: Hyperthermia is a well-accepted cancer therapy. Microwaves provide a very precise, targeted means of hyperthermia and are currently used to treat plantar warts caused by cutaneous-infective human papillomaviruses (HPVs). Other HPV genotypes infecting the anogenital mucosa cause genital warts or preneoplastic lesions or cervical cancer. Effective, non-ablative therapies for these morbid HPV-associated lesions are lacking. Methods: The molecular consequences of microwave treatment were investigated in in vitro cultured three-dimensional HPV-positive cervical tumour tissues, and tissues formed from HPV-infected normal immortalised keratinocytes. Microwave energy delivery to tissues was quantified. Quantitative reverse transcriptase PCR was used to quantify mRNA expression. Immunohistochemistry and fluorescence immunostaining was used to assess protein expression. Findings: Microwave energy deposition induced sustained, localised cell death at the treatment site. There was a downregulation in levels of HPV oncoproteins E6 and E7 alongside a reduction in cellular growth/proliferation and induction of apoptosis/autophagy. HSP70 expression confirmed hyperthermia, concomitant with induction of translational stress. Interpretation: The data suggest that microwave treatment inhibits tumour cell proliferation and allows the natural apoptosis of HPV-infected cells to resume. Precision microwave delivery presents a potential new treatment for treating HPV-positive anogenital precancerous lesions and cancers. Funding: Funding was through an Innovate UK Biomedical Catalyst grant (ID# 92138-556187), a Chief Scientist Office grant (TCS/19/11) and core support from Medical Research Council (MC_ UU_12014) core funding for the MRC-University of Glasgow Centre for Virus Research

Calicivirus VP2 forms a portal-like assembly following receptor engagement.

To initiate infection, many viruses enter their host cells by triggering endocytosis following receptor engagement. However, the mechanisms by which non-enveloped viruses escape the endosome are poorly understood. Here we present near-atomic-resolution cryo-electron microscopy structures for feline calicivirus both undecorated and labelled with a soluble fragment of its cellular receptor, feline junctional adhesion molecule A. We show that VP2, a minor capsid protein encoded by all caliciviruses1,2, forms a large portal-like assembly at a unique three-fold axis of symmetry, following receptor engagement. This assembly-which was not detected in undecorated virions-is formed of twelve copies of VP2, arranged with their hydrophobic N termini pointing away from the virion surface. Local rearrangement at the portal site leads to the opening of a pore in the capsid shell. We hypothesize that the portal-like assembly functions as a channel for the delivery of the calicivirus genome, through the endosomal membrane, into the cytoplasm of a host cell, thereby initiating infection. VP2 was previously known to be critical for the production of infectious virus3; our findings provide insights into its structure and function that advance our understanding of the Caliciviridae.IG is a Wellcome Senior Fellow (Ref: 207498/Z/17/Z). M.J.C. was supported by a PhD studentship from the UK Biotechnology and Biological Sciences Research Council (BBSRC WestBIO DTP: BB/J013854/1). D.B and M.M. are supported by the UK Medical Research Council (MC_UU_12014/7)

Helical ordering of envelope‐associated proteins and glycoproteins in respiratory syncytial virus

Human respiratory syncytial virus (RSV) causes severe respiratory illness in children and the elderly. Here, using cryogenic electron microscopy and tomography combined with computational image analysis and three-dimensional reconstruction, we show that there is extensive helical ordering of the envelope-associated proteins and glycoproteins of RSV filamentous virions. We calculated a 16 Å resolution sub-tomogram average of the matrix protein (M) layer that forms an endoskeleton below the viral envelope. These data define a helical lattice of M-dimers, showing how M is oriented relative to the viral envelope. Glycoproteins that stud the viral envelope were also found to be helically ordered, a property that was coordinated by the M-layer. Furthermore, envelope glycoproteins clustered in pairs, a feature that may have implications for the conformation of fusion (F) glycoprotein epitopes that are the principal target for vaccine and monoclonal antibody development. We also report the presence, in authentic virus infections, of N-RNA rings packaged within RSV virions. These data provide molecular insight into the organisation of the virion and the mechanism of its assembly

Seismic shear-wave structure of the upper mantle beneath the Mohns Ridge

M.S. University of Hawaii at Manoa 2011.Includes bibliographical references.Crust produced at mid-ocean ridges with full spreading rates less than ~20 mm/yr is observed to be only 0-4 km thick, well below the global average of 6-7 km for oceanic crust produced at faster spreading rates. The origin of this difference is unknown, but is speculated to result from a thicker thermal boundary layer at the axis of the slower spreading ridges that either inhibits shallow melting or melt extraction. We present an analysis of regional broadband data, predominately Love and Rayleigh waves, collected along the very slow-spreading Mohns Ridge (where crustal thickness is ~4 km) in the Norwegian-Greenland Sea. The seismic data constrain lithospheric and asthenospheric velocities for lithospheric ages from 0-25 Ma. We find lithospheric thickness to closely match the prediction of a simple ridge half-space thermal model (via the temperature and pressure effects on the seismic properties of mantle materials) and asthenospheric shear wave velocities to be consistent with this thermal model plus <2% melt at the youngest ages. Just at the top of the mantle, a thin zone with velocities intermediate between those of mantle and gabbroic rocks suggests that some melt is frozen into the mantle as might occur if a thin axial lithospheric lid inhibits melt extraction from the mantle and the melt is subsequently frozen into the mantle. While this explains the thin crust, the possibility that hydrothermal alteration produces the low mantle velocities cannot be ruled out