Characterization of Systemic Disease Development and Paw Inflammation in a Susceptible Mouse Model of Mayaro Virus Infection and Validation Using X-ray Synchrotron Microtomography

Mayaro virus (MAYV) is an emerging arthropod-borne virus endemic in Latin America and the causative agent of arthritogenic febrile disease. Mayaro fever is poorly understood; thus, we established an in vivo model of infection in susceptible type-I interferon receptor-deficient mice (IFNAR−/−) to characterize the disease. MAYV inoculations in the hind paws of IFNAR−/− mice result in visible paw inflammation, evolve into a disseminated infection and involve the activation of immune responses and inflammation. The histological analysis of inflamed paws indicated edema at the dermis and between muscle fibers and ligaments. Paw edema affected multiple tissues and was associated with MAYV replication, the local production of CXCL1 and the recruitment of granulocytes and mononuclear leukocytes to muscle. We developed a semi-automated X-ray microtomography method to visualize both soft tissue and bone, allowing for the quantification of MAYV-induced paw edema in 3D with a voxel size of 69 µm3. The results confirmed early edema onset and spreading through multiple tissues in inoculated paws. In conclusion, we detailed features of MAYV-induced systemic disease and the manifestation of paw edema in a mouse model extensively used to study infection with alphaviruses. The participation of lymphocytes and neutrophils and expression of CXCL1 are key features in both systemic and local manifestations of MAYV disease

Development and validation of fluorescence live-cell imaging approaches to study flavivirus infection kinetics in animal cells

The genus Flavivirus in the family Flaviviridae comprises many clinically important viruses, such as dengue virus (DENV), Zika virus (ZIKV), and yellow fever virus (YFV). The quest for therapeutic targets to combat flavivirus infections requires a better understanding of the kinetics of the virus-cell interplay during infections with wild-type viral strains. Nevertheless, this is hindered by limitations of the current cell-based systems for monitoring flavivirus infection by live-cell imaging. The present dissertation describes the development and validation of fluorescence-activatable sensors to detect the activity of flavivirus NS2B-NS3 serine proteases in living cells. The system consists of green fluorescent protein (GFP)-based reporters that become fluorescent upon cleavage by recombinant DENV-2/ZIKV proteases in vitro. A version of this sensor containing the flavivirus internal NS3 cleavage site linker (AAQRRGRIG) reported the highest fluorescence activation in stably transduced mammalian cells upon DENV-2/ZIKV infection. The onset of fluorescence correlated with viral protease activity. Moreover, a far-red version of this flavivirus sensor presented the best signal-to-noise ratio in a fluorescent Dulbecco’s plaque assay, leading to the construction of a multireporter platform combining the flavivirus sensor with DNA fluorescent dyes for the detection of virus-induced chromatin condensation and cell death (cytophatic effect labeling). This enabled studies of viral plaque formation with a single-cell resolution. This cytopathic effect labeling approach was conceptualized and validated during the present work. Finally, the application of the multireporter platform also enabled the study of kinetics of infection and cytophatic effect induction by DENV-2, ZIKV, and YFV in cell-subpopulations. We anticipate that future studies of viral infection kinetics with our reporter systems will enable basic investigations of virus-host cell interactions and will also facilitate the screening of antiviral drugs to manage flavivirus infections.El género Flavivirus de la familia Flaviviridae incluye muchos virus de importancia médica, como el virus del dengue (DENV), el virus Zika (ZIKV) y el virus de la fiebre amarilla (YFV). La búsqueda de blancos terapéuticos para combatir las afecciones causadas por flavivirus requiere un mejor entendimiento de la cinética de interacción virus-célula durante las infecciones con cepas virales silvestres. Sin embargo, esto se ve obstaculizado por las limitaciones de los sistemas celulares actuales para monitorear la infección por flavivirus mediante imagenología de células vivas. La presente tesis describe el desarrollo y validación de sensores fluorescentes activables para detectar la actividad de la serin proteasa flaviviral NS2B-NS3 en células vivas. El sistema consta de reporteros basados en la proteína verde fluorescente (GFP) que activan la fluorescencia al ser cortados por proteasas recombinantes de DENV-2/ZIKV in vitro. Tras la infección por DENV-2/ZIKV, una versión de este sensor que contiene el sitio de corte interno de la proteína NS3 de flavivirus (AAQRRGRIG) reportó la mayor activación de fluorescencia en células de mamífero transducidas de manera estable. La activación de la fluorescencia correlacionó con la actividad de la proteasa viral. Además, una versión de color rojo lejano de este sensor de flavivirus presentó la mejor relación señal/ruido en un ensayo de placas de Dulbecco fluorescentes, lo que llevó a la construcción de una plataforma multireportero que combina el sensor de flavivirus con sondas fluorescentes de intercalado en el ADN para la detección de condensación de cromatina y muerte celular inducida por el virus (marcaje del efecto citopático). Esto permitió realizar estudios de formación de placas virales con resolución a nivel de células individuales. Dicho abordaje para el marcaje del efecto citopático fue conceptualizado y validado durante el presente trabajo. Finalmente, la aplicación de la plataforma multireportero también posibilitó el estudio de la cinética de infección a nivel de subpoblaciones celulares, así como de la inducción del efecto citopático por DENV-2, ZIKV y YFV. Anticipamos que estudios futuros de la cinética de infección viral con nuestros sistemas reporteros permitirán investigaciones básicas de la interacción virus-célula huésped y facilitarán el tamizaje de fármacos antivirales para controlar las infecciones por flavivirus.International Centre for Genetic Engineering and Biotechnology (ICGEB)UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Centro de Investigación en Enfermedades Tropicales (CIET)UCR::Vicerrectoría de Docencia::Salud::Facultad de MicrobiologíaUCR::Vicerrectoría de Investigación::Sistema de Estudios de Posgrado::Interdisciplinarias::Doctorado Académico en Ciencia

A beta-herpesvirus with fluorescent capsids to study transport in living cells.

Fluorescent tagging of viral particles by genetic means enables the study of virus dynamics in living cells. However, the study of beta-herpesvirus entry and morphogenesis by this method is currently limited. This is due to the lack of replication competent, capsid-tagged fluorescent viruses. Here, we report on viable recombinant MCMVs carrying ectopic insertions of the small capsid protein (SCP) fused to fluorescent proteins (FPs). The FPs were inserted into an internal position which allowed the production of viable, fluorescently labeled cytomegaloviruses, which replicated with wild type kinetics in cell culture. Fluorescent particles were readily detectable by several methods. Moreover, in a spread assay, labeled capsids accumulated around the nucleus of the newly infected cells without any detectable viral gene expression suggesting normal entry and particle trafficking. These recombinants were used to record particle dynamics by live-cell microscopy during MCMV egress with high spatial as well as temporal resolution. From the resulting tracks we obtained not only mean track velocities but also their mean square displacements and diffusion coefficients. With this key information, we were able to describe particle behavior at high detail and discriminate between particle tracks exhibiting directed movement and tracks in which particles exhibited free or anomalous diffusion

3D Reconstruction of VZV Infected Cell Nuclei and PML Nuclear Cages by Serial Section Array Scanning Electron Microscopy and Electron Tomography

Varicella-zoster virus (VZV) is a human alphaherpesvirus that causes varicella (chickenpox) and herpes zoster (shingles). Like all herpesviruses, the VZV DNA genome is replicated in the nucleus and packaged into nucleocapsids that must egress across the nuclear membrane for incorporation into virus particles in the cytoplasm. Our recent work showed that VZV nucleocapsids are sequestered in nuclear cages formed from promyelocytic leukemia protein (PML) in vitro and in human dorsal root ganglia and skin xenografts in vivo. We sought a method to determine the three-dimensional (3D) distribution of nucleocapsids in the nuclei of herpesvirus-infected cells as well as the 3D shape, volume and ultrastructure of these unique PML subnuclear domains. Here we report the development of a novel 3D imaging and reconstruction strategy that we term Serial Section Array-Scanning Electron Microscopy (SSA-SEM) and its application to the analysis of VZV-infected cells and these nuclear PML cages. We show that SSA-SEM permits large volume imaging and 3D reconstruction at a resolution sufficient to localize, count and distinguish different types of VZV nucleocapsids and to visualize complete PML cages. This method allowed a quantitative determination of how many nucleocapsids can be sequestered within individual PML cages (sequestration capacity), what proportion of nucleocapsids are entrapped in single nuclei (sequestration efficiency) and revealed the ultrastructural detail of the PML cages. More than 98% of all nucleocapsids in reconstructed nuclear volumes were contained in PML cages and single PML cages sequestered up to 2,780 nucleocapsids, which were shown by electron tomography to be embedded and cross-linked by an filamentous electron-dense meshwork within these unique subnuclear domains. This SSA-SEM analysis extends our recent characterization of PML cages and provides a proof of concept for this new strategy to investigate events during virion assembly at the single cell level

Investigations on influenza A virus morphology

Clinical isolates of influenza A virus (IAV) typically form a pleomorphic population of virions that present as a continuum of morphologies broadly classified as filaments, bacilli, and spheres. Laboratory strains of IAV, however present mainly as spherical and bacilliform particles, suggesting a role for filaments in vivo. How these filaments form is not fully understood, but it has previously been shown that mutations in the viral matrix protein (M1) can be determinants of filament formation. In this work we show that filament formation also depends on multiple other genetic factors. To this end, we compared two IAV strains A/equine/Ohio/03 (O/2003) and A/equine/South Africa/4/03 (SA/2003) and found that SA/2003 could form filaments while O/2003 could not, despite no differences in their M1 sequences. To map the genetic basis of this difference, we generated reassortant viruses between O/2003 and SA/2003 and identified segments 1 (encoding polymerase basic protein 2, PB2), 4 (haemagglutinin, HA) and 6 (neuraminidase, NA) as determinants of morphology. We established that single mutations in segments 4 and 6, which alter the HA and NA proteins, alter virion morphology. To our surprise, we also identified three synonymous mutations in segment 1 of the virus that were determinants of filament formation despite not altering any known protein. We then extended this work to unravel the associated mechanisms of this change and found despite some differences in the activity of NA, contribution of HA to filament production, and differences in segment 1 RNA structure, there was no clear underlying mechanism. Given, that we were unable to identify the mechanisms associated with the change in morphology, we further extended this work to identify the factors involved in morphogenesis. To characterize IAV filament morphogenesis we employed cryogenic electron tomography (Cryo-ET) of vitrified equine fibroblasts (E. Derm). Although we were unable to identify any additional factors associated with IAV budding, we were able to generate a robust pipeline for studying filament formation. These results show that M1 is not the only determinant of IAV morphology, and that the ability to form filaments, a poorly studied but natural characteristic of IAV infection, is in fact modulated by multiple proteins and RNA determinants

Label-Free Digital Holo-tomographic Microscopy Reveals Virus-Induced Cytopathic Effects in Live Cells

Cytopathic effects (CPEs) are a hallmark of infections. CPEs are difficult to observe due to phototoxicity from classical light microscopy. We report distinct patterns of virus infections in live cells using digital holo-tomographic microscopy (DHTM). DHTM is label-free and records the phase shift of low-energy light passing through the specimen on a transparent surface with minimal perturbation. DHTM measures the refractive index (RI) and computes the refractive index gradient (RIG), unveiling optical heterogeneity in cells. We find that vaccinia virus (VACV), herpes simplex virus (HSV), and rhinovirus (RV) infections progressively and distinctly increased RIG. VACV infection, but not HSV and RV infections, induced oscillations of cell volume, while all three viruses altered cytoplasmic membrane dynamics and induced apoptotic features akin to those caused by the chemical compound staurosporine. In sum, we introduce DHTM for quantitative label-free microscopy in infection research and uncover virus type-specific changes and CPE in living cells with minimal interference. // IMPORTANCE This study introduces label-free digital holo-tomographic microscopy (DHTM) and refractive index gradient (RIG) measurements of live, virus-infected cells. We use DHTM to describe virus type-specific cytopathic effects, including cyclic volume changes of vaccinia virus infections, and cytoplasmic condensations in herpesvirus and rhinovirus infections, distinct from apoptotic cells. This work shows for the first time that DHTM is suitable to observe virus-infected cells and distinguishes virus type-specific signatures under noninvasive conditions. It provides a basis for future studies, where correlative fluorescence microscopy of cell and virus structures annotate distinct RIG values derived from DHTM

Artificial intelligence-based preventive, personalized and precision medicine for cardiovascular disease/stroke risk assessment in rheumatoid arthritis patients: a narrative review

The challenges associated with diagnosing and treating cardiovascular disease (CVD)/Stroke in Rheumatoid arthritis (RA) arise from the delayed onset of symptoms. Existing clinical risk scores are inadequate in predicting cardiac events, and conventional risk factors alone do not accurately classify many individuals at risk. Several CVD biomarkers consider the multiple pathways involved in the development of atherosclerosis, which is the primary cause of CVD/Stroke in RA. To enhance the accuracy of CVD/Stroke risk assessment in the RA framework, a proposed approach involves combining genomic-based biomarkers (GBBM) derived from plasma and/or serum samples with innovative non-invasive radiomic-based biomarkers (RBBM), such as measurements of synovial fluid, plaque area, and plaque burden. This review presents two hypotheses: (i) RBBM and GBBM biomarkers exhibit a significant correlation and can precisely detect the severity of CVD/Stroke in RA patients. (ii) Artificial Intelligence (AI)-based preventive, precision, and personalized (aiP3) CVD/Stroke risk AtheroEdge™ model (AtheroPoint™, CA, USA) that utilizes deep learning (DL) to accurately classify the risk of CVD/stroke in RA framework. The authors conducted a comprehensive search using the PRISMA technique, identifying 153 studies that assessed the features/biomarkers of RBBM and GBBM for CVD/Stroke. The study demonstrates how DL models can be integrated into the AtheroEdge™–aiP3 framework to determine the risk of CVD/Stroke in RA patients. The findings of this review suggest that the combination of RBBM with GBBM introduces a new dimension to the assessment of CVD/Stroke risk in the RA framework. Synovial fluid levels that are higher than normal lead to an increase in the plaque burden. Additionally, the review provides recommendations for novel, unbiased, and pruned DL algorithms that can predict CVD/Stroke risk within a RA framework that is preventive, precise, and personalized. © 2023, The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature

Mitochondrial dynamics: regulation of insulin secretion and novel quantification methods

The recent surge in Type 2 Diabetes (T2D) has renewed interest in the study of cellular metabolism – which mitochondria tightly control. Previous work has shown mitochondrial dysfunction plays a critical role in the development of metabolic diseases, such as T2D. The pancreatic β-cell synthesizes and secretes insulin in vivo in response to diverse fuel signals such as glucose, fatty acids, and amino acids; failure or loss of β-cell mass is a hallmark of T2D. Pancreatic β-cell mitochondria are dynamic organelles living a life of fusion, fission, and movement collectively called mitochondrial dynamics. Mitochondrial fusion is impaired in obesity and models of obesity, while basal secretion of insulin is elevated. Previous studies demonstrate that hyperinsulinemia alone is sufficient to induce insulin resistance, yet the relationship between mitochondrial morphology and basal insulin secretion has not yet been studied. Here, we investigated the link between loss of mitochondrial fusion and insulin secretion at basal glucose concentrations by reducing the expression of mitofusin 2 (Mfn2), which controls mitochondrial morphology and metabolism. We found that forced mitochondrial fragmentation caused increased insulin secretion at basal glucose concentrations. In addition, fragmentation of mitochondria enhanced the secretory response of islets to palmitate at nonstimulatory glucose concentrations and increased fatty acid uptake and oxidation in a cell model of pancreatic β-cells. We developed unique solutions to challenges posed by the measurement of mitochondrial dynamics via confocal microscopy by using novel image analysis techniques, including a novel method of mitochondrial segmentation. This technique also revealed novel biology of brown adipose tissue mitochondria dependent on their localization within the cell. Our findings demonstrate that changes to mitochondrial dynamics in the β-cell can lead to increased insulin secretion at basal glucose concentrations. These data support the possibility that hyperinsulinemia and the downstream outcome of insulin resistance can be initiated by altered mitochondrial function in the β-cell independently of other tissues. By uncovering a new process that governs basal insulin secretion, we provide novel targets for regulation, such as mitochondrial morphology or fatty acid induced insulin secretion that may present new approaches to treatment of diabetes