scholarly journals Modeling cell infection via virus-producing cells rather than free infectious virus significantly improves fits ofin vitroviral kinetic data

2019 ◽  
Author(s):  
Veronika Bernhauerová ◽  
Veronica V. Rezelj ◽  
Laura I. Levi ◽  
Marco Vignuzzi
Keyword(s):  
Mathematical Modeling ◽  
Experimental Data ◽  
Viral Load ◽  
Viral Replication ◽  
Mathematical Models ◽  
Zika Virus ◽  
Time Course ◽  
Replication Cycle ◽  
Infected Cells

AbstractChikungunya and Zika viruses are arthropod-borne viruses that pose significant threat to public health. Experimental data show that duringin vitroinfection both viruses exhibit qualitatively distinct replication cycle kinetics. Chikungunya viral load rapidly accumulates within the first several hours post infection whereas Zika virus begins to increase at much later times. We sought to characterize these qualitatively distinctin vitrokinetics of chikungunya and Zika viruses by fitting a family of mathematical models to time course viral load datasets. We demonstrate that the standard viral kinetic model, which considers that new infections result only from free virus penetrating susceptible cells, does not fit experimental data as well as a model in which the number of virus-infected cells is the primary determinant of infection rate. We provide biologically meaningful quantifications of the main viral kinetic parameters and show that our results support cell-to-cell or localized transmission as a significant contributor to viral infection with chikungunya and Zika viruses.ImportanceMathematical modeling has become a useful tool to tease out information about virus-host interactions and thus complements experimental work in characterizing and quantifying processes within viral replication cycle. Importantly, mathematical models can fill in incomplete data sets and identify key parameters of infection, provided the appropriate model is used. Thein vitrotime course dynamics of mosquito transmitted viruses, such as chikungunya and Zika, have not been studied by mathematical modeling and thus limits our knowledge about quantitative description of the individual determinants of viral replication cycle. This study employs dynamical modeling framework to show that the rate at which cells become virus-infected is proportional to the number or virus-infected cells rather than free extracellular virus in the milieu, a widely accepted assumption in models of viral infections. Using the refined mathematical model in combination with viral load data, we provide quantification of the main drivers of chikungunya and Zikain vitrokinetics. Together, our results bring quantitative understanding of the basic components of chikungunya and Zika virus dynamics.

scholarly journals Stage-Dependent Inhibition of HIV-1 Replication by Antiretroviral Drugs in Cell Culture

2009 ◽  
Vol 54 (3) ◽  
pp. 1047-1054 ◽  
Author(s):  
Daniel A. Donahue ◽  
Richard D. Sloan ◽  
Björn D. Kuhl ◽  
Tamara Bar-Magen ◽  
Susan M. Schader ◽  
...  
Keyword(s):  
Viral Load ◽  
Viral Replication ◽  
Reverse Transcription ◽  
Antiretroviral Drugs ◽  
Proteolytic Processing ◽  
Viral Inhibition ◽  
Dependent Inhibition ◽  
Infected Cells ◽  
Hiv 1

ABSTRACT Recent clinical trials have shown that the use of the HIV-1 integrase (IN) inhibitor raltegravir (RAL) results in drops in the viral load that are more rapid than those achieved by use of the reverse transcriptase (RT) inhibitor efavirenz. Previously, mathematical modeling of viral load decay that takes into account the stage of viral replication targeted by a drug has yielded data that closely approximate the clinical trial results. This model predicts greater inhibition of viral replication by drugs that act later in the viral replication cycle. In the present study, we have added drugs that target entry, reverse transcription, integration, or proteolytic processing to acutely infected cells and have shown modest viral inhibition by entry inhibitors, intermediate levels of inhibition by RT and IN inhibitors, and high levels of inhibition by protease inhibitors relative to the levels of growth for the no-drug controls. When dual or triple combinations of these drugs were added to acutely infected cells, we found that the levels of inhibition achieved by any given combination were comparable to those achieved by the latest-acting drug in the combination. In single-round infections in which the kinetics of reverse transcription and integration had been determined by quantitative PCR, addition of IN inhibitors at various times postinfection resulted in levels of inhibition equal to or greater than those achieved by addition of RT inhibitors. Collectively, our data provide in vitro evidence of the stage-dependent inhibition of HIV-1 by clinically relevant drugs. We discuss how stage-dependent inhibition helps to explain the unique viral load decay dynamics observed clinically with RAL.

scholarly journals Revisiting Ehrlichia ruminantium Replication Cycle Using Proteomics: The Host and the Bacterium Perspectives

2021 ◽  
Vol 9 (6) ◽  
pp. 1144
Author(s):  
Isabel Marcelino ◽  
Philippe Holzmuller ◽  
Ana Coelho ◽  
Gabriel Mazzucchelli ◽  
Bernard Fernandez ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Host Cell ◽  
Cell Metabolism ◽  
Replication Cycle ◽  
Host Cells ◽  
Ehrlichia Ruminantium ◽  
Host Interaction ◽  
Infected Cells ◽  
Related Proteins

The Rickettsiales Ehrlichia ruminantium, the causal agent of the fatal tick-borne disease Heartwater, induces severe damage to the vascular endothelium in ruminants. Nevertheless, E. ruminantium-induced pathobiology remains largely unknown. Our work paves the way for understanding this phenomenon by using quantitative proteomic analyses (2D-DIGE-MS/MS, 1DE-nanoLC-MS/MS and biotin-nanoUPLC-MS/MS) of host bovine aorta endothelial cells (BAE) during the in vitro bacterium intracellular replication cycle. We detect 265 bacterial proteins (including virulence factors), at all time-points of the E. ruminantium replication cycle, highlighting a dynamic bacterium–host interaction. We show that E. ruminantium infection modulates the expression of 433 host proteins: 98 being over-expressed, 161 under-expressed, 140 detected only in infected BAE cells and 34 exclusively detected in non-infected cells. Cystoscape integrated data analysis shows that these proteins lead to major changes in host cell immune responses, host cell metabolism and vesicle trafficking, with a clear involvement of inflammation-related proteins in this process. Our findings led to the first model of E. ruminantium infection in host cells in vitro, and we highlight potential biomarkers of E. ruminantium infection in endothelial cells (such as ROCK1, TMEM16K, Albumin and PTPN1), which may be important to further combat Heartwater, namely by developing non-antibiotic-based strategies.

scholarly journals Akt Interacts with Usutu Virus Polymerase, and Its Activity Modulates Viral Replication

Pathogens ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 244
Author(s):  
Laura Albentosa-González ◽  
Rosario Sabariegos ◽  
Armando Arias ◽  
Pilar Clemente-Casares ◽  
Antonio Mas
Keyword(s):  
Public Health ◽  
Confocal Microscopy ◽  
Viral Replication ◽  
Insufficient Data ◽  
Health Concerns ◽  
Usutu Virus ◽  
A Cell ◽  
Infected Cells ◽  
Specific Inhibitors

Usutu virus (USUV) is a flavivirus that mainly infects wild birds through the bite of Culex mosquitoes. Recent outbreaks have been associated with an increased number of cases in humans. Despite being a growing source of public health concerns, there is yet insufficient data on the virus or host cell targets for infection control. In this work we have investigated whether the cellular kinase Akt and USUV polymerase NS5 interact and co-localize in a cell. To this aim, we performed co-immunoprecipitation (Co-IP) assays, followed by confocal microscopy analyses. We further tested whether NS5 is a phosphorylation substrate of Akt in vitro. Finally, to examine its role in viral replication, we chemically silenced Akt with three inhibitors (MK-2206, honokiol and ipatasertib). We found that both proteins are localized (confocal) and pulled down (Co-IP) together when expressed in different cell lines, supporting the fact that they are interacting partners. This possibility was further sustained by data showing that NS5 is phosphorylated by Akt. Treatment of USUV-infected cells with Akt-specific inhibitors led to decreases in virus titers (>10-fold). Our results suggest an important role for Akt in virus replication and stimulate further investigations to examine the PI3K/Akt/mTOR pathway as an antiviral target.

scholarly journals New Isolates of Pandoraviruses: Contribution to the Study of Replication Cycle Steps

2018 ◽  
Vol 93 (5) ◽  
Author(s):  
Ana Cláudia dos Santos Pereira Andrade ◽  
Paulo Victor de Miranda Boratto ◽  
Rodrigo Araújo Lima Rodrigues ◽  
Talita Machado Bastos ◽  
Bruna Luiza Azevedo ◽  
...  
Keyword(s):  
Time Course ◽  
Replication Cycle ◽  
Host Cells ◽  
Comprehensive Description ◽  
Genomic Features ◽  
New Information ◽  
Viral Particles ◽  
Infected Cells ◽  
Giant Viruses ◽  
Electron Lucent

ABSTRACT Giant viruses are complex members of the virosphere, exhibiting outstanding structural and genomic features. Among these viruses, the pandoraviruses are some of the most intriguing members, exhibiting giant particles and genomes presenting at up to 2.5 Mb, with many genes having no known function. In this work, we analyzed, by virological and microscopic methods, the replication cycle steps of three new pandoravirus isolates from samples collected in different regions of Brazil. Our data indicate that all analyzed pandoravirus isolates can deeply modify the Acanthamoeba cytoplasmic environment, recruiting mitochondria and membranes into and around the electron-lucent viral factories. We also observed that the viral factories start forming before the complete degradation of the cellular nucleus. Various patterns of pandoravirus particle morphogenesis were observed, and the assembly of the particles seemed to be started either by the apex or by the opposite side. On the basis of the counting of viral particles during the infection time course, we observed that pandoravirus particles could undergo exocytosis after their morphogenesis in a process that involved intense recruitment of membranes that wrapped the just-formed particles. The treatment of infected cells with brefeldin affected particle exocytosis in two of the three analyzed strains, indicating biological variability among isolates. Despite such particle exocytosis, the lysis of host cells also contributed to viral release. This work reinforces knowledge of and reveals important steps in the replication cycle of pandoraviruses. IMPORTANCE The emerging Pandoraviridae family is composed of some of the most complex viruses known to date. Only a few pandoravirus isolates have been described until now, and many aspects of their life cycle remain to be elucidated. A comprehensive description of the replication cycle is pivotal to a better understanding of the biology of the virus. For this report, we describe new pandoraviruses and used different methods to better characterize the steps of the replication cycle of this new group of viruses. Our results provide new information about the diversity and biology of these giant viruses.

scholarly journals Super Resolution Microscopy and Deep Learning Identify Zika Virus Reorganization of the Endoplasmic Reticulum

2020 ◽  
Author(s):  
Rory K. M. Long ◽  
Kathleen P. Moriarty ◽  
Ben Cardoen ◽  
Guang Gao ◽  
A. Wayne Vogl ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Deep Learning ◽  
Viral Replication ◽  
Zika Virus ◽  
Deep Neural Networks ◽  
Super Resolution ◽  
Zikv Infection ◽  
Subcellular Organelle ◽  
Infected Cells ◽  
Super Resolution Microscopy

AbstractThe endoplasmic reticulum (ER) is a complex subcellular organelle composed of diverse structures such as tubules, sheets and tubular matrices. Flaviviruses such as Zika virus (ZIKV) induce reorganization of endoplasmic reticulum (ER) membranes to facilitate viral replication. Here, using 3D super resolution microscopy, ZIKV infection is shown to induce the formation of dense tubular matrices associated with viral replication in the central ER. Viral non-structural proteins NS4B and NS2B associate with replication complexes within the ZIKV-induced tubular matrix and exhibit distinct ER distributions outside this central ER region. Deep neural networks trained to identify ZIKV-infected versus mock-infected cells successfully identified ZIKV-induced central ER tubular matrices as a determinant of viral infection. Super resolution microscopy and deep learning are therefore able to identify and localize morphological features of the ER and may be of use to screen for inhibitors of infection by ER-reorganizing viruses.

scholarly journals Genetic diversity of Collaborative Cross mice controls viral replication, clinical severity and brain pathology induced by Zika virus infection, independently of Oas1b

2019 ◽  
Author(s):  
Caroline Manet ◽  
Etienne Simon-Lorière ◽  
Grégory Jouvion ◽  
David Hardy ◽  
Matthieu Prot ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Viral Load ◽  
Viral Replication ◽  
Clinical Severity ◽  
Brain Pathology ◽  
Zikv Infection ◽  
Infected Cells ◽  
Host Genetic ◽  
Host Genes

ABSTRACTThe explosive spread of Zika virus (ZIKV) has been associated with major variations in severe disease and congenital afflictions among infected populations, suggesting an influence of host genes. We investigated how genome-wide variants could impact susceptibility to ZIKV infection in mice. We first describe that the susceptibility of Ifnar1 knockout mice is largely influenced by their genetic background. We then show that the broad genetic diversity of Collaborative Cross mice, which receptor to type I interferon (IFNAR) was blocked by anti-IFNAR antibody, expressed phenotypes ranging from complete resistance to severe symptoms and death with large variations in the peak and rate of decrease of plasma viral load, in brain viral load, in brain histopathology and in viral replication rate in infected cells. Differences of susceptibility between CC strains were correlated between Zika, Dengue and West Nile viruses. We identified highly susceptible and resistant mouse strains as new models to investigate the mechanisms of human ZIKV disease and other flavivirus infections. Genetic analyses revealed that phenotypic variations are driven by multiple genes with small effects, reflecting the complexity of ZIKV disease susceptibility in human population. Notably, our results rule out a role of the Oas1b gene in the susceptibility to ZIKV. Altogether, this study emphasizes the role of host genes in the pathogeny of ZIKV infection and lays the foundation for further genetic and mechanistic studies.IMPORTANCEIn recent outbreaks, ZIKV has infected millions of people and induced rare but potentially severe complications, including Guillain-Barré syndrome and encephalitis in adults. While several viral sequence variants were proposed to enhance the pathogenicity of ZIKV, the influence of host genetic variants in the clinical heterogeneity remains mostly unexplored. We have addressed this question using a mouse panel which models the genetic diversity of human population and a ZIKV strain from a recent clinical isolate. Through a combination of in vitro and in vivo approaches, we demonstrate that multiple host genetic variants determine viral replication in infected cells, and clinical severity, kinetics of blood viral load and brain pathology in mice. We describe new mouse models expressing high susceptibility or resistance to ZIKV and to other flaviviruses. These models will facilitate the identification and mechanistic characterization of host genes that influence ZIKV pathogenesis.

scholarly journals Transcriptional role of p53 in interferon-mediated antiviral immunity

2008 ◽  
Vol 205 (8) ◽  
pp. 1929-1938 ◽  
Author(s):  
César Muñoz-Fontela ◽  
Salvador Macip ◽  
Luis Martínez-Sobrido ◽  
Lauren Brown ◽  
Joseph Ashour ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Viral Replication ◽  
Viral Infections ◽  
Suppressor Gene ◽  
Central Component ◽  
Type I ◽  
Infected Cells

Tumor suppressor p53 is activated by several stimuli, including DNA damage and oncogenic stress. Previous studies (Takaoka, A., S. Hayakawa, H. Yanai, D. Stoiber, H. Negishi, H. Kikuchi, S. Sasaki, K. Imai, T. Shibue, K. Honda, and T. Taniguchi. 2003. Nature. 424:516–523) have shown that p53 is also induced in response to viral infections as a downstream transcriptional target of type I interferon (IFN) signaling. Moreover, many viruses, including SV40, human papillomavirus, Kaposi's sarcoma herpesvirus, adenoviruses, and even RNA viruses such as polioviruses, have evolved mechanisms designated to abrogate p53 responses. We describe a novel p53 function in the activation of the IFN pathway. We observed that infected mouse and human cells with functional p53 exhibited markedly decreased viral replication early after infection. This early inhibition of viral replication was mediated both in vitro and in vivo by a p53-dependent enhancement of IFN signaling, specifically the induction of genes containing IFN-stimulated response elements. Of note, p53 also contributed to an increase in IFN release from infected cells. We established that this p53-dependent enhancement of IFN signaling is dependent to a great extent on the ability of p53 to activate the transcription of IFN regulatory factor 9, a central component of the IFN-stimulated gene factor 3 complex. Our results demonstrate that p53 contributes to innate immunity by enhancing IFN-dependent antiviral activity independent of its functions as a proapoptotic and tumor suppressor gene.

scholarly journals Zika virus is transmitted in neural progenitor cells via cell-to-cell spread and infection is inhibited by the autophagy inducer trehalose

2020 ◽  
pp. JVI.02024-20
Author(s):  
Alex E Clark ◽  
Zhe Zhu ◽  
Florian Krach ◽  
Jeremy N Rich ◽  
Gene W. Yeo ◽  
...  
Keyword(s):  
Viral Replication ◽  
Progenitor Cells ◽  
Zika Virus ◽  
Neural Progenitor Cells ◽  
Neutralizing Antibody ◽  
Neural Progenitor ◽  
Health Concern ◽  
Free Virus ◽  
Zikv Infection ◽  
Infected Cells

Zika virus (ZIKV) is a mosquito-borne human pathogen that causes congenital Zika syndrome and neurological symptoms in some adults. There are currently no approved treatments or vaccines for ZIKV, and exploration of therapies targeting host processes could avoid viral development of drug resistance. The purpose of our study was to determine if the non-toxic and widely used disaccharide trehalose, which showed antiviral activity against Human Cytomegalovirus (HCMV) in our previous work, could restrict ZIKV infection in clinically relevant neural progenitor cells (NPCs). Trehalose is known to induce autophagy, the degradation and recycling of cellular components. Whether autophagy is proviral or antiviral for ZIKV is controversial and depends on cell type and specific conditions used to activate or inhibit autophagy. We show here that trehalose treatment of NPCs infected with recent ZIKV isolates from Panama and Puerto Rico significantly reduces viral replication and spread. In addition, we demonstrate that ZIKV infection in NPCs spreads primarily cell-to-cell as an expanding infectious center, and NPCs are infected via contact with infected cells far more efficiently than by cell-free virus. Importantly, ZIKV was able to spread in NPCs in the presence of neutralizing antibody.Importance Zika virus causes birth defects and can lead to neurological disease in adults. While infection rates are currently low, ZIKV remains a public health concern with no treatment or vaccine available. Targeting a cellular pathway to inhibit viral replication is a potential treatment strategy that avoids development of antiviral resistance. We demonstrate in this study that the non-toxic autophagy-inducing disaccharide trehalose reduces spread and output of ZIKV in infected neural progenitor cells (NPCs), the major cells infected in the fetus. We show that ZIKV spreads cell-to-cell in NPCs as an infectious center and that NPCs are more permissive to infection by contact with infected cells than by cell-free virus. We find that neutralizing antibody does not prevent the spread of the infection in NPCs. These results are significant in demonstrating anti-ZIKV activity of trehalose and in clarifying the primary means of Zika virus spread in clinically relevant target cells.

scholarly journals Atlastin Endoplasmic Reticulum-Shaping Proteins Facilitate Zika Virus Replication

2019 ◽  
Vol 93 (23) ◽  
Author(s):  
Blandine Monel ◽  
Maaran Michael Rajah ◽  
Mohamed Lamine Hafirassou ◽  
Samy Sid Ahmed ◽  
Julien Burlaud-Gaillard ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Viral Replication ◽  
Zika Virus ◽  
Antiviral Treatment ◽  
Viral Proteins ◽  
Neurological Complications ◽  
Cytopathic Effects ◽  
Replication Sites ◽  
Infected Cells ◽  
Er Membrane

ABSTRACT The endoplasmic reticulum (ER) is the site for Zika virus (ZIKV) replication and is central to the cytopathic effects observed in infected cells. ZIKV induces the formation of ER-derived large cytoplasmic vacuoles followed by “implosive” cell death. Little is known about the nature of the ER factors that regulate flavivirus replication. Atlastins (ATL1, -2, and -3) are dynamin-related GTPases that control the structure and the dynamics of the ER membrane. We show here that ZIKV replication is significantly decreased in the absence of ATL proteins. The appearance of infected cells is delayed, the levels of intracellular viral proteins and released virus are reduced, and the cytopathic effects are strongly impaired. We further show that ATL3 is recruited to viral replication sites and interacts with the nonstructural viral proteins NS2A and NS2B3. Thus, proteins that shape and maintain the ER tubular network ensure efficient ZIKV replication. IMPORTANCE Zika virus (ZIKV) is an emerging virus associated with Guillain-Barré syndrome, and fetal microcephaly as well as other neurological complications. There is no vaccine or specific antiviral treatment against ZIKV. We found that endoplasmic reticulum (ER)-shaping atlastin proteins (ATL1, -2, and -3), which induce ER membrane fusion, facilitate ZIKV replication. We show that ATL3 is recruited to the viral replication site and colocalize with the viral proteins NS2A and NS2B3. The results provide insights into host factors used by ZIKV to enhance its replication.

scholarly journals The aminobisphosphonate pamidronate controls influenza pathogenesis by expanding a γδ T cell population in humanized mice

2011 ◽  
Vol 208 (7) ◽  
pp. 1511-1522 ◽  
Author(s):  
Wenwei Tu ◽  
Jian Zheng ◽  
Yinping Liu ◽  
Sin Fun Sia ◽  
Ming Liu ◽  
...  
Keyword(s):  
Influenza Virus ◽  
T Cell ◽  
Viral Replication ◽  
Mononuclear Cells ◽  
Immunodeficient Mice ◽  
H5n1 Influenza ◽  
Infected Cells ◽  
Peripheral Mononuclear Cells ◽  
Vγ9vδ2 T Cells

There are few antiviral drugs for treating influenza, and the emergence of antiviral resistance has further limited the available therapeutic options. Furthermore, antivirals are not invariably effective in severe influenza, such as that caused by H5N1 viruses. Thus, there is an urgent need to develop alternative therapeutic strategies. Here, we show that human Vγ9Vδ2 T cells expanded by the aminobisphosphonate pamidronate (PAM) kill influenza virus–infected cells and inhibit viral replication in vitro. In Rag2−/−γc−/− immunodeficient mice reconstituted with human peripheral mononuclear cells (huPBMCs), PAM reduces disease severity and mortality caused by human seasonal H1N1 and avian H5N1 influenza virus, and controls the lung inflammation and viral replication. PAM has no such effects in influenza virus–infected Rag2−/−γc−/− mice reconstituted with Vγ9Vδ2 T cell–depleted huPBMCs. Our study provides proof-of-concept of a novel therapeutic strategy for treating influenza by targeting the host rather than the virus, thereby reducing the opportunity for the emergence of drug-resistant viruses. As PAM has been commonly used to treat osteoporosis and Paget’s disease, this new application of an old drug potentially offers a safe and readily available option for treating influenza.

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