Faculty Opinions recommendation of Characterization of the early events in dengue virus cell entry by biochemical assays and single-virus tracking.

2007 ◽  
Author(s):  
Scott Halstead
Keyword(s):  
Dengue Virus ◽  
Cell Entry ◽  
Biochemical Assays ◽  
Single Virus Tracking

scholarly journals Characterization of the Early Events in Dengue Virus Cell Entry by Biochemical Assays and Single-Virus Tracking

2007 ◽  
Vol 81 (21) ◽  
pp. 12019-12028 ◽  
Author(s):  
Hilde M. van der Schaar ◽  
Michael J. Rust ◽  
Barry-Lee Waarts ◽  
Heidi van der Ende-Metselaar ◽  
Richard J. Kuhn ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Dengue Virus ◽  
Membrane Fusion ◽  
High Ratio ◽  
Cell Entry ◽  
Viral Membrane ◽  
Virus Particles ◽  
Biochemical Assays ◽  
Particle Ratio ◽  
Infectious Unit

ABSTRACT In this study, we investigated the cell entry characteristics of dengue virus (DENV) type 2 strain S1 on mosquito, BHK-15, and BS-C-1 cells. The concentration of virus particles measured by biochemical assays was found to be substantially higher than the number of infectious particles determined by infectivity assays, leading to an infectious unit-to-particle ratio of approximately 1:2,600 to 1:72,000, depending on the specific assays used. In order to explain this high ratio, we investigated the receptor binding and membrane fusion characteristics of single DENV particles in living cells using real-time fluorescence microscopy. For this purpose, DENV was labeled with the lipophilic fluorescent probe DiD (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt). The surface density of the DiD dye in the viral membrane was sufficiently high to largely quench the fluorescence intensity but still allowed clear detection of single virus particles. Fusion of the viral membrane with the cell membrane was evident as fluorescence dequenching. It was observed that DENV binds very inefficiently to the cells used, explaining at least in part the high infectious unit-to-particle ratio. The particles that did bind to the cells showed different types of transport behavior leading to membrane fusion in both the periphery and perinuclear regions of the cell. Membrane fusion was observed in 1 out of 6 bound virus particles, indicating that a substantial fraction of the virus has the capacity to fuse. DiD dequenching was completely inhibited by ammonium chloride, demonstrating that fusion occurs exclusively from within acidic endosomes.

scholarly journals Genomic investigation of a dengue virus outbreak in Thiès, Senegal, in 2018

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Amy Gaye ◽  
Tolla Ndiaye ◽  
Mouhamad Sy ◽  
Awa B. Deme ◽  
Alphonse B. Thiaw ◽  
...  
Keyword(s):  
West Africa ◽  
Dengue Virus ◽  
Clinical Signs ◽  
Health Concern ◽  
Public Health Concern ◽  
Local Circulation ◽  
Viral Genomes ◽  
Genomic Characterization ◽  
The City

AbstractDengue virus is a major and rapidly growing public health concern in tropic and subtropic regions across the globe. In late 2018, Senegal experienced its largest dengue virus outbreak to date, covering several regions. However, little is known about the genetic diversity of dengue virus (DENV) in Senegal. Here we report complete viral genomes from 17 previously undetected DENV cases from the city of Thiès. In total we identified 19 cases of DENV in a cohort of 198 individuals with fever collected in October and November 2018. We detected 3 co-circulating serotypes; DENV 3 was the most frequent accounting for 11/17 sequences (65%), 4 (23%) were DENV2 and 2 (12%) were DENV1. Sequences were most similar to recent sequences from West Africa, suggesting ongoing local circulation of viral populations; however, detailed inference is limited by the scarcity of available genomic data. We did not find clear associations with reported clinical signs or symptoms, highlighting the importance of testing for diagnosing febrile diseases. Overall, these findings expand the known range of DENV in Senegal, and underscore the need for better genomic characterization of DENV in West Africa.

scholarly journals Characterization of Lassa Virus Cell Entry and Neutralization with Lassa Virus Pseudoparticles

2009 ◽  
Vol 83 (7) ◽  
pp. 3228-3237 ◽  
Author(s):  
François-Loic Cosset ◽  
Philippe Marianneau ◽  
Geraldine Verney ◽  
Fabrice Gallais ◽  
Noel Tordo ◽  
...  
Keyword(s):  
Immune Response ◽  
Humoral Immune Response ◽  
Neutralizing Antibodies ◽  
Cell Attachment ◽  
Low Ph ◽  
Cell Entry ◽  
Lassa Virus ◽  
Ph Optimum ◽  
Humoral Immune

ABSTRACT The cell entry and humoral immune response of the human pathogen Lassa virus (LV), a biosafety level 4 (BSL4) Old World arenavirus, are not well characterized. LV pseudoparticles (LVpp) are a surrogate model system that has been used to decipher factors and routes involved in LV cell entry under BSL2 conditions. Here, we describe LVpp, which are highly infectious, with titers approaching those obtained with pseudoparticles displaying G protein of vesicular stomatitis virus and their the use for the characterization of LV cell entry and neutralization. Upon cell attachment, LVpp utilize endocytic vesicles for cell entry as described for many pH-dependent viruses. However, the fusion of the LV glycoproteins is activated at unusually low pH values, with optimal fusion occurring between pH 4.5 and 3, a pH range at which fusion characteristics of viral glycoproteins have so far remained largely unexplored. Consistent with a shifted pH optimum for fusion activation, we found wild-type LV and LVpp to display a remarkable resistance to exposure to low pH. Finally, LVpp allow the fast and quantifiable detection of neutralizing antibodies in human and animal sera and will thus facilitate the study of the humoral immune response in LV infections.

scholarly journals Functional Characterization of a Redundant Plasmodium TRAP Family Invasin, TRAP-Like Protein, by Aldolase Binding and a Genetic Complementation Test

2008 ◽  
Vol 7 (6) ◽  
pp. 1062-1070 ◽  
Author(s):  
Kirsten Heiss ◽  
Hui Nie ◽  
Sumit Kumar ◽  
Thomas M. Daly ◽  
Lawrence W. Bergman ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Cell Entry ◽  
Host Cells ◽  
Complementation Test ◽  
Type I ◽  
Cycle Progression ◽  
Targeted Gene Disruption ◽  
Specific Host ◽  
Host Cell Entry

ABSTRACT Efficient and specific host cell entry is of exquisite importance for intracellular pathogens. Parasites of the phylum Apicomplexa are highly motile and actively enter host cells. These functions are mediated by type I transmembrane invasins of the TRAP family that link an extracellular recognition event to the parasite actin-myosin motor machinery. We systematically tested potential parasite invasins for binding to the actin bridging molecule aldolase and complementation of the vital cytoplasmic domain of the sporozoite invasin TRAP. We show that the ookinete invasin CTRP and a novel, structurally related protein, termed TRAP-like protein (TLP), are functional members of the TRAP family. Although TLP is expressed in invasive stages, targeted gene disruption revealed a nonvital role during life cycle progression. This is the first genetic analysis of TLP, encoding a redundant TRAP family invasin, in the malaria parasite.

scholarly journals Characterization of SARS-CoV-2 variants B.1.617.1 (Kappa), B.1.617.2 (Delta) and B.1.618 on cell entry, host range, and sensitivity to convalescent plasma and ACE2 decoy receptor

2021 ◽  
Author(s):  
Wenlin Ren ◽  
Xiaohui Ju ◽  
Mingli Gong ◽  
Jun Lan ◽  
Yanying Yu ◽  
...  
Keyword(s):  
Host Range ◽  
Viral Entry ◽  
Neutralizing Antibodies ◽  
Cell Entry ◽  
Decoy Receptor ◽  
Host Tropism ◽  
Entry Inhibitors ◽  
Rapid Spread ◽  
Entry Efficiency

ABSTRACTRecently, highly transmissible SARS-CoV-2 variants B.1.617.1 (Kappa), B.1.617.2 (Delta) and B.1.618 were identified in India with mutations within the spike proteins. The spike protein of Kappa contains four mutations E154K, L452R, E484Q and P681R, and Delta contains L452R, T478K and P681R, while B.1.618 spike harbors mutations Δ145-146 and E484K. However, it remains unknown whether these variants have altered in their entry efficiency, host tropism, and sensitivity to neutralizing antibodies as well as entry inhibitors. In this study, we found that Kappa, Delta or B.1.618 spike uses human ACE2 with no or slightly increased efficiency, while gains a significantly increased binding affinity with mouse, marmoset and koala ACE2 orthologs, which exhibits limited binding with WT spike. Furthermore, the P618R mutation leads to enhanced spike cleavage, which could facilitate viral entry. In addition, Kappa, Delta and B.1.618 exhibits a reduced sensitivity to neutralization by convalescent sera owning to the mutation of E484Q, T478K, Δ145-146 or E484K, but remains sensitive to entry inhibitors-ACE2-lg decoy receptor. Collectively, our study revealed that enhanced human and mouse ACE2 receptor engagement, increased spike cleavage and reduced sensitivity to neutralization antibodies of Kappa, Delta and B.1.618 may contribute to the rapid spread of these variants and expanded host range. Furthermore, our result also highlighted that ACE2-lg could be developed as broad-spectrum antiviral strategy against SARS-CoV-2 variants.

scholarly journals A versatile reporter system to monitor virus infected cells and its application to dengue virus and SARS-CoV-2

2020 ◽  
Author(s):  
Felix Pahmeier ◽  
Christoper J Neufeldt ◽  
Berati Cerikan ◽  
Vibhu Prasad ◽  
Costantin Pape ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Viral Replication ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Fluorescent Protein ◽  
Live Cell ◽  
Reporter System ◽  
Infected Cells ◽  
Positive Strand Rna

ABSTRACTPositive-strand RNA viruses have been the etiological agents in several major disease outbreaks over the last few decades. Examples of that are flaviviruses, such as dengue virus and Zika virus that cause millions of yearly infections and spread around the globe, and coronaviruses, such as SARS-CoV-2, which is the cause of the current pandemic. The severity of outbreaks caused by these viruses stresses the importance of virology research in determining mechanisms to limit virus spread and to curb disease severity. Such studies require molecular tools to decipher virus-host interactions and to develop effective interventions. Here, we describe the generation and characterization of a reporter system to visualize dengue virus and SARS-CoV-2 replication in live cells. The system is based on viral protease activity causing cleavage and nuclear translocation of an engineered fluorescent protein that is expressed in the infected cells. We show the suitability of the system for live cell imaging and visualization of single infected cells as well as for screening and testing of antiviral compounds. Given the modular building blocks, the system is easy to manipulate and can be adapted to any virus encoding a protease, thus offering a high degree of flexibility.IMPORTANCEReporter systems are useful tools for fast and quantitative visualization of viral replication and spread within a host cell population. Here we describe a reporter system that takes advantage of virus-encoded proteases that are expressed in infected cells to cleave an ER-anchored fluorescent protein fused to a nuclear localization sequence. Upon cleavage, the fluorescent protein translocates to the nucleus, allowing for rapid detection of the infected cells. Using this system, we demonstrate reliable reporting activity for two major human pathogens from the Flaviviridae and the Coronaviridae families: dengue virus and SARS-CoV-2. We apply this reporter system to live cell imaging and use it for proof-of-concept to validate antiviral activity of a nucleoside analogue. This reporter system is not only an invaluable tool for the characterization of viral replication, but also for the discovery and development of antivirals that are urgently needed to halt the spread of these viruses.

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