Detection and quantification of dengue virus using a novel biosensor system based on dengue NS3 protease activity

Hsieh, Ming-Shu; Chen, Meiyu; Hsieh, Chun-Hsiang; Pan, Chien-Hsiung; Yu, Guann-Yi; Chen, Hsin–Wei

doi:10.1371/journal.pone.0188170

Cited by 17 publications

(15 citation statements)

References 51 publications

(46 reference statements)

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“…Four serotypes of DENV (DENV-1, Hawaii strain; DENV-2, 16681 strain; DENV-3, H87 strain; DENV-4, H241 strain) were propagated in C6/36 cells, and viral titers were determined by focus-forming assay in BHK-21 cells as described previously (37). The ZIKV PRVABC59 strain was amplified in C6/36 cells, and viral titers were measured by plaque-forming assays in Vero cells as described previously (38,39). The anti-inflammatory compounds 1h, 2d, 2j, and 2l were synthesized as described previously (30) .…”

Section: Methodsmentioning

confidence: 99%

Anti-inflammatory Compound Shows Therapeutic Safety and Efficacy against Flavivirus Infection

Chuang

Huang

Liao

et al. 2019

Antimicrob Agents Chemother

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Flaviviruses comprise several medically important viruses, including Japanese encephalitis virus, West Nile virus, dengue virus (DENV), yellow fever virus, and Zika virus (ZIKV). A large outbreak of DENV and ZIKV occurred recently, leading to many cases of illness and death. However, despite decades of effort, we have no clinically specific therapeutic drugs against DENV and ZIKV. Previous studies showed that inflammatory responses play a critical role in dengue and Zika virus pathogenesis. Thus, in this study, we examined a series of novel anti-inflammatory compounds and found that treatment with compound 2d could dose dependently reduce viral protein expression and viral progeny production in HEK-293 and Raw264.7 cells infected with four serotypes of DENV and ZIKV. In addition, considering medication safety, compound 2d could not suppress cyclooxygenase-1 (COX-1) enzymatic activities and thus could prevent the side effect of bleeding. Moreover, compound 2d significantly inhibited COX-2 enzymatic activities and prostaglandin E2 levels, associated with viral replication, compared to results with a selective COX-2 inhibitor, celecoxib. Furthermore, administering 5 mg/kg compound 2d to DENV-2-infected AG129 mice prolonged survival and reduced viremia and serum cytokine levels. Overall, compound 2d showed therapeutic safety and efficacy in vitro and in vivo and could be further developed as a potential therapeutic agent for flavivirus infection.

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Section: Methodsmentioning

confidence: 99%

Anti-inflammatory Compound Shows Therapeutic Safety and Efficacy against Flavivirus Infection

Chuang

Huang

Liao

et al. 2019

Antimicrob Agents Chemother

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“…An alternative approach is the use of engineered fluorescent reporter proteins stably expressed in cells that are altered upon virus infection ( 19 – 21 ). Building on this idea, we established a reporter system based on an ER-anchored green fluorescent protein (GFP) that, upon recognition and cleavage of a specific linker region by a viral protease, is released from the ER and translocated to the nucleus.…”

Section: Introductionmentioning

confidence: 99%

“…A possible explanation for this could be that unspecific cleavage of the linker region might be mediated by cellular proteases due to high levels of expression of the reporter construct upon transient transduction.Reporter constructs for detection of flavivirus infection have been described previously which were either cytosolic or employed viral non-structural proteins as ER anchors(19)(20)(21)40). Most of these reporter systems rely on the expression of large fragments of viral proteins(19)(20)(21) which can alter the physiological stoichiometry of the viral proteins and induce undesired pleiotropic effects. Indeed, even expression of single NS proteins can affect cellular functions, such as alteration of mitochondrial morphodynamics by NS4B (45).…”

mentioning

confidence: 99%

A Versatile Reporter System To Monitor Virus-Infected Cells and Its Application to Dengue Virus and SARS-CoV-2

Pahmeier

Neufeldt

Cerikan

et al. 2021

J Virol

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Positive-strand RNA viruses have been the etiological agents in several major disease outbreaks over the last few decades. Examples of this include flaviviruses, such as dengue virus and Zika virus that cause millions of yearly infections around the globe, and coronaviruses, such as SARS-CoV-2, the source of the current pandemic. The severity of outbreaks caused by these viruses stresses the importance of research aimed at determining methods to limit virus spread and to curb disease severity. Such studies require molecular tools to decipher virus-host interactions and to develop effective treatments. Here, we describe the generation and characterization of a reporter system that can be used to visualize and identify cells infected with dengue virus or SARS-CoV-2. This system is based on viral protease activity that mediates cleavage and nuclear translocation of an engineered fluorescent protein stably expressed in cells. We show the suitability of this system for live cell imaging, for visualization of single infected cells, and for screening and testing of antiviral compounds. With the integrated modular building blocks, this system is easy to manipulate and can be adapted to any virus encoding a protease, thus offering a high degree of flexibility. IMPORTANCE Reporter systems are useful tools for fast and quantitative visualization of virus infected cells 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 GFP moiety 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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“…An alternative approach is the use of engineered fluorescent reporter proteins stably expressed in cells and altering their subcellular distribution upon viral infection (21)(22)(23). Building on this idea, here we established a reporter system based on an ERanchored green fluorescent protein (GFP) that upon cleavage by a viral protease is released from the ER and translocated into the nucleus.…”

Section: Introductionmentioning

confidence: 99%

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

Pahmeier

Neufeldt

Cerikan

et al. 2020

Preprint

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Positive-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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Detection and quantification of dengue virus using a novel biosensor system based on dengue NS3 protease activity

Cited by 17 publications

References 51 publications

Anti-inflammatory Compound Shows Therapeutic Safety and Efficacy against Flavivirus Infection

Anti-inflammatory Compound Shows Therapeutic Safety and Efficacy against Flavivirus Infection

A Versatile Reporter System To Monitor Virus-Infected Cells and Its Application to Dengue Virus and SARS-CoV-2

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

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