Fluorescent poliovirus for flow cytometric cell surface binding studies

Freistadt, Marion S.; Eberle, Karen E.

doi:10.1016/j.jviromet.2005.08.011

Cited by 9 publications

(4 citation statements)

References 13 publications

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“…Different brightness may be achieved by varying the concentration of dye used. However, increasing the dye concentration can reduce virus viability 7,10 . One possible limitation is the interference in receptor binding due to the blockade of access by the fluorophores.…”

Section: Discussionmentioning

confidence: 99%

Visualizing Dengue Virus through Alexa Fluor Labeling

Zhang

Tan

Ooi

2011

JoVE

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The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors that influence this interaction could lead to improved understanding of disease pathogenesis and thus influence vaccine or therapeutic design. Hence, the development of methods to probe this interaction would be useful. Recent advancements in fluorophores development 1-3 and imaging technology 4 can be exploited to improve our current knowledge on dengue pathogenesis and thus pave the way to reduce the millions of dengue infections occurring annually.The enveloped dengue virus has an external scaffold consisting of 90 envelope glycoprotein (E) dimers protecting the nucleocapsid shell, which contains a single positive strand RNA genome 5 . The identical protein subunits on the virus surface can thus be labeled with an amine reactive dye and visualized through immunofluorescent microscopy. Here, we present a simple method of labeling of dengue virus with Alexa Fluor succinimidyl ester dye dissolved directly in a sodium bicarbonate buffer that yielded highly viable virus after labeling. There is no standardized procedure for the labeling of live virus and existing manufacturer's protocol for protein labeling usually requires the reconstitution of dye in dimethyl sulfoxide. The presence of dimethyl sulfoxide, even in minute quantities, can block productive infection of virus and also induce cell cytotoxicity 6 . The exclusion of the use of dimethyl sulfoxide in this protocol thus reduced this possibility. Alexa Fluor dyes have superior photostability and are less pH-sensitive than the common dyes, such as fluorescein and rhodamine 2 , making them ideal for studies on cellular uptake and endosomal transport of the virus. The conjugation of Alexa Fluor dye did not affect the recognition of labeled dengue virus by virus-specific antibody and its putative receptors in host cells 7 . This method could have useful applications in virological studies.

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Section: Discussionmentioning

confidence: 99%

Visualizing Dengue Virus through Alexa Fluor Labeling

Zhang

Tan

Ooi

2011

JoVE

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“…While the applications of flow cytometry are many and varied, it has not been well-studied for detection of enterovirus infection from culture. Previous studies have focused on using flow cytometry to quantitate poliovirus infection in neuronal cells (Daley et al, 2005), characterize enterovirus binding to host cell surfaces (Freistadt and Eberle, 2006;Mbida et al, 1991;Triantafilou et al, 2001), confirm cytomegalovirus infection of tissue culture cells with a genetically engineered fluorescence reporter system (Kung et al, 2000), and serotype human immunodeficiency virus type 1 (Zolla-Pazner et al, 1995). This is the first report of a clini- Table 1 Kinetic study of coxsackievirus B1-infected PMK cells as demonstrated by flow cytometric analysis, IFA, and observation of CPE.…”

Section: Discussionmentioning

confidence: 99%

Flow cytometric detection and serotyping of enterovirus for the clinical laboratory

She

Preobrazhensky

Taggart

et al. 2009

Journal of Virological Methods

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Culture and serotyping of human enteroviruses by fluorescence microscopy are time-consuming and labor-intensive. Flow cytometry has the potential of being more rapid, sensitive, and objective but has not been used for these purposes in a clinical laboratory. Primary rhesus monkey kidney (PMK) cells were inoculated with several enterovirus serotypes and stained with enterovirus-specific antibodies for flow cytometry and indirect fluorescence antibody testing (IFA). Kinetic studies of coxsackievirus B1 and echovirus 30 infection of PMK cells were performed on days 1-4 after inoculation. Flow cytometry results for echovirus 6, 9, 11, and 30 and coxsackievirus B1 correlated with IFA in all cases. Coxsackievirus B1 and echovirus 30 infections were detected 1 day earlier by flow cytometry than IFA. Flow cytometry can be effectively used for detecting enterovirus-infected cells in a clinical laboratory with the advantages of better quantitation of low levels of infection and earlier detection of virally infected cells in culture systems.

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“…To achieve successful virus labeling, fluorescent reagents must be either physically incorporated into viral particles or bioconjugated onto viruses via covalent/noncovalent reactions, such as carbodiimide coupling reaction, biotin–avidin affinity system, and click chemistry . However, direct fluorescent labeling may compromise viral infectivity due to the chemical modifications of viral capsid and envelope, which are crucial for viral infection . Moreover, incorporated fluorophores can also occupy the surface recognition sites of viruses, thereby dampening the adsorption of virus on the viral receptor and entry into host cells .…”

Section: Introductionmentioning

confidence: 99%

“…Moreover, incorporated fluorophores can also occupy the surface recognition sites of viruses, thereby dampening the adsorption of virus on the viral receptor and entry into host cells . Although the amount of fluorescence on viruses can be titrated to diminish the potential interference of direct labeling on viral infectivity in vitro, whether those approaches could be applied in vivo is not yet known.…”

Section: Introductionmentioning

confidence: 99%

In Situ Bioorthogonal Metabolic Labeling for Fluorescence Imaging of Virus Infection In Vivo

Pan

et al. 2017

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Optical fluorescence imaging is an important strategy to explore the mechanism of virus-host interaction. However, current fluorescent tag labeling strategies often dampen viral infectivity. The present study explores an in situ fluorescent labeling strategy in order to preserve viral infectivity and precisely monitor viral infection in vivo. In contrast to pre-labeling strategy, mice are first intranasally infected with azide-modified H5N1 pseudotype virus (N -H5N1p), followed by injection of dibenzocyclooctyl (DBCO)-functionalized fluorescence 6 h later. The results show that DBCO dye directly conjugated to N -H5N1p in lung tissues through in vivo bioorthogonal chemistry with high specificity and efficacy. More remarkably, in situ labeling rather than conventional prelabeling strategy effectively preserves viral infectivity and immunogenicity both in vitro and in vivo. Hence, in situ bioorthogonal viral labeling is a promising and reliable strategy for imaging and tracking viral infection in vivo.

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Fluorescent poliovirus for flow cytometric cell surface binding studies

Cited by 9 publications

References 13 publications

Visualizing Dengue Virus through Alexa Fluor Labeling

Visualizing Dengue Virus through Alexa Fluor Labeling

Flow cytometric detection and serotyping of enterovirus for the clinical laboratory

In Situ Bioorthogonal Metabolic Labeling for Fluorescence Imaging of Virus Infection In Vivo

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