Live-Cell Imaging of Flaviviridae Family Virus Infections: Progress and Challenges
Abstract
1. Introduction
2. The Orthoflavivirus and Hepacivirus Genome Organisation and Life Cycle
3. Monitoring Viral Replication and Spread Using Viral Protease-Dependent Reporters
4. Genetic Tagging of Viral Genomes for Analysis of Viral Protein Traffic and Virus–Host Interactions
5. Visualisation of Virus Binding and Entry Using Fluorescently Labelled Virus Particles
6. Imaging Viral RNA Localisation and Traffic Using Genetically Tagged and Wildtype Viral Genomes
7. Considerations in Live-Cell Imaging Approaches
8. Future Directions
Author Contributions
Funding
Conflicts of Interest
References
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Approach | Labelling Strategy | Stage of Viral Life Cycle Investigated | Virus, Tag Location, and References |
---|---|---|---|
Monitoring Viral Replication and Spread Using Viral Protease-Dependent Reporters | Viral protease-dependent translocation of GFP from the ER or mitochondria to the nucleus of infected cells. | Monitoring viral replication and spread | DENV [7,8], ZIKV [9], HCV [10] and Flavivirus [11] |
Viral protease-dependent translocation of a transactivator or Cre recombinase to the nucleus of infected cells to trigger GFP or mCherry expression. | Monitoring viral replication and spread | DENV [12] and HCV [13] | |
Genetic Tagging of Viral Genomes for Analysis of Viral Protein Traffic and Virus–Host Interactions | Genetically encoded fluorescent proteins (GFP, mScarlet, and mCherry) | Replication | DENV NS1 [14] |
Replication and RC biogenesis | HCV NS5A [15,16,17] | ||
Virus particle assembly and release | HCV E1 [18] | ||
Genetically encoded tetracysteine (TC) tag | Entry | ZIKV Capsid [19] | |
Replication | HCV NS5A [16] | ||
Virus particle assembly and release | HCV Core [20,21] | ||
Self-labelling enzyme tags (SNAP-tag) | Replication and RC biogenesis | HCV NS5A [16,22] | |
Dual genetic tags (two fluorescent proteins or one fluorescent protein and one TC tag) | Spatiotemporal organisation of replication and virus particle assembly | HCV NS5A and Core [16], NS5A and E1 [18], or NS5A and E2 [23] | |
Visualisation of Virus Binding and Entry Using Fluorescently Labelled Virus Particles | Lipophilic dyes (DiD, Dil, and R18) | Attachment and entry | DENV [24], YFV [25], JEV-VLP [25], and HCV [26,27] lipid bilayer membrane |
Amine-reactive succinimidyl ester fluorescent dyes (Alexa Fluor 594 NHS ester and Atto647N-NHS ester) | Attachment and entry | DENV [28] and ZIKV [29] envelope | |
Imaging Viral RNA Localisation and Traffic Using Genetically Tagged and Wildtype Viral Genomes | Genetically encoded MS2 binding sites | Replication | 3′ UTR of the TBEV [30] or HCV [31] genome |
Approach | Strengths | Limitations |
---|---|---|
Monitoring Viral Replication and Spread Using Viral Protease-Dependent Reporters |
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Genetic Tagging of Viral Genomes for Analysis of Viral Protein Traffic and Virus–Host Interactions |
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Visualisation of Virus Binding and Entry Using Fluorescently Labelled Virus Particles |
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Imaging Viral RNA Localisation and Traffic Using Genetically Tagged and Wildtype Viral Genomes |
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Label | Labelling Strategy | Strengths | Limitations |
---|---|---|---|
Fluorescent proteins (e.g., GFP, mScarlet, mCherry) | Genetically encoded. | Highly specific labelling. | Large size, RNA perturbation, variable photostability and brightness, spectral overlap, and maturation time. |
Self-labelling enzymes (SNAP-tag) | Genetically encoded. Catalyse the covalent attachment of membrane-permeant fluorescent dyes. | Highly specific labelling, better photostability, high brightness, flexible, and immediate fluorescence after fluorescent dye labelling. | Large size and the timing of dye labelling requires consideration. |
Tetracysteine (TC) tag | Genetically encoded. Bound by membrane-permeant biarsenical dyes (FIAsH and ReAsH). | Small size, flexible, self-labelling, and immediate fluorescence after dye binding. | Moderate brightness and photostability, few dyes available, and requires the application of harsh reducing agents to achieve specific labelling and improve SNR. |
Lipophilic dyes (DiD, Dil, R18) | Hydrophobic insertion into lipid membrane. | Applicable for the study of virus attachment, entry, and lipid bilayer fusion events. | Variable photostability and brightness and the specific labelling of virus particles requires multiple laborious steps. |
Amine-reactive succinimidyl ester fluorescent dyes (Alexa Fluor 594 NHS ester, Atto647N-NHS ester) | NHS ester covalently binds amine groups of proteins. | Applicable to the study of virus attachment and entry, high labelling stability, and high brightness and photostability. | Potential for off-target labelling and impacts on virus–host protein interactions and specific labelling requires multiple laborious steps. |
MS2 binding sites | Genetically encoded. Bound by MS2 fusions to fluorescent proteins or self-labelling enzymes. | Highly specific labelling. | Large size, high background fluorescence, and variable photostability and brightness. |
RNA aptamers | Hybridised to target RNA. Bound by membrane-permeant fluorophore–quencher pair. | Low background fluorescence. | Large size and variable photostability and brightness. |
Imaging Modality | Best Spatial Resolution (Lateral: XY and Axial: Z) | Advantage (s) | Limitation (s) |
---|---|---|---|
Wide-field | XY: ~250 nm Z: ~500 nm | Inexpensive, high temporal resolution, and low photobleaching/phototoxicity. | Low spatial resolution. |
Laser Scanning Confocal | XY: ~200 nm Z: ~500 nm | High spatial resolution. | Low temporal resolution and photobleaching/ phototoxicity. |
Spinning Disc Confocal | XY: ~200 nm Z: ~500 nm | High spatial and temporal resolution. | Moderate photobleaching/ phototoxicity. |
Lattice Lightsheet | XY: ~250 nm Z: ~300 nm | High temporal resolution, very low photobleaching/phototoxicity, long-term volumetric live-cell imaging, and near-isotopic resolution. | Moderate spatial resolution. |
SIM | XY: ~100 nm Z: ~250 nm | Super resolution and low photobleaching/phototoxicity. | Moderately intensive data processing. |
STED | XY: ~50 nm Z: ~100 nm | Super resolution. | Low temporal resolution and photobleaching/ phototoxicity. |
dSTORM / PALM | XY: ~20 nm Z: ~60 nm | Super resolution with single molecule sensitivity. | Very low temporal resolution, photobleaching/phototoxicity, complex sample preparation (dSTORM), and intensive data processing. |
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Centofanti, S.M.; Eyre, N.S. Live-Cell Imaging of Flaviviridae Family Virus Infections: Progress and Challenges. Viruses 2025, 17, 847. https://doi.org/10.3390/v17060847
Centofanti SM, Eyre NS. Live-Cell Imaging of Flaviviridae Family Virus Infections: Progress and Challenges. Viruses. 2025; 17(6):847. https://doi.org/10.3390/v17060847
Chicago/Turabian StyleCentofanti, Siena M., and Nicholas S. Eyre. 2025. "Live-Cell Imaging of Flaviviridae Family Virus Infections: Progress and Challenges" Viruses 17, no. 6: 847. https://doi.org/10.3390/v17060847
APA StyleCentofanti, S. M., & Eyre, N. S. (2025). Live-Cell Imaging of Flaviviridae Family Virus Infections: Progress and Challenges. Viruses, 17(6), 847. https://doi.org/10.3390/v17060847