Live Imaging and Quantification of Viral Infection in K18 hACE2 Transgenic Mice Using Reporter-Expressing Recombinant SARS-CoV-2
November 5th, 2021
2.7K views
This protocol describes the dynamics of viral infections using luciferase- and fluorescence-expressing recombinant (r)SARS-CoV-2 and an in vivo imaging systems (IVIS) in K18 hACE2 transgenic mice to overcome the need of secondary approaches required to study SARS-CoV-2 infections in vivo.
Overview
This protocol describes the use of fluorescent or luminescent-expressing recombinant SARS-CoV-2 for in vivo and ex vivo live imaging of viral detection and tracking in K18 human ACE2 transgenic mice. This technique allows for effective evaluation of therapeutics for SARS-CoV-2 infection.
Key Study Components
Area of Science
- Virology
- In vivo imaging
- Transgenic mouse models
Background
- Understanding SARS-CoV-2 infection dynamics is crucial for therapeutic development.
- In vivo imaging systems (IVIS) facilitate real-time monitoring of viral behavior.
- K18 hACE2 transgenic mice are a relevant model for studying SARS-CoV-2.
- Fluorescent and luminescent reporters enhance detection capabilities.
Purpose of Study
- To develop a protocol for tracking SARS-CoV-2 infections in vivo.
- To assess pathogenicity, viral replication, and trophism using imaging techniques.
- To improve therapeutic evaluation methods for SARS-CoV-2.
Methods Used
- Use of luciferase- and fluorescence-expressing recombinant SARS-CoV-2.
- In vivo imaging with IVIS after substrate injection.
- Monitoring bioluminescence signal post-imaging setup.
- Shaving mice to enhance imaging clarity.
Main Results
- Effective tracking of SARS-CoV-2 in the K18 hACE2 transgenic mouse model.
- Demonstrated feasibility of in vivo and ex vivo imaging for viral studies.
- Provided insights into viral dynamics and therapeutic responses.
- Established a reliable method for assessing SARS-CoV-2 pathogenicity.
Conclusions
- The protocol offers a robust approach for studying SARS-CoV-2 infections.
- In vivo imaging enhances understanding of viral behavior and treatment efficacy.
- This technique can be adapted for other viral studies in transgenic models.
Frequently Asked Questions
What is the significance of using K18 hACE2 transgenic mice?
These mice are a relevant model for studying SARS-CoV-2 infections due to their expression of human ACE2, the virus's entry receptor.
How does the imaging technique improve therapeutic evaluation?
It allows for real-time monitoring of viral dynamics and treatment responses, providing valuable insights into therapeutic efficacy.
What are the advantages of using luminescent reporters?
Luminescent reporters enhance the sensitivity and specificity of viral detection in live subjects.
Why is shaving the mice recommended?
Shaving improves the clarity of the bioluminescence signal, enhancing imaging results.
Can this protocol be adapted for other viruses?
Yes, the imaging techniques can be modified for studying various viral infections in transgenic models.
What is the role of IVIS in this study?
IVIS is used for in vivo imaging to monitor viral infections and track their progression in real-time.
Executive Industry Relevance
Live imaging of viral infection using reporter-expressing SARS-CoV-2 enables real-time, quantitative monitoring of pathogen dynamics in vivo, supporting early-stage therapeutic evaluation. This approach reduces reliance on endpoint assays and enhances predictive confidence in antiviral screening by providing longitudinal infection tracking. The K18 hACE2 transgenic mouse model combined with bioluminescent or fluorescent reporters offers a translationally relevant system for de-risking antiviral candidates before costly preclinical studies.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables direct visualization of viral infection kinetics to validate therapeutic targets in a physiologically relevant model.
- Operational Value: Supports mechanistic de-risking by correlating reporter signal with viral load and tissue tropism.
- Predictive Value: Facilitates target confirmation through longitudinal monitoring of infection progression and response to intervention.
Screening & Assay Development
- Scientific Value: Provides quantitative, non-invasive readouts for high-throughput assessment of antiviral compounds in vivo.
- Operational Value: Enables standardized, reproducible imaging workflows compatible with IVIS platforms for longitudinal studies.
- Assay Readiness: Generates scalable flux and radiant efficiency metrics that support dose-response and time-course analyses.
Translational & Preclinical Research
- Translational Continuity: Bridges in vitro findings to in vivo efficacy by tracking viral spread and clearance in relevant tissues.
- Disease Relevance: Uses K18 hACE2 mice to model human-like SARS-CoV-2 pathogenesis, including weight loss and mortality.
- Risk-Adjusted Advancement: Allows early go/no-go decisions based on measurable reductions in bioluminescent or fluorescent signal.
Pipeline & Workflow Integration
This imaging method fits within the antiviral discovery continuum from target validation through lead optimization to preclinical efficacy, enabling data-driven decisions at each stage.
- Discovery Biology: Supports hypothesis testing by visualizing real-time viral replication and tissue-specific infection patterns.
- Screening: Delivers quantitative bioluminescence and fluorescence outputs that allow comparison of compound effects across time and dose.
- Analytics: Provides measurable flux and radiant efficiency values that enable statistical comparison of infection levels between groups.
- Translational Research: Connects antiviral activity to reduction in viral signal in lungs and other organs, mirroring clinical endpoints.
- Enterprise Reuse: Establishes a reusable imaging platform applicable to multiple viral pathogens and therapeutic modalities.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence by reducing variability associated with endpoint harvesting and enabling within-subject longitudinal tracking.
- Operational Value: Enhances reproducibility through standardized imaging protocols and quantifiable signal normalization to mock-infected controls.
- Strategic Value: Improves capital efficiency by identifying ineffective candidates earlier, reducing late-stage failure risk.
- Portfolio Impact: Supports risk-adjusted prioritization by delivering early, measurable evidence of target engagement and antiviral effect.
Implementation Considerations
- Requires expertise in biosafety level 3 procedures, IVIS operation, and fluorescent/bioluminescent imaging optimization.
- Dependent on access to IVIS systems with heating chambers, ROI analysis tools, and substrate injection capabilities.
- Necessitates standardization across teams for shaving, anesthesia, and timing of substrate administration to minimize signal variability.
- Involves adaptation considerations for different reporter proteins (Nluc vs Venus) and tissue types (lungs, organs) in ex vivo analysis.
- Includes practical limitations such as signal attenuation in deep tissues and the need for ex vivo validation to confirm in vivo findings.
Biopharma FAQs
Why does longitudinal bioluminescence tracking matter for target validation?
Longitudinal bioluminescence tracking enables repeated measurement of viral load in the same animal, reducing variability and increasing statistical power in target validation studies. This approach supports mechanistic de-risking by linking target modulation to measurable changes in infection kinetics over time.
How does isolating the viral replication signal improve discovery pipeline efficiency?
Isolating the viral replication signal through reporter expression allows specific quantification of SARS-CoV-2 activity independent of host background noise. This enables accurate assessment of antiviral effects in vivo, improving hit-to-lead progression by providing clear, quantifiable endpoints for compound screening.
What quantitative measurements enable predictive confidence in antiviral screening?
Quantitative measurements such as radiant efficiency and flux values from ROI analysis provide objective, numerical readouts of viral burden that can be compared across treatment groups. These metrics support dose-response modeling and help establish exposure-response relationships critical for predictive confidence in lead selection.
Why do replication requirements matter for cross-functional collaboration?
Replication requirements ensure that imaging results are consistent across experiments, sites, and operators, which is essential for building confidence in data shared between discovery, preclinical, and translational teams. Standardized protocols for shaving, substrate dosing, and imaging timing enhance reproducibility and facilitate technology transfer.
What statistical analysis capabilities are required before implementing this imaging method?
Before implementation, teams must establish capabilities for longitudinal data analysis, including mixed-effects models to account for repeated measures and group comparisons using ANOVA or t-tests with appropriate corrections. These analyses are needed to determine statistically significant differences in viral signal between control and treatment groups across time points.
Chapters in this video
0:05
Introduction
1:15
Bioluminescence Monitoring K18 hACE2 Transgenic Mice Infected with rSARS-CoV-2/Nluc
2:24
Fluorescence Analysis in K18 hACE2 Transgenic Mice Infected with rSARS-CoV-2/Venus
3:49
Viral Titrations
5:48
Results: rSARS-CoV-2Nluc and rSARS-CoV-2/Venus Expression in Infected K18 Hace2 Transgenic Mice
8:12
Conclusion
