Construction of a recombinant rhinovirus accommodating fluorescent marker expression

Area of Science:

• Virology
• Molecular Biology
• Immunology

Background:

• Rhinovirus (RV) is a common cause of respiratory infections and asthma exacerbations.
• The RV genome consists of a 7.3 kb single-strand positive-sense RNA.

Purpose of the Study:

• To engineer a recombinant Rhinovirus type 1A (RV1A) with fluorescent marker expression for infection tracking.
• To assess the utility of engineered RV1A constructs in studying viral pathogenesis and antiviral efficacy.

Main Methods:

• Engineered recombinant RV1A infectious cDNA clones expressing green fluorescent protein (GFP), Renilla luciferase, or iLOV.
• Detected RV-infected cells using flow cytometry, immunohistochemistry, and immunofluorescence microscopy.
• Evaluated RV1A-iLOV in cell culture and a mouse model to assess viral load, cytokine expression, and antiviral effects.

Main Results:

• RV1A-GFP and RV1A-Renilla luciferase constructs failed due to deletion; RV1A-iLOV showed stable expression in infected cells and mice.
• RV1A-iLOV facilitated examination of bafilomycin's antiviral effect and demonstrated similar viral loads and cytokine mRNA levels in mice compared to parental virus.
• Direct fluorescent imaging of iLOV in fixed lung tissue was unsuccessful; antibody-based detection identified iLOV signal in airway epithelial cells and lung macrophages.

Conclusions:

• RV1A-iLOV serves as a valuable molecular tool for Rhinovirus pathogenesis research.
• The construction strategy for RV1A-iLOV is adaptable to other RV serotypes.
• Detection of iLOV-expressing RV in fixed tissues necessitates anti-iLOV antibody use, which limits the construct's direct imaging utility.