Construction of a recombinant rhinovirus accommodating fluorescent marker expression

Han, Mingyuan; Rajput, Charu; Hinde, Joanna L.; Wu, Qian; Lei, Jing; Ishikawa, Tomoko; Bentley, J. Kelley; Hershenson, Marc B.

BACKGROUND: Rhinovirus (RV) causes the common cold and asthma exacerbations. The RV genome is a 7.3 kb single‐strand positive‐sense RNA. OBJECTIVE: Using minor group RV1A as a backbone, we sought to design and generate a recombinant RV1A accommodating fluorescent marker expression, thereby allowing tracking of viral infection. METHOD: Recombinant RV1A infectious cDNA clones harboring the coding sequence of green fluorescent protein (GFP), Renilla luciferase, or iLOV (for light, oxygen, or voltage sensing) were engineered and constructed. RV‐infected cells were determined by flow cytometry, immunohistochemistry, and immunofluorescence microscopy. RESULTS: RV1A‐GFP showed a cytopathic effect in HeLa cells but failed to express GFP or Renilla luciferase due to deletion. The smaller fluorescent protein construct, RV1A‐iLOV, was stably expressed in infected cells. RV1A‐iLOV expression was used to examine the antiviral effect of bafilomycin in HeLa cells. Compared to parental virus, RV1A‐iLOV infection of BALB/c mice yielded a similar viral load and level of cytokine mRNA expression. However, imaging of fixed lung tissue failed to reveal a fluorescent signal, likely due to the oxidation and bleaching of iLOV‐bound flavin mononucleotide. We therefore employed an anti‐iLOV antibody for immunohistochemical and immunofluorescence imaging. The iLOV signal was identified in airway epithelial cells and CD45+ CD11b+ lung macrophages. CONCLUSIONS: These results suggest that RV1A‐iLOV is a useful molecular tool for studying RV pathogenesis. The construction strategy for RV1A‐iLOV could be applied to other RV serotypes. However, the detection of iLOV‐expressing RV in fixed tissue required the use of an anti‐iLOV antibody, limiting the value of this construct.