Abstract
BackgroundRhinovirus (RV) causes the common cold and asthma exacerbations. The RV genome is a 7.3 kb single‐strand positive‐sense RNA.ObjectiveUsing 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.MethodRecombinant 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.ConclusionsThese 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.
Highlights
Rhinovirus (RV) is the most frequent viral infectious agent of the respiratory tract in humans and is the predominant cause of the common cold.[1]
RV is placed in the Picornaviridae family, genus Enterovirus, with three species based on phylogenetic sequence criteria.[5,6]
To detect RV in the tissues, investigators have employed the monoclonal antibody R16-7.9,13,14 This antibody, originally developed by Wai-M ing Lee at the University of Wisconsin, binds to the VP2 capsid protein of the closely related RV-A16 and RV-A1 strains,[6] but not to RV-A2, RV-B14, or RV-A49.15 Because the presence of more than 100 different RV serotypes makes it infeasible to develop a cross-reactive antibody for RV, we sought to develop a recombinant virus with a fluorescent marker that could be used for tracking of RV infection in vivo
Summary
Rhinovirus (RV) is the most frequent viral infectious agent of the respiratory tract in humans and is the predominant cause of the common cold.[1]. RV is placed in the Picornaviridae family, genus Enterovirus, with three species based on phylogenetic sequence criteria.[5,6] Clinical specimens collected from in the 1960s and 1970s yielded The smaller fluorescent protein construct, RV1A-iLOV, was stably expressed in infected cells. The detection of iLOV-expressing RV in fixed tissue required the use of an anti-iLOV antibody, limiting the value of this construct
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