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Construction of a recombinant rhinovirus accommodating fluorescent marker expression.

ABSTRACT: 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.

SUBMITTER: Han M

PROVIDER: S-EPMC6185886 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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Construction of a recombinant rhinovirus accommodating fluorescent marker expression.

Han Mingyuan M Rajput Charu C Hinde Joanna L JL Wu Qian Q Lei Jing J Ishikawa Tomoko T Bentley J Kelley JK Hershenson Marc B MB

Influenza and other respiratory viruses 20180906 6

<h4>Background</h4>Rhinovirus (RV) causes the common cold and asthma exacerbations. The RV genome is a 7.3 kb single-strand positive-sense RNA.<h4>Objective</h4>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.<h4>Method</h4>Recombinant RV1A infectious cDNA clones harboring the coding sequence of green fluorescent protein (GFP), Renilla luciferase, or iLOV (for light ...[more]

PMID: 30120824

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Construction of a recombinant rhinovirus accommodating fluorescent marker expression.

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Construction of a recombinant rhinovirus accommodating fluorescent marker expression.

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