Abstract
The severity of influenza-related illness is mediated by many factors, including in vivo cell tropism, timing and magnitude of the immune response, and presence of pre-existing immunity. A direct way to study cell tropism and virus spread in vivo is with an influenza virus expressing a reporter gene. However, reporter gene-expressing influenza viruses are often attenuated in vivo and may be genetically unstable. Here, we describe the generation of an influenza A virus expressing GFP from a tri-cistronic NS segment. To reduce the size of this engineered gene segment, we used a truncated NS1 protein of 73 amino acids combined with a heterologous dimerization domain to increase protein stability. GFP and nuclear export protein coding information were fused in frame with the truncated NS1 open reading frame and separated from each other by 2A self-processing sites. The resulting PR8-NS1(1-73)GFP virus was successfully rescued and replicated as efficiently as the parental PR8 virus in vitro and was slightly attenuated in vivo. Flow cytometry-based monitoring of cells isolated from PR8-NS1(1-73)GFP virus infected BALB/c mice revealed that GFP expression peaked on day two in all cell types tested. In particular respiratory epithelial cells and myeloid cells known to be involved in antigen presentation, including dendritic cells (CD11c+) and inflammatory monocytes (CD11b+ GR1+), became GFP positive following infection. Prophylactic treatment with anti-M2e monoclonal antibody or oseltamivir reduced GFP expression in all cell types studied, demonstrating the usefulness of this reporter virus to analyze the efficacy of antiviral treatments in vivo. Finally, deep sequencing analysis, serial in vitro passages and ex vivo analysis of PR8-NS1(1-73)GFP virus, indicate that this virus is genetically and phenotypically stable.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Antibodies, Monoclonal / administration & dosage*
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Antibodies, Monoclonal / therapeutic use
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Antiviral Agents / administration & dosage
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Antiviral Agents / pharmacology
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Cells, Cultured
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Dendritic Cells / metabolism
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Dendritic Cells / virology
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Dogs
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Green Fluorescent Proteins / genetics
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Green Fluorescent Proteins / metabolism*
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Influenza A virus / drug effects
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Influenza A virus / genetics
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Influenza A virus / physiology*
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Madin Darby Canine Kidney Cells
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Mice
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Monocytes / metabolism
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Monocytes / virology
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Orthomyxoviridae Infections / metabolism
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Orthomyxoviridae Infections / prevention & control*
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Orthomyxoviridae Infections / virology
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Oseltamivir / administration & dosage
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Oseltamivir / pharmacology
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Protein Stability
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Recombinant Fusion Proteins / chemistry
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / metabolism
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Viral Matrix Proteins / antagonists & inhibitors
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Viral Nonstructural Proteins / genetics
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Viral Nonstructural Proteins / metabolism*
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Viral Tropism / drug effects*
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Virus Replication / drug effects
Substances
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Antibodies, Monoclonal
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Antiviral Agents
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INS1 protein, influenza virus
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M2 protein, Influenza A virus
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Recombinant Fusion Proteins
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Viral Matrix Proteins
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Viral Nonstructural Proteins
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Green Fluorescent Proteins
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Oseltamivir
Associated data
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SRA/SRR1752132
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SRA/SRR1766133
Grants and funding
This work was supported by Agentschap voor innovatie door wetenschap en techniek (IWT) grants 81050 and 83050 to SDB, by Fonds wetenschappelijk onderzoek Vlaanderen (FWO-Vlaanderen) grants to BS and SVdH, by a FWO14/KAN/042 grant to BS, by IUAP BELVIR project p7/45 and by Ghent University Special Research Grant BOF12/GOA/014 to JV. KR is currently supported by EC FP7 project FLUNIVAC. The Flow Cytometer core facility at IRC is supported by a Methusalem grant (BOF09/01M00709) from Ghent University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.