title: A GFP expressing influenza A virus to report in vivo tropism and protection by a matrix protein 2 ectodomain-specific monoclonal antibody.
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.
authors: Department of Medical Protein Research, VIB, Ghent, Belgium; Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium.
authors: Sarah
authors: S
authors: De Baets
authors: Department of Medical Protein Research, VIB, Ghent, Belgium; Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium.
authors: Judith
authors: J
authors: Verhelst
authors: Department of Medical Protein Research, VIB, Ghent, Belgium; Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium.
authors: Silvie
authors: S
authors: Van den Hoecke
authors: Department of Medical Protein Research, VIB, Ghent, Belgium; Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium.
authors: Anouk
authors: A
authors: Smet
authors: Department of Medical Protein Research, VIB, Ghent, Belgium; Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium.
authors: Michael
authors: M
authors: Schotsaert
authors: Department of Medical Protein Research, VIB, Ghent, Belgium; Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium.
authors: Emma R
authors: ER
authors: Job
authors: Department of Medical Protein Research, VIB, Ghent, Belgium; Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium.
authors: Kenny
authors: K
authors: Roose
authors: Department of Medical Protein Research, VIB, Ghent, Belgium; Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium.
authors: Bert
authors: B
authors: Schepens
authors: Department of Medical Protein Research, VIB, Ghent, Belgium; Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium.
authors: Walter
authors: W
authors: Fiers
authors: Department of Medical Protein Research, VIB, Ghent, Belgium; Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium.
authors: Xavier
authors: X
authors: Saelens
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000008:administration & dosage* / Q000627:therapeutic use; D000998:Antiviral Agents / Q000008:administration & dosage / Q000494:pharmacology; D002478:Cells, Cultured; D003713:Dendritic Cells / Q000378:metabolism / Q000821:virology; D004285:Dogs; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D009980:Influenza A virus / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D061985:Madin Darby Canine Kidney Cells; D051379:Mice; D009000:Monocytes / Q000378:metabolism / Q000821:virology; D009976:Orthomyxoviridae Infections / Q000378:metabolism / Q000517:prevention & control* / Q000821:virology; D053139:Oseltamivir / Q000008:administration & dosage / Q000494:pharmacology; D055550:Protein Stability; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014763:Viral Matrix Proteins / Q000037:antagonists & inhibitors; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*; D056189:Viral Tropism / Q000187:drug effects*; D014779:Virus Replication / Q000187:drug effects; D000086082:Viroporin Proteins
chemical_list: D000911:Antibodies, Monoclonal; D000998:Antiviral Agents; D049452:Green Fluorescent Proteins; D053139:Oseltamivir; D011993:Recombinant Fusion Proteins; D014763:Viral Matrix Proteins; D017361:Viral Nonstructural Proteins; C085820:INS1 protein, influenza virus; C488937:M2 protein, Influenza A virus; D000086082:Viroporin Proteins