title: Optimisations and Challenges Involved in the Creation of Various Bioluminescent and Fluorescent Influenza A Virus Strains for In Vitro and In Vivo Applications.
abstract: Bioluminescent and fluorescent influenza A viruses offer new opportunities to study influenza virus replication, tropism and pathogenesis. To date, several influenza A reporter viruses have been described. These strategies typically focused on a single reporter gene (either bioluminescent or fluorescent) in a single virus backbone. However, whilst bioluminescence is suited to in vivo imaging, fluorescent viruses are more appropriate for microscopy. Therefore, the idea l reporter virus varies depending on the experiment in question, and it is important that any reporter virus strategy can be adapted accordingly. Herein, a strategy was developed to create five different reporter viruses in a single virus backbone. Specifically, enhanced green fluorescent protein (eGFP), far-red fluorescent protein (fRFP), near-infrared fluorescent protein (iRFP), Gaussia luciferase (gLUC) and firefly luciferase (fLUC) were inserted into the PA gene segment of A/PR/8/34 (H1N1). This study provides a comprehensive characterisation of the effects of different reporter genes on influenza virus replication and reporter activity. In vivo reporter gene expression, in lung tissues, was only detected for eGFP, fRFP and gLUC expressing viruses. In vitro, the eGFP-expressing virus displayed the best reporter stability and could be used for correlative light electron microscopy (CLEM). This strategy was then used to create eGFP-expressing viruses consisting entirely of pandemic H1N1, highly pathogenic avian influenza (HPAI) H5N1 and H7N9. The HPAI H5N1 eGFP-expressing virus infected mice and reporter gene expression was detected, in lung tissues, in vivo. Thus, this study provides new tools and insights for the creation of bioluminescent and fluorescent influenza A reporter viruses.
authors.affiliation: Department of Viroscience, Erasmus Medical Centre, Rotterdam, the Netherlands.
authors.forename: Monique I
authors.initials: MI
authors.lastname: Spronken
authors.affiliation: Department of Viroscience, Erasmus Medical Centre, Rotterdam, the Netherlands; School of Biomedical Sciences, University of Queensland, Brisbane, Australia.
authors.forename: Kirsty R
authors.initials: KR
authors.lastname: Short
authors.affiliation: Department of Viroscience, Erasmus Medical Centre, Rotterdam, the Netherlands.
authors.forename: Sander
authors.initials: S
authors.lastname: Herfst
authors.affiliation: Department of Viroscience, Erasmus Medical Centre, Rotterdam, the Netherlands.
authors.forename: Theo M
authors.initials: TM
authors.lastname: Bestebroer
authors.affiliation: Department of Viroscience, Erasmus Medical Centre, Rotterdam, the Netherlands.
authors.forename: Vincent P
authors.initials: VP
authors.lastname: Vaes
authors.affiliation: Department of Molecular Cell Biology, Section Electron Microscopy, Leiden University Medical Centre, Leiden, the Netherlands.
authors.forename: Barbara
authors.initials: B
authors.lastname: van der Hoeven
authors.affiliation: Department of Molecular Cell Biology, Section Electron Microscopy, Leiden University Medical Centre, Leiden, the Netherlands.
authors.forename: Abraham J
authors.initials: AJ
authors.lastname: Koster
authors.affiliation: Erasmus Optical Imaging Centre, Department of Pathology, Erasmus University Medical Centre, Rotterdam, the Netherlands.
authors.forename: Gert-Jan
authors.initials: GJ
authors.lastname: Kremers
authors.affiliation: Rocky Mountain Veterinary Branch, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT, United States of America.
authors.forename: Dana P
authors.initials: DP
authors.lastname: Scott
authors.affiliation: Department of Viroscience, Erasmus Medical Centre, Rotterdam, the Netherlands; Leiden Institute of Advanced Computer Science, Leiden University, Leiden, the Netherlands.
authors.forename: Alexander P
authors.initials: AP
authors.lastname: Gultyaev
authors.affiliation: Department of Viroscience, Erasmus Medical Centre, Rotterdam, the Netherlands; Milken Institute School of Public Health, Department of Health Policy and Management, George Washington University, Washington, DC, United States of America.
authors.forename: Erin M
authors.initials: EM
authors.lastname: Sorell
authors.affiliation: Department of Viroscience, Erasmus Medical Centre, Rotterdam, the Netherlands.
authors.forename: Miranda
authors.initials: M
authors.lastname: de Graaf
authors.affiliation: Department of Molecular Cell Biology, Section Electron Microscopy, Leiden University Medical Centre, Leiden, the Netherlands.
authors.forename: Montserrat
authors.initials: M
authors.lastname: Bárcena
authors.affiliation: Department of Viroscience, Erasmus Medical Centre, Rotterdam, the Netherlands.
authors.forename: Guus F
authors.initials: GF
authors.lastname: Rimmelzwaan
authors.affiliation: Department of Viroscience, Erasmus Medical Centre, Rotterdam, the Netherlands.
authors.forename: Ron A
authors.initials: RA
authors.lastname: Fouchier
mesh_terms: D000369:Aged, 80 and over; D000818:Animals; D003673:Defective Viruses / Q000235:genetics / Q000502:physiology; D004285:Dogs; D005260:Female; D005453:Fluorescence; D017930:Genes, Reporter; D005818:Genetic Engineering / Q000379:methods; D006801:Humans; D066298:In Vitro Techniques; D009980:Influenza A virus / Q000235:genetics* / Q000472:pathogenicity / Q000502:physiology; D000069416:Intravital Microscopy; D008163:Luminescent Measurements; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics*; D008168:Lung / Q000648:ultrastructure / Q000821:virology; D061985:Madin Darby Canine Kidney Cells; D051379:Mice; D008807:Mice, Inbred BALB C; D008854:Microscopy, Electron; D009154:Mutation; D030781:Organisms, Genetically Modified; D009976:Orthomyxoviridae Infections / Q000473:pathology / Q000821:virology; D011401:Promoter Regions, Genetic / Q000235:genetics; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis; D019562:Viral Load; D014764:Viral Proteins / Q000235:genetics*; D014773:Virology / Q000379:methods*; D014779:Virus Replication
chemical_list: D008164:Luminescent Proteins; C061290:PA protein, influenza viruses; D011994:Recombinant Proteins; D014764:Viral Proteins; D012324:RNA-Dependent RNA Polymerase