Giuseppe
Emeritus
Analysis of in vivo dynamics of influenza virus infection in mice using a GFP reporter virus (Proc Natl Acad Sci USA, abstract, edited)
[Source: PNAS, <cite cite="http://www.pnas.org/content/107/25/11531.short?rss=1">Analysis of in vivo dynamics of influenza virus infection in mice using a GFP reporter virus ? PNAS</cite>. Abstract, edited.]
Analysis of in vivo dynamics of influenza virus infection in mice using a GFP reporter virus
Balaji Manicassamy a,b, Santhakumar Manicassamy c, Alan Belicha-Villanueva a,b, Giuseppe Pisanelli a,d, Bali Pulendran c, and Adolfo Garc?a-Sastre a,b,e,1
Author Affiliations
a) Department of Microbiology, b) Global Health and Emerging Pathogens Institute, and e) Department of Medicine, Division of Infectious Diseases, Mount Sinai School of Medicine, New York, NY 10029; c) Emory Vaccine Center, Emory University, Atlanta, GA 30329; and d) Department of Pathology and Animal Health, School of Biotechnological Sciences, University of Naples ?Federico II?, 80137 Naples, Italy
Edited by Rafi Ahmed, Emory University, Atlanta, GA, and approved May 12, 2010 (received for review December 30, 2009)
Abstract
Influenza A virus is being extensively studied because of its major impact on human and animal health. However, the dynamics of influenza virus infection and the cell types infected in vivo are poorly understood. These characteristics are challenging to determine, partly because there is no efficient replication-competent virus expressing an easily traceable reporter gene. Here, we report the generation of a recombinant influenza virus carrying a GFP reporter gene in the NS segment (NS1-GFP virus). Although attenuated when compared with wild-type virus, the NS1-GFP virus replicates efficiently in murine lungs and shows pathogenicity in mice. Using whole-organ imaging and flow cytometry, we have tracked the dynamics of influenza virus infection progression in mice. Imaging of murine lungs shows that infection starts in the respiratory tract in areas close to large conducting airways and later spreads to deeper sections of the lungs. In addition to epithelial cells, we found GFP-positive antigen-presenting cells, such as CD11b+CD11c−, CD11b−CD11c+, and CD11b+CD11c+, as early as 24 h after intranasal infection. In addition, a significant proportion of NK and B cells were GFP positive, suggesting active infection of these cells. We next tested the effects of the influenza virus inhibitors oseltamivir and amantadine on the kinetics of in vivo infection progression. Treatment with oseltamivir dramatically reduced influenza infection in all cell types, whereas, surprisingly, amantadine treatment more efficiently blocked infection in B and NK cells. Our results demonstrate high levels of immune cells harboring influenza virus antigen during viral infection and cell-type?specific effects upon treatment with antiviral agents, opening additional avenues of research in the influenza virus field.
* antivirals * pathogenesis * recombinant influenza virus * GFP virus * cell tropism
Footnotes
1) To whom correspondence should be addressed. E-mail: adolfo.garcia-sastre@mssm.edu.
Author contributions: B.M., S.M., A.B.-V., G.P., B.P., and A.G.-S. designed research; B.M., S.M., A.B.-V., and G.P. performed research; B.M., B.P., and A.G.-S. contributed new reagents/analytic tools; B.M., S.M., A.B.-V., G.P., B.P., and A.G.-S. analyzed data; and B.M., S.M., and A.G.-S. wrote the paper.
Conflict of interest statement: Mount Sinai School of Medicine has filed a patent application covering the use of recombinant influenza viruses expressing a reporter gene.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.0914994107/-/DCSupplemental.
Freely available online through the PNAS open access option.
-
------<cite cite="http://www.pnas.org/content/107/25/11531.short?rss=1"></cite>
[Source: PNAS, <cite cite="http://www.pnas.org/content/107/25/11531.short?rss=1">Analysis of in vivo dynamics of influenza virus infection in mice using a GFP reporter virus ? PNAS</cite>. Abstract, edited.]
Analysis of in vivo dynamics of influenza virus infection in mice using a GFP reporter virus
Balaji Manicassamy a,b, Santhakumar Manicassamy c, Alan Belicha-Villanueva a,b, Giuseppe Pisanelli a,d, Bali Pulendran c, and Adolfo Garc?a-Sastre a,b,e,1
Author Affiliations
a) Department of Microbiology, b) Global Health and Emerging Pathogens Institute, and e) Department of Medicine, Division of Infectious Diseases, Mount Sinai School of Medicine, New York, NY 10029; c) Emory Vaccine Center, Emory University, Atlanta, GA 30329; and d) Department of Pathology and Animal Health, School of Biotechnological Sciences, University of Naples ?Federico II?, 80137 Naples, Italy
Edited by Rafi Ahmed, Emory University, Atlanta, GA, and approved May 12, 2010 (received for review December 30, 2009)
Abstract
Influenza A virus is being extensively studied because of its major impact on human and animal health. However, the dynamics of influenza virus infection and the cell types infected in vivo are poorly understood. These characteristics are challenging to determine, partly because there is no efficient replication-competent virus expressing an easily traceable reporter gene. Here, we report the generation of a recombinant influenza virus carrying a GFP reporter gene in the NS segment (NS1-GFP virus). Although attenuated when compared with wild-type virus, the NS1-GFP virus replicates efficiently in murine lungs and shows pathogenicity in mice. Using whole-organ imaging and flow cytometry, we have tracked the dynamics of influenza virus infection progression in mice. Imaging of murine lungs shows that infection starts in the respiratory tract in areas close to large conducting airways and later spreads to deeper sections of the lungs. In addition to epithelial cells, we found GFP-positive antigen-presenting cells, such as CD11b+CD11c−, CD11b−CD11c+, and CD11b+CD11c+, as early as 24 h after intranasal infection. In addition, a significant proportion of NK and B cells were GFP positive, suggesting active infection of these cells. We next tested the effects of the influenza virus inhibitors oseltamivir and amantadine on the kinetics of in vivo infection progression. Treatment with oseltamivir dramatically reduced influenza infection in all cell types, whereas, surprisingly, amantadine treatment more efficiently blocked infection in B and NK cells. Our results demonstrate high levels of immune cells harboring influenza virus antigen during viral infection and cell-type?specific effects upon treatment with antiviral agents, opening additional avenues of research in the influenza virus field.
* antivirals * pathogenesis * recombinant influenza virus * GFP virus * cell tropism
Footnotes
1) To whom correspondence should be addressed. E-mail: adolfo.garcia-sastre@mssm.edu.
Author contributions: B.M., S.M., A.B.-V., G.P., B.P., and A.G.-S. designed research; B.M., S.M., A.B.-V., and G.P. performed research; B.M., B.P., and A.G.-S. contributed new reagents/analytic tools; B.M., S.M., A.B.-V., G.P., B.P., and A.G.-S. analyzed data; and B.M., S.M., and A.G.-S. wrote the paper.
Conflict of interest statement: Mount Sinai School of Medicine has filed a patent application covering the use of recombinant influenza viruses expressing a reporter gene.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.0914994107/-/DCSupplemental.
Freely available online through the PNAS open access option.
-
------<cite cite="http://www.pnas.org/content/107/25/11531.short?rss=1"></cite>