SARS-CoV-2 Reverse Genetics Reveals a Variable Infection Gradient in the Respiratory Tract

Yixuan J Hou; Kenichi Okuda; Caitlin E Edwards; David R Martinez; Takanori Asakura; Kenneth H Dinnon 3rd; Takafumi Kato; Rhianna E Lee; Boyd L Yount; Teresa M Mascenik; Gang Chen; Kenneth N Olivier; Andrew Ghio; Longping V Tse; Sarah R Leist; Lisa E Gralinski; Alexandra Schäfer; Hong Dang; Rodney Gilmore; Satoko Nakano; Ling Sun; M Leslie Fulcher; Alessandra Livraghi-Butrico; Nathan I Nicely; Mark Cameron; Cheryl Cameron; David J Kelvin; Aravinda de Silva; David M Margolis; Alena Markmann; Luther Bartelt; Ross Zumwalt; Fernando J Martinez; Steven P Salvatore; Alain Borczuk; Purushothama R Tata; Vishwaraj Sontake; Adam Kimple; Ilona Jaspers; Wanda K O'Neal; Scott H Randell; Richard C Boucher; Ralph S Baric

doi:10.1016/j.cell.2020.05.042

SARS-CoV-2 Reverse Genetics Reveals a Variable Infection Gradient in the Respiratory Tract

Cell. 2020 Jul 23;182(2):429-446.e14. doi: 10.1016/j.cell.2020.05.042. Epub 2020 May 27.

Authors

Yixuan J Hou¹, Kenichi Okuda², Caitlin E Edwards¹, David R Martinez¹, Takanori Asakura², Kenneth H Dinnon 3rd³, Takafumi Kato², Rhianna E Lee², Boyd L Yount¹, Teresa M Mascenik², Gang Chen², Kenneth N Olivier⁴, Andrew Ghio⁵, Longping V Tse¹, Sarah R Leist¹, Lisa E Gralinski¹, Alexandra Schäfer¹, Hong Dang², Rodney Gilmore², Satoko Nakano², Ling Sun², M Leslie Fulcher², Alessandra Livraghi-Butrico², Nathan I Nicely⁶, Mark Cameron⁷, Cheryl Cameron⁸, David J Kelvin⁹, Aravinda de Silva³, David M Margolis¹⁰, Alena Markmann¹¹, Luther Bartelt¹¹, Ross Zumwalt¹², Fernando J Martinez¹³, Steven P Salvatore¹⁴, Alain Borczuk¹⁴, Purushothama R Tata¹⁵, Vishwaraj Sontake¹⁵, Adam Kimple¹⁶, Ilona Jaspers¹⁷, Wanda K O'Neal², Scott H Randell², Richard C Boucher¹⁸, Ralph S Baric¹⁹

Affiliations

¹ Department of Epidemiology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
² Marsico Lung Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
³ Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
⁴ Laboratory of Chronic Airway Infection, Pulmonary Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA.
⁵ National Health and Environmental Effects Research Laboratory, Environmental Protection Agency, Chapel Hill, NC, USA.
⁶ Protein Expression and Purification Core, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
⁷ Department of Population and Quantitative Health Science, Case Western Reserve University, Cleveland, OH, USA.
⁸ Department of Nutrition, Case Western Reserve University, Cleveland, OH, USA.
⁹ Department of Microbiology and Immunology, Canadian Center for Vaccinology, Dalhousie University, Halifax, NS, Canada; Laboratory of Immunology, Shantou University Medical College, Shantou, Guangdong, China.
¹⁰ Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; HIV Cure Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
¹¹ Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
¹² Department of Pathology, University of New Mexico, Albuquerque, NM, USA.
¹³ Division of Pulmonary and Critical Care Medicine, Joan & Sanford I. Weill Medical College of Cornell University, New York, NY, USA.
¹⁴ Department of Pathology, Joan & Sanford I. Weill Medical College of Cornell University, New York, NY, USA.
¹⁵ Department of Cell Biology, Regeneration Next Initiative, Duke University Medical Center, Durham, NC, USA.
¹⁶ Department of Otolaryngology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
¹⁷ Department of Pediatrics, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
¹⁸ Marsico Lung Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA. Electronic address: richard_boucher@med.unc.edu.
¹⁹ Department of Epidemiology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA. Electronic address: rbaric@email.unc.edu.

Abstract

The mode of acquisition and causes for the variable clinical spectrum of coronavirus disease 2019 (COVID-19) remain unknown. We utilized a reverse genetics system to generate a GFP reporter virus to explore severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogenesis and a luciferase reporter virus to demonstrate sera collected from SARS and COVID-19 patients exhibited limited cross-CoV neutralization. High-sensitivity RNA in situ mapping revealed the highest angiotensin-converting enzyme 2 (ACE2) expression in the nose with decreasing expression throughout the lower respiratory tract, paralleled by a striking gradient of SARS-CoV-2 infection in proximal (high) versus distal (low) pulmonary epithelial cultures. COVID-19 autopsied lung studies identified focal disease and, congruent with culture data, SARS-CoV-2-infected ciliated and type 2 pneumocyte cells in airway and alveolar regions, respectively. These findings highlight the nasal susceptibility to SARS-CoV-2 with likely subsequent aspiration-mediated virus seeding to the lung in SARS-CoV-2 pathogenesis. These reagents provide a foundation for investigations into virus-host interactions in protective immunity, host susceptibility, and virus pathogenesis.

Keywords: ACE2; COVID-19; SARS-CoV-2; infectious clone; nasal infection; neutralization assay; primary cells; reporter virus; respiratory tropism; reverse genetics.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Aged
Angiotensin-Converting Enzyme 2
Animals
Antibodies, Monoclonal / immunology
Antibodies, Neutralizing / immunology
Betacoronavirus / genetics*
Betacoronavirus / immunology
Betacoronavirus / pathogenicity
COVID-19
COVID-19 Serotherapy
Cell Line
Cells, Cultured
Chlorocebus aethiops
Coronavirus Infections / immunology
Coronavirus Infections / pathology*
Coronavirus Infections / therapy
Coronavirus Infections / virology*
Cystic Fibrosis / pathology
DNA, Recombinant
Female
Furin / metabolism
Humans
Immunization, Passive
Lung / metabolism
Lung / pathology
Lung / virology
Male
Middle Aged
Nasal Mucosa / metabolism
Nasal Mucosa / pathology
Nasal Mucosa / virology
Pandemics
Peptidyl-Dipeptidase A / metabolism
Pneumonia, Viral / immunology
Pneumonia, Viral / pathology*
Pneumonia, Viral / virology*
Respiratory System / pathology
Respiratory System / virology*
Reverse Genetics / methods*
SARS-CoV-2
Serine Endopeptidases / metabolism
Vero Cells
Virulence
Virus Replication

Substances

Antibodies, Monoclonal
Antibodies, Neutralizing
DNA, Recombinant
Peptidyl-Dipeptidase A
ACE2 protein, human
Angiotensin-Converting Enzyme 2
Serine Endopeptidases
TMPRSS2 protein, human
Furin

Abstract

Publication types

MeSH terms

Substances

Grants and funding