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 III, 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
Cell, doi:10.1016/j.cell.2020.05.042
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.
SUPPLEMENTAL INFORMATION Supplemental Information can be found online at https://doi.org/10.1016/j. cell.2020.05.042.
DECLARATION OF INTERESTS The authors declare no competing financial interests. Assembly of SARS-CoV-2 WT and reporter cDNA constructs Seven cDNA fragments covering the entire SARS-CoV-2 WA1 genome were amplified by RT-PCR using PrimeSTAR GXL HiFi DNA polymerase (TaKaRa). Junctions between each fragment contain non-palindromic sites BsaI (GGTCTCN^NNNN) or BsmBI (CGTCTCN^NNNN) with unique four-nucleotide cohesive ends. Fragment E and F contains two BsmBI sites at both termini, while other fragments harbor BsaI sites at the junction. Four-nucleotide cohesive ends of each fragment are indicated in Figure 1A . To assist the transcription of full-length viral RNA, we introduced a T7 promoter sequence into the upstream of fragment A, as well as a 25nt poly-A tail into the downstream of the fragment G. Each fragment was cloned into high-copy vector pUC57 and verified by Sanger sequencing. A silent mutation T15102A was introduced into a conserved region in nsp12 in plasmid D as a genetic marker. To enhance the efficiency of recovering SARS-CoV-2 virus in the cell culture, a sgRNA-N construct, encoding a 75nt leader sequence, N gene, 3 0 UTR, and a 25nt poly-A tail, was assembled under the control of a T7 promoter. Two reporter viruses, one containing GFP and the other harboring, a GFP-fused nLuc gene, were generated by replacing the ORF7 gene with the reporter..
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