








key: cord-350855-gofzhff7
authors: Hou, Yixuan J.; Okuda, Kenichi; Edwards, Caitlin E.; Martinez, David R.; Asakura, Takanori; Dinnon, Kenneth H.; Kato, Takafumi; Lee, Rhianna E.; Yount, Boyd L.; Mascenik, Teresa M.; Chen, Gang; Olivier, Kenneth N.; Ghio, Andrew; Tse, Longping V.; Leist, Sarah R.; Gralinski, Lisa E.; Schäfer, Alexandra; Dang, Hong; Gilmore, Rodney; Nakano, Satoko; Sun, Ling; Fulcher, M. Leslie; Livraghi-Butrico, Alessandra; Nicely, Nathan I.; Cameron, Mark; Cameron, Cheryl; Kelvin, David J.; de Silva, Aravinda; Margolis, David M.; Markmann, Alena; Bartelt, Luther; Zumwalt, Ross; Martinez, Fernando J.; Salvatore, Steven P.; Borczuk, Alain; Tata, Purushothama R.; Sontake, Vishwaraj; Kimple, Adam; Jaspers, Ilona; O’Neal, Wanda K.; Randell, Scott H.; Boucher, Richard C.; Baric, Ralph S.
title: SARS-CoV-2 Reverse Genetics Reveals a Variable Infection Gradient in the Respiratory Tract
date: 2020-05-27
journal: Cell
DOI: 10.1016/j.cell.2020.05.042
sha: 
doc_id: 350855
cord_uid: gofzhff7

Summary The mode of acquisition and causes for the variable clinical spectrum of COVID-19 remain unknown. We utilized a reverse genetics system to generate a GFP reporter virus to explore 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 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) vs 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.

We measured the relative infectivity of the SARS-CoV-2 GFP virus in primary 283 cells based on the average peak titers and observed that infectivity exhibited the same 284 pattern as the ACE2 expression levels from the upper to lower respiratory tract ( Figure  285 6Bi-6Biv). The icSARS-CoV-2-GFP virus replicated efficiently in HNE and LAE, with 286 peak viral titers significantly higher than the titers in SAE, AT2-like and AT1-like cultures 287 ( Figure 6Bv ). Although the viral peak titers were similar, the icSARS-CoV-2-GFP 288 infection in HNE culture resulted in significantly higher titers than LAE at 24h, 48h and 289 96h post-infection, suggesting more robust replication in the primary nasal cells ( Figure  290 6Bvi). Collectively, these data indicate that virus infectivity/replication efficiency varies 291 markedly from proximal airway to alveolar respiratory regions. 292

Whole mount immunohistochemistry of HNE and LAE cultures was utilized to 293 identify cell types infected by SARS-CoV-2 ( Figure 6C , S4A). The ciliated cell was 294 routinely infected and extruded. In contrast, the other major cell type facing the airway 295 lumen, i.e., the MUC5B+ club cell, was not infected, nor was the MUC5AC+ metaplastic 296 goblet cell. We did note a cell type co-expressing the ciliated cell marker tubulin and 297 MUC5B was rarely infected in HNE, a finding consistent with infection of a 298 secretory/club cell transitioning to a ciliated cell phenotype. 299

There is debate whether AT2 and/or AT1 cells express sufficient ACE2 to 300 mediate infection and whether AT2, AT1, or both cell types are infectable. Previous 301 studies reported 2003 SARS-CoV infects AT2 but not AT1 pneumocytes (Mossel et al., 302 14 standard AT2/AT1 cell cultures and a novel cell culture approach that well preserves 304 AT2 and AT1 cell populations over the infection/GFP expression interval were tested. 305

As shown in Figure 6A and S4B, AT2 cells appeared to be preferentially infected. Second, to further characterize the infectivity of LAE vs SAE, replication rates of 317 three SARS-CoV-2 viruses in LAE and SAE cultures from the same donor were 318 compared. All three viruses replicated more slowly in SAE than LAE cells. The GFP 319 virus replicated modestly less effectively than the clinical isolate or WT virus in the two 320 regions ( Figure 6E ). This observation differs from the equivalent replication noted in the 321 Vero-E6 cells (Figures 2A and 2B) , suggesting an intact ORF7 gene contributes to 322 SARS-CoV-2 replication, and perhaps virulence, in human tissues. 323

Third, the replication of SARS-CoV and SARS-CoV-2 in LAE cells were 324 compared. SARS-Urbani WT and GFP viruses, in parallel with the three SARS-CoV-2 325 viruses, were administered to LAE cultures from the same donor. GFP signals were 326 detected in LAE cultures for both viruses, but the SARS-CoV-2-GFP exhibited delayed 327 and less intense signals than SARS-CoV-Urbani-GFP ( Figure S4D ). This phenotype is 328 consistent with the growth curve in which a lower titer of SARS-CoV-2 was recorded at 329 24h. 330 331

We utilized RNA-ISH/IHC to localize virus in four lungs from SARS-CoV-2-333 infected deceased subjects (Table S1) were also infected. RNA in situ and IHC co-localization of an AT2 cell marker, SPC 342 (SFTPC) and AT1 cell marker (AGER) with SARS-CoV-2 indicated that AT2 cells and 343 AT1 cells (or AT2 cells that had transitioned to AT1 cells) were infected ( Figure 7C We generated a SARS-CoV-2 reverse genetics system, characterized virus RNA 362 transcription profiles, evaluated the impact of ectopically expressed proteases on virus 363 growth, and used reporter viruses to characterize virus tropisms, ex vivo replication, and 364 to develop a high-throughput neutralizing assay. These reagents were utilized to 365 explore aspects of early infectivity and disease pathogenesis relevant to SARS-CoV-2 366 respiratory infections. 367

Our RNAscope/cytospin technology extended the description of ACE2 in 368 respiratory epithelia based on scRNAseq data (Sungnak et al., 2020) . RNA/cytospin 369 detected ~20% of upper respiratory cells expressing ACE2 vs ~4% for scRNAseq 370 ( Figure 4F ). Most of the RNA-ISH-detected ACE2-expressing cells were ciliated cells, 371 not normal MUC5B+ secretory (club) cells or goblet cells. Notably, the nose contained 372 the highest percentage of ACE2-expressing ciliated cells in the proximal airways ( Figure  373 4G). The higher nasal ACE2 expression-level findings were confirmed by qPCR data 374 comparing nasal to bronchial airway epithelia. qPCR data also revealed that ACE2 375 levels further waned in the more distal bronchiolar and alveolar regions. Importantly, 376 these ACE2 expression patterns were paralleled by high SARS-CoV-2 infectivity of 377 nasal epithelium with a gradient in infectivity characterized by a marked reduction in the 378 distal lung (bronchioles, alveoli) ( Figures 6A and 6B) . 379

Multiple aspects of the variability in SARS-CoV-2 infection of respiratory epithelia 380

were notable in these studies. First, significant donor variations in virus infectivity and 381 replication efficiency were observed. Notably, the variability was less in the nose than 382 lower airways. The reason(s) for the differences in lower airway susceptibility are 383 important but remain unclear (Cockrell et al., 2018) . We identified variations in ACE2 384 receptor expression (Figures 4A-D) but not numbers of ciliated cells as potential 385 variables ( Figure 6D ). Second, variation in infectivity of a single cell type, i.e., the 386 ciliated cell, was noted with only a fraction of ciliated cells having access to virus 387 infected at 72 h ( Figure 6A ). Third, the dominant secretory cell, i.e., the MUC5B+ club 388 cell, was not infected in vitro or in vivo, despite detectable ACE2 and TMPRSS2 389 expression ( Figures 4G-4I ). Collectively, these data suggest that measurements of 390 ACE2/TMPRSS2 expression do not fully describe cell infectivity and that a description 391 of other variables that mediate susceptibility to infection, including the innate immune 392 system(s), is needed (Menachery et al., 2014) . 393

The ACE2 receptor gradient in the normal lung raised questions focused on the 394 initial sites of respiratory tract virus infection, the mechanisms that seed infection into 395 the deep lung, and the virus-host interaction networks that attenuate or augment intra-396 regional virus growth in the lung to produce severe disease, especially in vulnerable 397 patients experiencing chronic lung or inflammatory diseases (Guan et al., 2020; Leung 398 et al., 2020) . 399

We speculate that nasal surfaces may be the dominant initial site for SARS-CoV- In summary, our studies have quantitated differences in ACE2 receptor 525 expression and SARS-CoV-2 infectivity in the nose (high) vs the peripheral lung (low). 526

These studies should provide valuable reference data for future animal models 527 development and expand the pool of tissues, e.g., nasal, for future study of disease 528 pathogenesis and therapy. While speculative, if the nasal cavity is the initial site 529 mediating seeding of the lung via aspiration, these studies argue for the widespread use 530 of masks to prevent aerosol, large droplet, and/or mechanical exposure to the nasal 531 passages. Complementary therapeutic strategies that reduce viral titer in the nose early 532 24 in the disease, e.g., nasal lavages, topical antivirals, or immune modulation, may be 533 beneficial. Finally, our studies provide key reagents and strategies to identify type 534 specific and highly conserved neutralizing antibodies that can be assessed most easily 535 in the nasal cavity as well as in the blood and lower airway secretions. 536 537 Acknowledgments 538

We would like to acknowledge the following funding sources from the National Allergy 

Further information and requests for resources and reagents should be directed to and 689 will be fulfilled by the Lead Contact, Ralph S. Baric (rbaric@email.unc.edu). 690

Material and reagents generated in this study will be made available upon installment of 693 a material transfer agreement (MTA). 

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present a reverse genetics system for SARS-CoV-2, which is then used to make reporter viruses to quantify the ability of patient sera and antibodies to neutralize infectious virus and to examine viral tropism along the human respiratory tract

sequence, N gene, 3'UTR, and a 25nt poly-A tail, was assembled under the control of a 1020 T7 promoter. Two reporter viruses, one containing GFP and the other harboring, a GFP-1021 fused nLuc gene, were generated by replacing the ORF7 gene with the reporter genes. 1022 1023

Seven genomic cDNA fragments were digested with appropriate endonucleases, 1025 resolved on 0.8% agarose gels, excised and purified using a QIAquick Gel Extraction kit 1026 (Qiagen). A full-length genomic cDNA was obtained by ligating seven fragments in an 1027 equal molar ratio with T4 DNA ligase (NEB). We then purified the ligated cDNA with 1028 chloroform and precipitated it in isopropanol. The full-length viral RNA or SARS-CoV-2 1029 sgRNA-N were synthesized using the T7 mMESSAGE mMACHINE T7 transcription kit 1030 (Thermo Fisher) at 30℃ for 4h. The full-length SARS-CoV-2 transcript and sgRNA-N 1031 were mixed and electroporated into 8×10 6 of Vero E6 cells. The cells were cultured as 1032 usual in the medium for two to three days. the lumen, not in SMG. (Ciii-iv) H&E (iii) and dual-immunofluorescence staining using 1215 acetylated alpha tubulin (red) and anti-SARS-CoV-2 rabbit polyclonal antibody (green) 1216 (iv) from the trachea of a separate autopsy. Related to Figure 7B and S5Di. (D) 1217Regional distribution of SARS-CoV-2 RNA from trachea to alveoli identified by RNA-ISH 1218 in one SARS-2-CoV autopsy lung (in i and ii, viral staining is red; in iii, viral staining is 1219 turquoise). RNA-ISH dual color images demonstrate SARS-CoV-2 RNA and SFTPC 1220 mRNA (alveolar type 2 cell marker) localization in alveoli of a SARS-CoV-2 autopsy 1221 55 lung. SARS-CoV-2 (turquoise) was identified in a SFTPC (red)-positive (iii, arrow) and a

