key: cord-337973-djqzgc1k
authors: Hao, Siyuan; Ning, Kang; Kuz, Cagla Aksu; Vorhies, Kai; Yan, Ziying; Qiu, Jianming
title: Long Period Modeling SARS-CoV-2 Infection of in Vitro Cultured Polarized Human Airway Epithelium
date: 2020-08-28
journal: bioRxiv
DOI: 10.1101/2020.08.27.271130
sha: 
doc_id: 337973
cord_uid: djqzgc1k

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replicates throughout human airways. The polarized human airway epithelium (HAE) cultured at an airway-liquid interface (HAE-ALI) is an in vitro model mimicking the in vivo human mucociliary airway epithelium and supports the replication of SARS-CoV-2. However, previous studies only characterized short-period SARS-CoV-2 infection in HAE. In this study, continuously monitoring the SARS-CoV-2 infection in HAE-ALI cultures for a long period of up to 51 days revealed that SARS-CoV-2 infection was long lasting with recurrent replication peaks appearing between an interval of approximately 7-10 days, which was consistent in all the tested HAE-ALI cultures derived from 4 lung bronchi of independent donors. We also identified that SARS-CoV-2 does not infect HAE from the basolateral side, and the dominant SARS-CoV-2 permissive epithelial cells are ciliated cells and goblet cells, whereas virus replication in basal cells and club cells was not detectable. Notably, virus infection immediately damaged the HAE, which is demonstrated by dispersed Zonula occludens-1 (ZO-1) expression without clear tight junctions and partial loss of cilia. Importantly, we identified that SARS-CoV-2 productive infection of HAE requires a high viral load of 2.5 × 105 virions per cm2 of epithelium. Thus, our studies highlight the importance of a high viral load and that epithelial renewal initiates and maintains a recurrent infection of HAE with SARS-CoV-2.

Introduction observed a cytoplasmic expression and a weak junction expression of ZO-1 at 30 dpi (Fig. 5A ) 230 and 21 dpi (SFig. 5A) for infected HAE-ALI B4-20 and HAE-ALI B9-20 , respectively. These results 231 demonstrate that a high viral load (at least >100 pfu (~8.2 ×10 4 vgc) to an epithelium of 0.33 232 cm 2 , which contains with ~5 ×10 5 epithelial cells, is necessary to initiate a productive infection. 233 234 Ciliated and goblet cells are permissive to SARS-CoV-2 but not the basal and club cells. 235 We next examined SARS-CoV-2 infection in which the inoculation of a high MOI of 2 236 was applied to the basolateral side in HAE-ALI B4-20 . The results showed there were no 237 detectable infectious virions released from both the apical and basolateral sides (Fig. 6A) . The 238 TEER of the infected HAE displayed no significant changes over the course of 24 days (Fig.  239   6B) , which was also evidenced for the well-preserved tight junctions (Fig. 6C) , as well as the 240 rich cilia expression (Fig. 6D) . Importantly, NP+ cells were not detected for as long as 23 dpi. positive anti-β-tubulin IV staining. Consistent with previous imaging results (Fig. 3) , most of the 252 NP+ cells were also positive with anti-β-tubulin IV staining (Fig. 7A) , whereas almost all the 253 CKRT5+ basal were negative for anti-NP staining (Fig. 7C) . Probing secretoglobin family 1A 254 member 1 (SCGB1A1) expression for club cells and mucin 5AC (MUC5AC) expression for goblet cells 41 revealed that the secretory cells were less abundant sub-populations in the 256 infected HAE-ALI cultures. While we could not locate any club cells stained positively for NP 257 expression (Fig. 7D) , we found some NP+/MUC5AC+ goblet cells (Fig. 7B) . Importantly, we 258 observed that in the infected HAE-ALI, a subset of CYKT5+ basal cells are found associated 259 with the expression of Ki67; however, in the mock-infected HAE-ALI, we did not found the cells 260 co-expressing both Ki67 and CYKT5 (Fig. 8B) . As Ki67 is a marker of cell proliferation 42 , this 261 result suggested that SARS-CoV-2 infection activates basal cells towards proliferation. 262

Taking these lines of evidence together, our results confirmed SARS-CoV-2 mainly 263 infects ciliated cells of HAE, as well as the goblet cells, despite its lower abundance in HAE-ALI 264 cultures. Our study suggests that basal and club cells are not permissive to SARS-CoV-2. Our observation that SARS-CoV-2 was unable to infect epithelial cells from the 299 basolateral side supports that the viral entry receptor ACE2 is polarly expressed at the apical 300 side 30, 31 . We believe the finding that CKRT5+ basal cells are largely not infected by SARS-301

CoV-2 is important to understand the course of SARS-CoV-2 infection in HAE. The airway basal 302 cells are the epithelial cell type not presenting on the surface of the airway lumen, thus, they are 303 not usually accessible to the virus on the apical side. However, when the infection commences 304 and the epithelial damage occurs, the destructive mucosal lesions (and the death of the infected 305 ciliated and goblet cells) would allow the virus to get access the basal cells (Fig. 8C) . Indeed, 306 the detectable virus shedding to the basolateral chamber (Fig. 4A&C , SFig. 1A and SFig. 3A) indicates a possible window to expose the basal cells to SARS-CoV-2. Notably, these time 308 points also represent the peaks of the release of virus progeny. However, none of the CKRT5+ 309 cells that was also NP-positive was found from the cytospins prepared from SARS-COV-2 310 infected HAE at 9 dpi when the infection appeared at the lowest level. We hypothesize that the 311 non-permissive nature of basal cells to SARS-CoV-2 is due to the negligible expression of 312 ACE2 49 or TMPRESS-2 43 . 313

The epithelial cell lining of the airways provides an efficient barrier against microbes and 314 aggressive molecules through interdependent functions, including mechanical clearance of the 315 mucus executed by movements of the cilia, a cellular barrier function by means of intercellular 316 epithelial junctions formed by a set of tight junction associated proteins such as ZO1, and 317 homeostasis of ion transport 40 . At the airway epithelial cellular level, the tight junction-318 associated proteins, such as ZO1, occludin, and claudins, play a central part in the epithelial 319 cytoprotection by maintaining a physical selective barrier between external and internal 320 environments. The tight junction proteins are highly labile structures whose formation and 321 structure may be very rapidly altered after airway injury, for example, airway inflammation. 322

Proinflammatory cytokines have a drastic effect on tight junction expression and barrier 323 functions, which significantly alter the epithelial barrier permeability 50-52 . SARS-CoV-2 infection 324 distorted the ZO-1 expression, and thereafter causes barrier dysfunction (TEER decrease). The 325

infection not only alters the ZO-1 expression of infected (NP1+ cells) but also uninfected cells 326 (NP-cells) (Fig. 3) . This is also true for the cilia loss. We believe that SARS-CoV-2 infection 327 produces inflammatory cytokines as an innate immunity response upon virus infection 53 , which 328 either disturbs ZO-1 and tubulin expression or alters their structures. The innate immunity 329 response also limits virus infection at the front line. In fact, SARS-CoV-2 requires a high viral 330 load (>300 pfu/cm 2 of HAE) to initiate a productive infection (Fig. 4) . Of note, the infectious titer 331 (pfu) was determined by plaque assay in Vero-E6 cells, which are interferon-deficient 54 . We 332 determined that 1 pfu of SARS-CoV-2 in Vero-E6 cells has a particle (viral genome copy) 333 number of 820, suggesting that a load of 2.46 x 10 5 particles is required to productively infect 1 334 cm 2 of the airway epithelium, which is much higher than the small DNA virus parvovirus human 335 bocavirus 1 (HBoV1) we studied 55 . HBoV1 can infect HAE at an MOI of as low as 0.001 336 genome copies per cell, which equals 1.5 x 10 3 particles per 1 cm 2 of the airway epithelium. 337

Epithelial regeneration involves migration of the basal cells that neighbor the acute 338 injured area (e.g., virus-infected area), active dividing and squamous metaplasia, rapid 339 redifferentiation to preciliated cells, and finally ciliogenesis towards a complete pseudostratified 340 mucociliary epithelium 56 . Airway epithelium repair is critical for the maintenance of the barrier 341 function and the limitation of airway hyperreactivity. In a biopsy study of fresh tracheas and 342 lungs from five deceased COVID-19 patients, it was found that the epithelium was severely 343 damaged in some parts of the trachea, and extensive basal cell proliferation was observed in 344 the trachea, where ciliated cells were damaged, as well as in the intrapulmonary airways 37 . 345

These data support our conclusion that basal cells are not permissive to SARS-CoV-2. As a 346 response to these previous findings, our study observed that a subset of proliferating basal cells 347 in the SARS-CoV-2 infected HAE-ALI, but not in the mock infected HAE-ALI (Fig. 8B) . Thus, we 348 hypothesize that SARS-CoV-2 infection induces basal cell proliferation, which accounts for the 349 observed long-lasting infections with recurrent peaks of viral replication, which warrants future 350 investigation. 351

Overall, we propose a model of SARS-CoV-2-infection of HAE (Fig. 8C) and then cytocentrifuged at 18,000 rom for 3 min onto slides using a Shandon Cytospin 3 453 cytocentrifuge. After cytospun, the slides were fixed overnight in 4% paraformaldehyde at 4°C. 454

The fixed HAE or dissociated cells were permeabilized with 0.2% Triton X-100 for 15 min 455 at room temperature. Then, the slide was incubated with primary antibody in PBS with 2% FBS 456 for 1 h at 37ºC. After washing, the membrane was incubated with fluorescein isothiocyanate-457 and rhodamine-conjugated secondary antibodies, followed by staining of the nuclei with DAPI 458 (4',6-diamidino-2-phenylindole). 459 

Online ahead of print

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Confocal images were taken at a magnification of x 40 on the indicated days post-infection (dpi)

Nuclei were stained with DAPI (blue)

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-2-infected HAE-ALI B9-20 cultures at 15 dpi were co-stained with 756

or with anti-NP and anti-β-tubulin IV antibodies (B), or co-757 stained anti-NP and anti-ZO-1 antibodies (B). A set of confocal images were taken at a 758 magnification of x 40 from the stained pierce of epithelium

image as shown in each channel of fluorescence. Nuclei were stained with DAPI (blue)

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Transepithelial electrical resistance measurement. The TEER of mock-and SARS-CoV-2-771

HAE-ALI culture were measured using an epithelial Volt-Ohm Meter (Millipore) at the 772 indicated dpi. The TEER values were normalized to the TEER measured on the day of infection, 773 which is set as 1.0. Values represent the mean of relative TEER +/-standard deviations

HAE-ALI B4-20 cultures were infected with SARS-CoV-2 at an MOI from 0.2 to 0.00002. At 779 30 dpi, both virus and mock infected HAE were co-stained with anti-NP and anti-ZO-1 780 antibodies (A), or co-stained with anti-NP and anti-β-tubulin IV antibodies (B). Confocal images 781 were taken at a magnification of x 40. Nuclei were stained with DAPI (blue).