Characterization and treatment of SARS-CoV-2 in nasal and bronchial human airway epithelia

Andrés Pizzorno; Blandine Padey; Thomas Julien; Sophie Trouillet-Assant; Aurélien Traversier; Elisabeth Errazuriz-Cerda; Julien Fouret; Julia Dubois; Alexandre Gaymard; François-Xavier Lescure; Victoria Dulière; Pauline Brun; Samuel Constant; Julien Poissy; Bruno Lina; Yazdan Yazdanpanah; Olivier Terrier; Manuel Rosa-Calatrava

In this study, we initially isolated and amplified in VeroE6 cells a SARS-CoV-2 virus directly form a nasal swab from one of the first hospitalized patients with confirmed in France (11) . The complete genome sequence of the isolated SARS-CoV-2 virus was deposited 75 in the GISAID EpiCoVTM database under the reference BetaCoV/France/IDF0571/2020 (accession ID EPI_ISL_411218). Phylogenetic analysis confirmed that the isolated virus is representative of currently circulating strains (12) . We first characterized the replicative capacities of this viral strain in VeroE6 cells at different multiplicities of infection (MOIs) (Fig. 1A) , using both classic infectious titer determination in cell culture (TCID50) and molecular semi-quantitative 80 methods, the latter based on ORF1b-nsp14-specific primers and probes designed by the School of Public Health/University of Hong Kong (details in Supplementary Materials). This double approach was facilitated by the appearance of clearly observable characteristic cytopathic effect from 48 hpi (Fig. 1B) , and enabled the validation of a large interval (range 1-8 log10(TCID50)) with high correlation (R-squared 0.94) between molecular and infectious viral titers (Fig. 1C) . 85 In parallel, we successfully inoculated nasal MucilAirâ„¢ HAE on the apical surface directly with nasal swab samples, as confirmed by transmission electron microscopy observations (Fig. S1 ). Characteristic features of coronavirus-induced cell ultrastructure remodeling were easily distinguishable in both the apical and basal sides of the HAE at 48 hpi, notably the high accumulation of progeny virions in mucus-producer goblet cells. Then, we advantageously 90 exploited the MucilAirâ„¢ HAE model and in-house adapted protocols previously optimized for different respiratory viruses (13) to perform experimental infections with SARS-CoV-2. Viral . CC-BY-NC-ND 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.31.017889 doi: bioRxiv preprint replication was monitored through repeated sampling and TCID50 titration at the apical surface of HAE (Fig. 1D) . Trans-epithelial electrical resistance (TEER), considered as a surrogate of epithelium integrity, was also measured during the time-course of infection (Fig. 1E) . In parallel, 95 comparative molecular viral genome quantification was performed at the three levels of the air/liquid HAE interphase: in apical washes (Fig. 1F, Apical) , total cellular RNA (Fig. 1G , Intracellular) and basal medium (Fig. 1H, Basal) . SARS-CoV-2 viral production at the epithelial apical surface increased sharply at 48 hpi, reaching 5.8 and 6.3 log10 TCID50/mL in nasal and bronchial HAE, respectively. The peak of viral replication was reached earlier in bronchial (48-72 100 hpi) than in nasal HAE, in which a progressive increase in infectious viral titers was observed until at least 96 hpi (Fig. 1D) . This replication kinetics was validated by molecular viral genome quantification at the apical pole ( Fig. 1F) . High viral replication correlated with a reduction in epithelium integrity at 48 hpi, reflected by more than 2.8-and 4-fold decreases in bronchial and nasal HAE TEER values, respectively, followed by a partial recovery in the case of bronchial HAE 105 (Fig. 1E) . Moreover, viral production at the apical pole was well correlated with intracellular viral genome detection during infec