Dynamics of interactions between three major respiratory pathogens in reconstituted human epithelium
Dynamics of interactions between three major respiratory pathogens in reconstituted human epithelium
The SARS-CoV-2 strain used in this study was isolated from a patient enrolled in a French clinical cohort of COVID-19 patients (ClinicalTrials.gov identifier NCT04262921; The sequence is available in the GISAID EpiCoV™ database under the accession BetaCoV/France/IDF0571/2020 (EPI_ISL_411218). Viral stocks were amplified and titrated by determination of the 50% tissue culture infectious dose (TCID₅₀/mL) on Vero cells (ATCC CRL-1586). The influenza A/Lyon/969/2009 (H1N1) strain was propagated and titrated on MDCK cells (ATCC CCL-34). A recombinant RSV A2 strain expressing mCherry (RSV-mCherry, kindly provided by Jean-François Eléouët, INRAE, Jouy-en-Josas, France) was propagated and quantified on LLC-MK2 cells (ATCC CCL-7). For infection, HAE were gently washed twice at the apical surface with Opti-MEM (Life Technologies) and subsequently mock-infected (Opti-MEM) or infected with SARS-CoV-2, IAV, or RSV at multiplicities of infection (MOI) of 0.01, 0.1, and 0.2, respectively. At defined time points, apical washes and basal media were collected and stored at −80°C for further analyses. Cells were lysed for total RNA extraction, which was used for RT-qPCR and RNA-seq. All experiments involving infectious SARS-CoV-2 material were performed in a biosafety level 3 (BSL-3) facility, while infections involving only IAV and/or RSV were conducted in a biosafety level 2 (BSL-2) laboratory. Briefly, HAE were lysed in RLT buffer (Qiagen RNeasy® Plus Mini Kit, ref. 74136) and scraped before collection. Total RNA was extracted using the same kit according to the manufacturer's instructions. RNA library preparation was realized following the manufacturer's recommendations (QuantSeq 3' mRNA-Seq V2 Library Prep Kit from LEXOGEN). 