biostudies-arrayexpressVINCENT CAVALRhinolophus ferrumequinumhttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-16878Transcriptome analysis Rhinolophus ferrumequinum (Rfe) cells upon poly-I:C transfection. Rfe cell expressing human ACE2 and TMPRSS2 (Rfe-AT) and a SARS-Cov-2 / BANAL-236 successible clonal Rfe-AT cell line (Rfe-ATC) were transfected with poly-I:C or PBS and transcriptome was analysed using RNA sequencing.biostudies-arrayexpressNucleic Acid Extraction - Total RNA was extracted from cell lysates using the NucleoSpin RNA II kit (Macherey-Nagel) and eluted in waterSample Treatment - For polyI:C stimulation, RFe cells were plated in 12-well plates. The next day, they were transfected with 100 ng polyI:C (InvivoGen) or PBS, respectively, using INTERFERin (Polyplus Transfection) transfection reagentSequencing - The RNA-seq analysis was performed with Sequana 0.18.0 [71]. We used the RNA-seq pipeline 0.20.0 (https://github.com/sequana/sequana_rnaseq) built on top of Snakemake 7.32.4 [72]. Briefly, reads were trimmed from adapters using Fastp 0.23.2 [73] then mapped to the corresponding genome using STAR 2.7.10a [74]. Rhinolophus ferrumequinum RFe-AT and RFe-TC samples were mapped to GCF_004115265.2 assembly with annotation GCF_004115265.2-RS_2023_02 from NCBI. FeatureCounts 2.0.1 [75] was used to produce the count matrix, assigning reads to features using corresponding annotation with strand-specificity information. Quality control statistics were summarized using MultiQC 1.17 [76]. Statistical analysis on the count matrix was performed to identify differentially regulated genes. Clustering of transcriptomic profiles were assessed using a Principal Component Analysis (PCA). Differential expression testing on each strain separately was conducted using DESeq2 library 1.38.3 [77] scripts indicating the significance (Benjamini-Hochberg adjusted p-values, false discovery rate FDR < 0.05) and the effect size (fold-change) for each comparison. DESeq2 output files (Table S1) was used to filter and plot Differentially Expressed Genes (DEG) (Log2 fold change ≥ 1.5, adjusted p-value < 0.05).Sample Collection - Total RNA was extracted from cell lysates using the NucleoSpin RNA II kit (Macherey-Nagel) and eluted in waterLibrary Construction - cDNA libraries were prepared from 100-200 ng RNA using a Illumina Stranded mRNA library Preparation Kit (Illumina, USA) following the manufacturer’s protocol whereby adaptors are added to cDNA by A-tail mediated ligation. Index barcodes were added by PCR of 16 cycles. Unbound adaptors and indexes were eliminated by purification on AMPure magnetic beads (Beckman-Coulter). The resulting library featured an electrophoretic profile of 200-1000 bp, with a major peak of 375 bp, as visualized on a 3500 Fragment Analyzer (Agilent). A NextSeq 2000 sequencing system and a P4 50c flowcell (Illumina) were used to obtain 67-nt single-end double-indexed readsOrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - The processed data file provided represents raw counts matrix.UnknownTranscriptomicsGenomicsProteomicsNextSeq 2000Asian Rhinolophus bats are considered the natural reservoirs of an ancestral SARS-CoV-2. However, the biology of SARS-CoV-2-related viruses in bat cells is not well understood. Here, we investigated the replication of BANAL-236, the only bat-derived SARS-CoV-2 relative isolated to date, in Rhinolophus cells. BANAL-236 did not replicate in wild-type Rhinolophus cell lines. Entry assays using pseudoviruses expressing the spike proteins (S) of SARS-CoV-2, BANAL-236, and BANAL-52 revealed that efficient S-mediated entry depends on the expression of human ACE2 (hACE2) and human TMPRSS2 (hTMPRSS2) in human and Rhinolophus cells. Expression of Rhinolophus entry factors, either alone or in combination, did not facilitate SARS-CoV-2 or BANAL-236 entry in human cells, suggesting that the S protein of BANAL-236 interacts more efficiently with hACE2 than with its Rhinolophus counterpart (rACE2). Through biochemical, virological, and electron microscopy analyses, we showed that BANAL-236 and SARS-CoV-2 completed their replication cycles in a Rhinolophus cell line engineered to express high levels of hACE2 and hTMPRSS2. Despite efficient viral replication in modified Rhinolophus and human cells, no induction of interferon (IFN)-stimulated genes was detected. Using a screening approach, we identified several BANAL-236 proteins that antagonize IFN production and signaling in human cells. Our findings thus show that BANAL-236 possesses critical features that enabled zoonotic spillover: hACE2 usage and potent evasion of human IFN responses. The Rhinolophus cellular model we established offers a platform for further investigating the interactions between bat coronaviruses and their reservoir hosts. <h4>Author summary</h4> Bats are known reservoirs for viruses that cause severe diseases in humans, such as coronaviruses and filoviruses. Bat species naturally or experimentally infected with these viruses rarely exhibit clinical symptoms, suggesting an evolved tolerance to viral infections. To elucidate the mechanisms underlying viral tolerance and to identify factors that could facilitate zoonotic spillover, it is essential to study the replication of bat-borne viruses in relevant bat cellular models. Here, we investigated the replication of BANAL-236, a SARS-CoV-2 related virus isolated from fecal samples of Rhinolophus bats in Northen Laos, in a novel cell line derived from Rhinolophus ferrumequinum lung fibroblasts. Our findings reveal that BANAL-236 can efficiently use human entry factors and potently evade the human innate immune response, two traits that may have contributed to its zoonotic transmission. Furthermore, the R. ferrumequinum cell lines we developed is a valuable model for investigating the molecular interactions between sarbecoviruses and their natural hosts.RNA-seq of coding RNARhinolophus ferrumequinumEntry, replication and innate immunity evasion of BANAL-236, a SARS-CoV-2-related bat virus, in Rhinolophus and human cellsVINCENT CAVALSégolène Gracias, Elodie Le Seac’h, Samuel Donaire-Carpio, Françoise Vuillier, Léa Vendramini, Adam Moundib, Sarah Temmam, Magdalena Rutkowska, Flora Donati, Anastasija Cupic, Javier Juste, Carles Martinez-Romero, Nathalie Morel, Olivier Schwartz, Nevan J Krogan, Lisa Miorin, Marcel A Müller, Caroline Demeret, Sandie Munier, Philippe Roingeard, Jyoti Batra, Adolfo Garcia-Sastre, Vincent Caval, Nolwenn JouvenetfalseTranscriptome analysis Rhinolophus ferrumequinum (Rfe) cells upon poly-I:C transfectionTranscriptome analysis Rhinolophus ferrumequinum (Rfe) cells upon poly-I:C transfection. Rfe cell expressing human ACE2 and TMPRSS2 (Rfe-AT) and a SARS-Cov-2 / BANAL-236 successible clonal Rfe-AT cell line (Rfe-ATC) were transfected with poly-I:C or PBS and transcriptome was analysed using RNA sequencing.2026-04-10T00:00:00Z2026-04-10T11:29:58.993Z2026-04-09T08:38:35.891ZE-MTAB-16878ERP191860EFO_0002944EFO_0004170EFO_0005518EFO_0003816EFO_0003738EFO_0004184EFO_000396910.1101/2025.09.29.679146