biostudies-arrayexpressMargot ZahmHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-16873The hepatitis E virus (HEV) is a leading cause of acute hepatitis worldwide. HEV infection can become chronic in immunocompromised individuals, in whom virus–host recombinant variants (VHRVs) can be detected. These variants often harbor host-derived insertions in the polyproline rich region (PPR), and most display enhanced replication in vitro. However, the mechanisms underlying this replicative advantage remain unclear. It is likely that genes of the infected cells are differentially expressed according to the replicative capacity of the strain. The host factors involved in the improvement of the replicative capacity of these VHRVs are yet to be identified. In this study, we analyzed the host transcriptional response to 9 VHRVs in HepG2/C3A cells using bulk RNA sequencing at 48h and 168hbiostudies-arrayexpressSample Treatment - Cells were infected for 6 h at 35.5°C with 7.5 x 106 UI of virus diluted in a medium consisting of high-glucose DMEM (4.5g/L) with 10% FBS, 2% dimethyl sulfoxide (DMSO), and 1% antibiotic added.Sample Collection - A volume of 140 µL of the resulting cell lysate was used for HEV RNA extraction.Library Construction - Stranded total RNA libraries were prepared from 1000ng or total RNA using the TruSeq® Stranded Total RNA Library Prep Gold Kit (Illumina), according to the manufacturer’s protocol with minor modifications. Ribosomal RNA (rRNA) and mitochondrial rRNA were depleted using Ribo-Zero™ Gold beads (Illumina). The remaining RNA was enzymatically fragmented, and reverse transcribed to generate cDNA while preserving strand orientation. After adapter ligation, libraries were amplified by PCR. All purification steps using AMPure XP beads (Beckman Coulter) were modified by reducing the bead drying time from 15 minutes to 2–6 minutes, assessed visually. Following the final amplification step, libraries were subjected to an additional purification and size selection step to improveSequencing - Libraries were equimolarly pooled and RNA sequencing was then performed on one S4 lane of the Illumina NovaSeq 6000 instrument (Illumina, San Diego, USA), on the GeT-PlaGe site of the Genome and Transcriptome core facility (GeT, Genotoul, Toulouse, France), using the NovaSeq 6000 S4 v1.5 Reagent Kit (300 cycles), and a paired-end 2 x 150 pb strategy.Growth Protocol - Following the infection period, the viral inoculum was removed and replaced with fresh culture medium. The following day cells were washed three times with phosphate-buffered saline (PBS). Half of the culture medium was changed every 2 days until cell lysis at 168h post-infection. For cells lysed 48h after infection, the culture medium was not changed. Supernatants at 48h and 168h were collected and subsequently clarified by centrifugation (1,000 x g for 5 min).Nucleic Acid Extraction - Total RNA was extracted from infected cells using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. HepG2/C3A were washed with PBS, trypsinized and pelleted by centrifugation at 300 x g for 5 min. Cell pellets were resuspended in 500 µL of the lysis buffer, then placed on a Qiashredder column to pretreat the cell lysates. Columns were centrifuged at maximum speed for 2 min. Eluted cell lysates were mixed with an equal volume of ethanol 70% and loaded onto RNeasy spin columns. After centrifugation at 8000 x g, columns were treated with DNase I (Qiagen) directly on the membrane to remove genomic DNA. Columns were washed with RW1 buffer to remove contaminants, then with RPE buffer which cleans RNA before elution. The RNA was eluted twice in 3OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesSequence Alignment - Raw sequencing reads were processed using the nf-core RNAseq pipeline v.3.12 developed using Nextflow. Briefly, this pipeline trims adapters and removes low quality sequences using Cutadapt v.3.4 wrapped with Trimgalore v.0.6.7. rRNAs are filtered out using SortMeRNA v.4.3.4. STAR v.2.7.9a then aligns trimmed reads to a chimeric assembly composed of the Homo sapiens GRCh38 assembly and the complete genome of Hepatitis E virus clone pSK-HEV-2 (GenBank: AF444002.1). Gene annotation includes Homo sapiens annotation from Ensembl release 110 and Hepatitis E virus ORFs. Finally, Salmon v.1.5.2 quantifies reads.Data Transformation - Normalized counts are in transcripts per million (TPM) and where generated using the Salmon tool: TPM_i = 10^6 * [ (NumReads_i / EffectiveLength_i) / (sum_j (NumReads_j / EffectiveLength_j)) ] Where NumReads_i is the expected number of reads arising from transcript i, EffectiveLength_i is its effective length, and likewise for j.MetabolomicsUnknownTranscriptomicsGenomicsProteomicsIllumina NovaSeq 6000RNA-seq of coding RNAHomo sapiensHost-interferon-stimulated gene response to virus-host recombinant variants of hepatitis E virus and enhanced viral replicationParonetto, Olivia; Allioux, Claire; Zahm, Margot; Gonzalez, Anne Alicia; Jeanne, Nicolas; Chaubet, Adeline; Collercandy, Nived; Abravanel, Florence; Lhuillier, Émeline; Chapuy-Regaud, Sabine; Izopet, Jacques; Lhomme, SébastienSébastien LhommeMargot ZahmfalseHost-Interferon-Stimulated Gene Response to Virus-Host Recombinant Variants of Hepatitis E Virus and Enhanced Viral ReplicationThe hepatitis E virus (HEV) is a leading cause of acute hepatitis worldwide. HEV infection can become chronic in immunocompromised individuals, in whom virus–host recombinant variants (VHRVs) can be detected. These variants often harbor host-derived insertions in the polyproline rich region (PPR), and most display enhanced replication in vitro. However, the mechanisms underlying this replicative advantage remain unclear. It is likely that genes of the infected cells are differentially expressed according to the replicative capacity of the strain. The host factors involved in the improvement of the replicative capacity of these VHRVs are yet to be identified. In this study, we analyzed the host transcriptional response to 9 VHRVs in HepG2/C3A cells using bulk RNA sequencing at 48h and 168h2026-09-01T00:00:00Z2026-09-01T07:30:02.526Z2026-04-09T08:29:51.418ZE-MTAB-16873ERP191850EFO_0002944EFO_0004170EFO_0003789EFO_0004917EFO_0005518EFO_0003816EFO_0003738EFO_0004184EFO_0003969https://dx.doi.org/10.1128/spectrum.00600-26