Human respiratory organoids sustained reproducible propagation of previously uncultivable human rhinovirus C

The lack of a robust system to reproducibly propagate HRV-C substantially hampered our understanding of the common respiratory virus. We sought to develop an organoid-based system to reproducibly propagate HRV-C and characterize virus-host interaction using the respiratory organoids established by our team. We demonstrated that airway organoids sustained serial virus passage with the aid of CYT-387-mediated immunosuppression; nasal organoids, an organoid model more closely simulating the human upper airway, achieved this without any intervention. Nasal organoids were more susceptible to HRV-C than airway organoids. Intriguingly, we observed a more intensive innate immune response in airway organoids than nasal organoids upon HRV-C infection, which was reproduced in a Poly (I:C) stimulation assay. Treatment with an anti-CDHR3 and two antivirals significantly reduced HRV-C viral growth in nasal organoids. An organoid-based immunofluorescence assay was established to titrate HRV-C infectious particles. Collectively, we developed an organoid-based system to reproducibly propagate the previously uncultivable HRV-C, which enabled an in-depth elucidation of HRV-C infection and innate immunity unprecedentedly. The organoid-based HRV-C infection model can be extended for developing antiviral strategies. More importantly, our study has paved a new avenue for propagating and studying other uncultivable human and animal viruses. To characterize the cellular response in airway and nasal organoids with HRV-C infection and CYT387 treatment, we infected airway and nasal organoids with or without CYT387 treatment. Two biological replicates of mock-treated, HRV-C infected, HRV-C infected with CYT387 treatment airway and nasal organoids were harvested for RNA extraction using RNeasy Mini Kit (Qiagen, Cas.74106). After a quality check with Bioanalyzer, RNA specimens were applied to library preparation by KAPA mRNA HyperPrep Kit. In brief, Poly-A containing mRNA was collected by using poly-T oligo-attached magnetic beads. The purified mRNA was fragmented to 200 ~ 300 bp by incubating at 94°C for 6 min in the presence of magnesium ions. The fragmented mRNA was then applied as a template to synthesize the first-strand cDNA by using random hexamer-primer and reverse transcriptase. In the second strand cDNA synthesis, the mRNA template was removed and a replacement strand was generated to form the blunt-end double-stranded (ds) cDNA. The ds cDNA underwent 3’ adenylation and indexed adaptor ligation. The adaptor-ligated libraries were enriched by 15 cycles of polymerase chain reaction (PCR). The libraries were denatured and diluted to optimal concentration. Illumina NovaSeq 6000 was used for Pair-End 151bp sequencing. Sequencing reads were assigned to individual samples. Each sample had an average throughput of 4.6 Gb and a total throughput of 54.8 Gb. Quality control of raw fastq data was carried out by FastQC v0.11.7 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and fastp [PMID: 30423086]. Then clean reads were aligned to the UCSC GRCh38 reference and the Rhinovirus C3 strain genome (GenBank: OK161378.1) using Hisat2 v2.2.1 [PMID: 31375807]. HTSeq v0.6.1 [PMID: 25260700] was used to generate raw read count for each gene. Differential expression analysis was performed using DESeq2 [PMID: 25516281]. Genes with log2|fold change| > 1 and adjusted p-value < 0.05 were considered significantly different. Volcano plot was generated using EnhancedVolcano R package (https://github.com/kevinblighe/EnhancedVolcano). Heatmaps of gene expression levels were constructing using the pheatmap R package (https://cran.r-project.org/web/packages/pheatmap/index.html). Gene set enrichment analysis (GSEA) was performed using fgsea R package (https://github.com/ctlab/fgsea).