Project description:Pathogenic mechanisms underlying severe SARS-CoV2 infection remain largely unelucidated. High-throughput sequencing technologies that capture genome and transcriptome information are key approaches to gain detailed mechanistic insights from infected cells. These techniques readily detect both pathogen and host-derived sequences, providing a means of studying host-pathogen interactions. Recent studies have reported the presence of host-virus chimeric (HVC) RNA in RNA-seq data from SARS-CoV2 infected cells and interpreted these findings as evidence of viral integration in the human genome as a pathogenic mechanism. Since SARS-CoV2 is a positive sense RNA virus that replicates in the cytoplasm it does not have a nuclear phase in its life cycle, so it is biologically unlikely to be in a location where splicing events could result in genome integration. Here, we investigated the biological authenticity of HVC events. In contrast to true biological events, e.g. mRNA splicing and genome rearrangement events, which generate reproducible chimeric sequencing fragments across different biological isolates, we found that HVC events across >100 RNA-seq libraries from patients with COVID-19 and SARS-CoV2 infected cell lines, were highly irreproducible. RNA-seq library preparation is inherently error-prone due to random template switching during reverse transcription of RNA to cDNA. By counting chimeric events observed when constructing an RNA-seq library from human RNA and spike-in RNA from an unrelated species, such as fruit-fly, we estimated that ~1% of RNA-seq reads are artifactually chimeric. In SARS-CoV2 RNA-seq we found that the frequency of HVC events were, in fact, no more frequent than this background “noise”. Finally, we developed a novel experimental approach to enrich SARS-CoV2 sequences from bulk RNA of infected cells. This method enriched viral sequences but did not enrich for HVC events, suggesting that the majority of HVC events are, in all likelihood, artifacts of library construction. In conclusion, our findings indicate that HVC events observed in RNA-sequencing libraries from SARS-CoV2 infected cells are extremely rare and are likely artifacts arising from either random template switching of reverse-transcriptase and/or sequence alignment errors. Therefore, the observed HVC events do not support SARS-CoV2 fusion to cellular genes and/or integration into human genomes.

Project description:The goal of this study was to determine changes in the expression of genes in monoctic myleoid derived suppressor cells (M-MDSC) as a result of SARS CoV2 infection. The study aimed to investigate if M-MDSC are functionally active and inhibit T cell function in response to SARS CoV2 antigens 5 months after first detection of the virus. Methods: Peripheral blood mononuclear cells (PBMC) were collected from CoV2 (-) and CoV2 (+) donors (N=5 each group). M-MDSC were isolated by flow cytometry, and RNA extracted for RNA-seq studies. Filtering low quality reads and removal of the 3’ adapter sequences were performed using the Trim Galore tool. Reads were mapped to the latest version of the human genome (build hg38) using HISAT2. Mapped reads were counted against the human GENCODE annotation (v37) using HT-Seq. The EdgeR library in the R computing environment was used for quality control of the RNA-Seq data, and ComBat-seq method for correction of batch effects. Differential gene expression analysis was conducted using EdgeR. Pathway enrichment analysis was performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.8. Results: An average of 34 million reads per sample were acquired and mapped to the human genome (build hg38). After applying filtering criteria, 9,217 human genes were identified with the HISAT2 and HTSeq workflow. Differential expression analysis was performed between CoV2 (+) and CoV2 (-) samples using EdgeR. A total of 188 differentially expressed genes (DEGs) were identified with nominal p-value <0.05; of which 63 were up- and 125 downregulated in CoV2 (+) samples. A total of 12 DEGs were identified with false discovery rate corrected p-value <0.05, of which 2 were up- and 10 downregulated. Pathway enrichment analysis identified pathways involved in immune response and innate immune signaling. Conclusion: The study demonstrated that CoV2 infection modulated the expression of genes involved in immune response and innate immune signaling. Most of the genes remained downregulated even after 5 months of first detection of SARS CoV2.

Project description:Purpose: The goals of this study are to monitor the evolution pattern of SARS-CoV2 in depending host cells by viral transcriptome sequencing analysis of Vero, A549, Caco2, and HRT18 cells infected with SARS-CoV2. Methods: SARS-CoV-2 isolate was passaged 4 time on Vero cells and used to extract RNA for the high-throughput sequencing. The 8×104 PFU of SARS-CoV2 stocks passaged on vero cells were inoculated to the monolayer of A549, CaCO2, and HRT-18 cell lines in 75T flask for 1hour at 37℃ in a 5% CO2 incubator with gentle shaking of 15 minutes interval. After that, the infected cells were washed two times with DPBS and incubated with the fresh maintenance medium for 3 days. The virus inoculation was performed in triplicate for each cell lines. In case of the first passage, the infected cell pellets were resuspended to 250µl with fresh medium, to extract RNA for the high-throughput sequencing. The cultured cell supernatant of the virus-infected A549, CaCO2, and HRT18 cells was centrifuged at 3,000g for 10min to use for the next passage, and stored at -80℃. The serial passage of SARS-CoV-2 on A549, CaCO2, and HRT18 cell lines were continued to passage 12 and the cultured cell supernatant of the infected cells in passage 12 was centrifuged at 3,000g for 10 min, and used to extract RNA for the high-throughput sequencing. The RNA samples were sequenced with illumine TruSeq Strand Total RNA LT kit and illumine NovaSeq6000 plaform form Macrogen, Inc (Seoul, Korea) for high throughput sequencing. The raw reads were trimmed with BBDuk and mapped the isolate SARS-CoV-2/human/KOR/KCDC03-NCCP43326/2020 (Genebank accession number. MW466791) with Bowtie 2 using Geneious program 2021.2.2 Result: Using SNP analysis workflow, our result showed the sequence variations pattern of SARS-CoV2 depending on host cell (A549, CaCO2, and HRT18 cell lines) and it was confirmed that a relatively large number of SNPs were commonly observed in spike protein. Some SNPs affect amino acid changes, and a common pattern of amino acid changes was observed the genomic sequence of SARS-CoV2 passaged in A549, CaCO2 and HRT18 cells. Conclusion: In this study, we tried to monitor the SARS-CoV-2 (GenBank accession No. MW466791 in 2020, Korea) evolution pattern in different host cells using high throughput sequencing analysis, and compare the selected mutations by each host cells with natural mutations found in currently circulating SARS-CoV-2 variants.

Dataset Information

Bulk RNAseq of SARS-COV2 infected human lung organoid

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