| Congresso Brasileiro de Microbiologia 2023 | Resumo: 457-1 | ||||
Resumo:Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the agent responsible for the coronavirus disease 2019 (COVID-19) pandemic. Generally, coronavirus particles are enveloped and contain a single-stranded, positive-sense RNA genome with a size of approximately 27-32 kbp. In our laboratory, we conducted high-quality sequencing of the SARS-CoV-2 genome and observed deletions of various sizes in the ORF7a gene in specific cases, including contacts, family members, and professional groups. Clinical samples were collected in collaboration with IBEX and CTD-UFRJ, and a total of 36 samples were analyzed. We identified three types of deletions in the ORF7a gene: ORF7a_del190 (deletion of 190 nucleotides), ORF7a_del339 (deletion of 339 nucleotides), and ORF7a_del365 (deletion of 365 nucleotides). Normally, the described size for the ORF7a gene in the SARS-CoV-2 reference sequence is 366 nucleotides, resulting in the production of a 121-amino acid protein. However, in these deletions, the carboxy-terminal transmembrane domain is lost, and the resulting region corresponds to only a portion of the ectodomain that interacts with host molecules. In this study, we aim to evaluate the impact of these ORF7a deletions on the transcriptomic profile of both the host and the virus during SARS-CoV-2 infection in Calu-3 cells. Our goal is to identify specific modulations of the viral transcriptome in different ORF7a deletions and compare this modulation in the absence, partial presence, and complete presence of ORF7a. Total RNA will be extracted from infected cultured cells using the miRVana kit (Thermo Fisher Scientific). Ribosomal RNA (rRNA) will be removed using the RiboMinus Human/Mouse Transcriptome Isolation kit (Thermo Fisher Scientific) following the manufacturer's instructions. The quality and concentration of RNA samples will be evaluated by microfluidic electrophoresis using an Agilent 2100 Bioanalyzer (Agilent Technologies). After the removal of rRNA and small RNAs, the RNA preparations will be used for RNA-seq library preparation using an Ion Total RNA-seq v2 kit (Thermo Fisher Scientific). Subsequent clonal amplification will be performed with an Ion Chef Instrument (Thermo Fisher Scientific). Sequencing will be carried out in the Ion GeneStudio S5 semiconductor sequencer (Thermo Fisher Scientific). The triplicate transcriptomic profiles obtained will be initially analyzed using Torrent Suite software (Thermo Fisher Scientific) and the CLC Genomics Workbench v.21 software package (Qiagen). High-quality sequences will be mapped to the human reference genome hg38 to identify mRNAs, while viral sequences will be identified by mapping to the SARS-CoV-2 reference genome. The sequences will be aggregated into transcribed regions and quantified using internal controls and reads per kilobase per million sequences (RPKM). For the analysis of differential gene expression and functional enrichment of mRNAs in cells infected with SARS-CoV-2 variants, transformations, normalizations, and analyses will be performed using CLC software and the edgeR software package. Parameters of fold-change of 2 and a p-value of less than 0.05 will be used.
Palavras-chave: ORF7a, RNA-seq, SARS-CoV-2, transcriptome Agência de fomento:Capes, CNPq, FAPERJ | |||||