Project description:Coronaviruses are commonly characterized by a unique discontinuous RNA transcriptional synthesis strategy guided by transcription-regulating sequences (TRSs). However, the details of RNA synthesis in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have not been fully elucidated. Here, we present a time-scaled, gene-comparable transcriptome of SARS-CoV-2, demonstrating that ACGAAC functions as a core TRS guiding the discontinuous RNA synthesis of SARS-CoV-2 from a holistic perspective. During infection, viral transcription, rather than genome replication, dominates all viral RNA synthesis activities. The most highly expressed viral gene is the nucleocapsid gene, followed by ORF7 and ORF3 genes, while the envelope gene shows the lowest expression. Host transcription dysregulation keeps exacerbating after viral RNA synthesis reaches a maximum. The most enriched host pathways are metabolism related. Two of them (cholesterol and valine metabolism) affect viral replication in reverse. Furthermore, the activation of numerous cytokines emerges before large-scale viral RNA synthesis. IMPORTANCE SARS-CoV-2 is responsible for the current severe global health emergency that began at the end of 2019. Although the universal transcriptional strategies of coronaviruses are preliminarily understood, the details of RNA synthesis, especially the time-matched transcription level of each SARS-CoV-2 gene and the principles of subgenomic mRNA synthesis, are not clear. The coterminal subgenomic mRNAs of SARS-CoV-2 present obstacles in identifying the expression of most genes by PCR-based methods, which are exacerbated by the lack of related antibodies. Moreover, SARS-CoV-2-related metabolic imbalance and cytokine storm are receiving increasing attention from both clinical and mechanistic perspectives. Our transcriptomic research provides information on both viral RNA synthesis and host responses, in which the transcription-regulating sequences and transcription levels of viral genes are demonstrated, and the metabolic dysregulation and cytokine levels identified at the host cellular level support the development of novel medical treatment strategies.

Project description:The spread of SARS-CoV-2 and the increasing mortality rates of COVID-19 create an urgent need for treatments, which are currently lacking. Although vaccines have been approved by the FDA for emergency use in the U.S., patients will continue to require pharmacologic intervention to reduce morbidity and mortality as vaccine availability remains limited. The rise of new variants makes the development of therapeutic strategies even more crucial to combat the current pandemic and future outbreaks. Evidence from several studies suggests the host immune response to SARS-CoV-2 infection plays a critical role in disease pathogenesis. Consequently, host immune factors are becoming more recognized as potential biomarkers and therapeutic targets for COVID-19. To develop therapeutic strategies to combat current and future coronavirus outbreaks, understanding how the coronavirus hijacks the host immune system during and after the infection is crucial. In this study, we investigated immunological patterns or characteristics of the host immune response to SARS-CoV-2 infection that may contribute to the disease severity of COVID-19 patients. We analyzed large bulk RNASeq and single cell RNAseq data from COVID-19 patient samples to immunoprofile differentially expressed gene sets and analyzed pathways to identify human host protein targets. We observed an immunological profile of severe COVID-19 patients characterized by upregulated cytokines, interferon-induced proteins, and pronounced T cell lymphopenia, supporting findings by previous studies. We identified a number of host immune targets including PERK, PKR, TNF, NF-kB, and other key genes that modulate the significant pathways and genes identified in COVID-19 patients. Finally, we identified genes modulated by COVID-19 infection that are implicated in oncogenesis, including E2F transcription factors and RB1, suggesting a mechanism by which SARS-CoV-2 infection may contribute to oncogenesis. Further clinical investigation of these targets may lead to bonafide therapeutic strategies to treat the current COVID-19 pandemic and protect against future outbreaks and viral escape variants.

Project description:Two doses of BNT162b2 mRNA vaccine induces a strong systemic SARS-CoV-2 specific humoral response. However, SARS-CoV-2 airborne transmission makes mucosal immune response a crucial first line of defense. Therefore, we characterized SARS-CoV-2-specific IgG responses induced by BNT162b2 vaccine, as well as IgG responses to other pathogenic and seasonal human coronaviruses in oral fluid and plasma from 200 UK healthcare workers who were naïve (N=62) or previously infected with SARS-CoV-2 (N=138) using a pan-coronavirus multiplex binding immunoassay (Meso Scale Discovery®). Additionally, we investigated the impact of historical SARS-CoV-2 infection on vaccine-induced IgG, IgA and neutralizing responses in selected oral fluid samples before vaccination, after a first and second dose of BNT162b2, as well as following a third dose of mRNA vaccine or breakthrough infections using the same immunoassay and an ACE2 inhibition assay. Prior to vaccination, we found that spike-specific IgG levels in oral fluid positively correlated with IgG levels in plasma from previously-infected individuals (Spearman r=0.6858, p<0.0001) demonstrating that oral fluid could be used as a proxy for the presence of plasma SARS-CoV-2 IgG. However, the sensitivity was lower in oral fluid (0.85, 95% CI 0.77-0.91) than in plasma (0.94, 95% CI 0.88-0.97). Similar kinetics of mucosal and systemic spike-specific IgG levels were observed following vaccination in naïve and previously-infected individuals, respectively. In addition, a significant enhancement of OC43 and HKU1 spike-specific IgG levels was observed in previously-infected individuals following one vaccine dose in oral fluid (OC43 S: p<0.0001; HKU1 S: p=0.0423) suggesting cross-reactive IgG responses to seasonal beta coronaviruses. Mucosal spike-specific IgA responses were induced by mRNA vaccination particularly in previously-infected individuals (71%) but less frequently in naïve participants (23%). Neutralizing responses to SARS-CoV-2 ancestral and variants of concerns were detected following vaccination in naïve and previously-infected participants, with likely contribution from both IgG and IgA in previously-infected individuals (correlations between neutralizing responses and IgG: Spearman r=0.5642, p<0.0001; IgA: Spearman r=0.4545, p=0.0001). We also observed that breakthrough infections or a third vaccine dose enhanced mucosal antibody levels and neutralizing responses. These data contribute to show that a previous SARS-CoV-2 infection tailors the mucosal antibody profile induced by vaccination.

Dataset Information

Differential host circRNA expression profiles in human lung epithelial cells infected with SARS-CoV-2.

Publications

Differential host circRNA expression profiles in human lung epithelial cells infected with SARS-CoV-2.

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