ABSTRACT
Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a new member of the Betacoronaviridae family, responsible for the recent pandemic outbreak of COVID-19. To start exploring the molecular events that follow host cell infection, we queried VirusCircBase and identified a circular RNA (circRNA) predicted to be synthesized by SARS-CoV-2, circ_3205, which we used to probe: (i) a training cohort comprised of two pools of cells from three nasopharyngeal swabs of SARS-CoV-2 infected (positive) or uninfected (negative, UCs) individuals; (ii) a validation cohort made up of 12 positive and 3 negative samples. The expression of circRNAs, miRNAs and miRNA targets was assayed through real-time PCR. CircRNA-miRNA interactions were predicted by TarpMiR, Analysis of Common Targets for circular RNAs (ACT), and STarMir tools. Enrichment of the biological processes and the list of predicted miRNA targets were retrieved from DIANA miRPath v3.0. Our results showed that the predicted SARS-CoV-2 circ_3205 was expressed only in positive samples and its amount positively correlated with that of SARS-CoV-2 Spike (S) mRNA and the viral load (r values = 0.80952 and 0.84867, Spearman's correlation test, respectively). Human (hsa) miR-298 was predicted to interact with circ_3205 by all three predictive tools. KCNMB4 and PRKCE were predicted as hsa-miR-298 targets. Interestingly, the function of both is correlated with blood coagulation and immune response. KCNMB4 and PRKCE mRNAs were upregulated in positive samples as compared to UCs (6 and 8.1-fold, p values = 0.049 and 0.02, Student's t test, respectively) and their expression positively correlated with that of circ_3205 (r values = 0.6 and 0.25, Spearman's correlation test, respectively). We propose that our results convincingly suggest that circ_3205 is a circRNA synthesized by SARS-CoV-2 upon host cell infection and that it may behave as a competitive endogenous RNA (ceRNA), sponging hsa-miR-298 and contributing to the upregulation of KCNMB4 and PRKCE mRNAs.
Subject(s)
COVID-19/genetics , COVID-19/metabolism , RNA, Circular/genetics , RNA, Viral , SARS-CoV-2/genetics , Computational Biology , Gene Expression Regulation, Viral , Gene Regulatory Networks , Humans , Large-Conductance Calcium-Activated Potassium Channel beta Subunits/genetics , MicroRNAs/genetics , MicroRNAs/metabolism , Nasopharynx/virology , Nerve Tissue Proteins/genetics , Protein Interaction Mapping , Protein Kinase C-epsilon/genetics , Reproducibility of ResultsABSTRACT
Reverse transcriptionquantitative polymerase chain reaction (RTqPCR) is the gold standard method for the diagnosis of COVID19 infection. Due to preanalytical and technical limitations, samples with low viral load are often misdiagnosed as falsenegative samples. Therefore, it is important to evaluate other strategies able to overcome the limits of RTqPCR. Blinded swab samples from two individuals diagnosed positive and negative for COVID19 were analyzed by droplet digital PCR (ddPCR) and RTqPCR in order to assess the sensitivity of both methods. Intercalation chemistries and a World Health Organization (WHO)/Center for Disease Control and Prevention (CDC)approved probe for the SARSCoV2 N gene were used. SYBRGreen RTqPCR is not able to diagnose as positive samples with low viral load, while, TaqMan Probe RTqPCR gave positive signals at very late Ct values. On the contrary, ddPCR showed higher sensitivity rate compared to RTqPCR and both EvaGreen and probe ddPCR were able to recognize the sample with low viral load as positive even at 10fold diluted concentration. In conclusion, ddPCR shows higher sensitivity and specificity compared to RTqPCR for the diagnosis of COVID19 infection in falsenegative samples with low viral load. Therefore, ddPCR is strongly recommended in clinical practice for the diagnosis of COVID19 and the followup of positive patients until complete remission.
Subject(s)
Betacoronavirus/genetics , Coronavirus Infections/diagnosis , Pneumonia, Viral/diagnosis , RNA, Viral/analysis , Real-Time Polymerase Chain Reaction/methods , Reverse Transcriptase Polymerase Chain Reaction/methods , COVID-19 , Coronavirus Nucleocapsid Proteins , Humans , Nucleocapsid Proteins/genetics , Pandemics , Phosphoproteins , Polyproteins , SARS-CoV-2 , Sensitivity and Specificity , Spike Glycoprotein, Coronavirus/genetics , Viral Proteins/geneticsABSTRACT
The emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) disease (COVID-19) has posed a serious threat to global health. As no specific therapeutics are yet available to control disease evolution, more in-depth understanding of the pathogenic mechanisms induced by SARS-CoV-2 will help to characterize new targets for the management of COVID-19. The present study identified a specific set of biological pathways altered in primary human lung epithelium upon SARS-CoV-2 infection, and a comparison with SARS-CoV from the 2003 pandemic was studied. The transcriptomic profiles were also exploited as possible novel therapeutic targets, and anti-signature perturbation analysis predicted potential drugs to control disease progression. Among them, Mitogen-activated protein kinase kinase (MEK), serine-threonine kinase (AKT), mammalian target of rapamycin (mTOR) and I kappa B Kinase (IKK) inhibitors emerged as candidate drugs. Finally, sex-specific differences that may underlie the higher COVID-19 mortality in men are proposed.