RNA-sequencing unravels unique host-response to infection with SARS-CoV-2

Introduction/Aim: As the causative agent of COVID-19, SARS-CoV-2 remains a global cause for concern. Compared to other highly pathogenic coronaviruses (SARS-CoV and MERS-CoV), SARS-CoV-2 exhibits stronger transmissibility but less lethality, indicating that SARS-CoV-2 displays unique characteristics, despite the partial genomic proximity. Thus, we aim to employ RNA sequencing to define transcriptional differences in epithelial responses following infection with SARS-CoV-2 compared to pathogenic SARS-CoV and MERS-CoV, and low pathogenic HCoV-229E. Method(s): Primary human bronchial epithelial cells (PBEC) were differentiated for 6 weeks at the air-liquid interface (ALI) before parallel infection by the 4 different coronaviruses (n = 4). After infection following apical application of coronaviruses at low dose (MOI 0.1), cells were harvested for bulk RNA sequencing. Gene were considered significant with a fold change (FC) > 2 and false discovery rate of FDR < 0.05. Inhibitor experiments were conducted on CALU-3 cells using DIM-C-pPhOH 10 muM (NR4A1 antagonist), Sp600125 10 muM (JNK inhibitor), T-5224 10 muM (AP-1 transcription factor inhibitor) and Cytosporone B (CsB 5 muM;NR4A1 agonist) preincubated for 1 h with these compounds and subsequently infected with SARS-CoV-2 or MERS-CoV (MOI of 1). Samples were collect 24 h later for PCR. Result(s): PCR and RNA-Seq demonstrated that all tested coronaviruses efficiently infected ALI-PBEC and replicated over 72 h (p < 0.05). RNA sequencing analysis revealed that infection with SARS-CoV, MERS-CoV and HCoV-229E resulted in largely similar transcriptional responses by the epithelial cells. However, whereas infection with these viruses was accompanied by an increased expression of genes associated with JNK/AP-1 signalling, including FOS, FOSB and NR4A1 (FC > 1, FDR < 0.05), no such increase was observed following SARS-CoV-2 infection. Further, we found that an NR4A1 antagonist reduced viral replication of MERS and SARs-CoV-2 100-fold in Calu-3 cells. Conclusion(s): In conclusion, these data suggest that SARS-CoV-2-infected ALI-PBEC exhibit a unique transcriptional response compared to other coronaviruses, which might relate to the pathogenicity of the virus.

RNA sequencing unravels unique host transcriptional responses to infection with SARS-CoV-2 in well differentiated primary human bronchial epithelial cells compared to other coronaviruses

As the causative agent of COVID-19, SARS-CoV-2 remains a global cause for concern. Compared to other highly pathogenic coronaviruses (SARS-CoV and MERS-CoV), SARS-CoV-2 exhibits stronger transmissibility but less lethality, indicating that SARS-CoV-2 displays unique characteristics, despite the partial genomic proximity. Thus, we aim to employ RNA sequencing to define transcriptional differences in epithelial responses following infection with SARS-CoV-2 compared to pathogenic SARS-CoV and MERS-CoV, and low pathogenic HCoV-229E. Primary human bronchial epithelial cells (PBEC) were differentiated for 6 weeks at the air-liquid interface (ALI) before parallel infection by the 4 different coronaviruses. After infection following apical application of coronaviruses at low dose, cells were harvested for bulk RNA sequencing. Results demonstrated that all tested coronaviruses efficiently infected ALI-PBEC. RNA sequencing analysis revealed that infection with SARS-CoV, MERS-CoV and HCoV-229E resulted in largely similar transcriptional responses by the epithelial cells. However, whereas infection with these viruses was accompanied by an increased expression of genes associated with JNK/AP-1 signalling, including FOS, FOSB and NR4A1, no such increase was observed following SARS-CoV-2 infection. Further, preliminary experiments indicated that an NR4A1 antagonist reduced viral replication in Calu-3 cells. In conclusion, these data suggest that SARS-CoV2-infected ALI-PBEC exhibit a unique transcriptional response compared to other coronaviruses, which might relate to the pathogenicity of the virus.

Assessment of Psychological Impact of Covid Pandemic on Frontline workers at Points of Entry,Pakistan : Nadia Noreen

Background The unprecedented public health crisis of the COVID-19 pandemic has caused heightened levels of stress and fear among health care workers.With the advent of COVID-19 in Pakistan,frontline workers of POEs have been under physical and psychological pressure including a high risk of infection, abnormal levels of workload, prolonged working hours, lack of personal protective equipment for safety from contagion, isolation, exhaustion, and lack of contact with family.The study aims to assess the impact of Covid-19 on the mental health of frontline healthcare workers. Methods A descriptive study was conducted among HCWs across points of entry from 1st October 2020 to 31st December 2020.Data was collected using a structured questionnaire.Depression, anxiety, and stress scale (DASS-21)was used for the assessment of depression, stress, anxiety. Descriptive analysis of socio-demographic and professional factors was done. Multivariable logistic regression analysis (MLRA) was performed using SPSS version 23.0. Results A total of 628 participants (586 males and 42 females) completed questionnaire.The mean age of the participants was 42.6 ± 45.9 years. The majority of the respondents were married (94.3%). The frequency of depression, anxiety, and stress in the HCWs was 12.1%, 42.3%, and 22.1 %, respectively. Multivariable logistic regression analysis found that the depression in HCWs was significantly associated with the profession and age (P < 0.001). The anxiety in HCWs was associated with their age and gender (P < 0.005). The stress in HCWs was significantly associated with their age (P < 0.05). Conclusions The HCWs at the Points of entry across Pakistan showed mild to moderate symptoms of DAS. The COVID-19 pandemic has caused a heavy psychological impact among the frontline healthcare professionals. Timely psychological counseling and early psychological intervention need to be implemented for HCWs to alleviate their anxiety and stress and improve their general mental health. Key messages • The COVID-19 pandemic has caused a heavy psychological impact. • Timely psychological counseling and early psychological intervention.

COMBINED VARIABLE IMMUNODEFICIENCY AND MULTISYSTEM DYSFUNCTION IN A 53 YEAR-OLD FEMALE: A CASE REPORT

Introduction: Common Variable Immunodeficiency (CVID) is a heterogenous disease causing insufficient activity of the humoral arm of adaptive immunity. It is characterized by a broad paucity of serum immunoglobulins. Patients are not only predisposed to recurrent infections, they also are more likely to develop certain cancers and autoimmune diseases. Case Description: We present a case of a 53 year-old female suffering from recurrent pulmonary infections and a past history of Non-Hodgkin’s Lymphoma who came for evaluation of drug allergies. By eliciting a thorough history, it was found that she had a poor response to the MMR and Varicella vaccines as a child, and was infected with COVID-19 twice in 2020. Additionally, longitudinal chest CT scans demonstrated bilateral pulmonary nodules, indicative of chronic inflammation. Testing of her antibody titers in order to determine suitability for S. Pneumoniae vaccination found an overall decrease in major immunoglobulin classes (IgG, IgM, and IgA). Hematological testing showed B-cells with normal morphology. The diagnosis of CVID was made, and prompt treatment with IVIG (intravenous immunoglobulins) was initiated, bringing her IgG levels from 282 to 680 (mg/dL) within three months. Discussion: Her constellation of symptoms and diseases likely arose as a result of immune dysfunction, highlighting the necessity of keeping immunodeficiencies like CVID on the differential when managing patients with seemingly unrelated medical problems. Early detection of immunopathologies like CVID should therefore be a priority for longitudinal care. This ideally ensures early detection and prompt management of the multi-system dysfunctions that pose a significant lifetime risk to such patients.

Animal and translational models of SARS-CoV-2 infection and COVID-19.

COVID-19 is causing a major once-in-a-century global pandemic. The scientific and clinical community is in a race to define and develop effective preventions and treatments. The major features of disease are described but clinical trials have been hampered by competing interests, small scale, lack of defined patient cohorts and defined readouts. What is needed now is head-to-head comparison of existing drugs, testing of safety including in the background of predisposing chronic diseases, and the development of new and targeted preventions and treatments. This is most efficiently achieved using representative animal models of primary infection including in the background of chronic disease with validation of findings in primary human cells and tissues. We explore and discuss the diverse animal, cell and tissue models that are being used and developed and collectively recapitulate many critical aspects of disease manifestation in humans to develop and test new preventions and treatments.

Exploring structurally diverse plant secondary metabolites as a potential source of drug targeting different molecular mechanisms of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) pathogenesis: An in silico approach (preprint)

Plants are endowed with a large pool of structurally diverse small molecules known as secondary metabolites. Present study aims to virtually screen these plant secondary metabolites (PMS) for their possible anti-SARS-CoV-2 properties targeting four protein/enzymes which determines viral pathogenesis. Results of molecular docking and data analysis revealed a unique pattern of structurally similar PSM interacting with the target protein. Among the top-ranked PSM with lower binding energy, >50% were triterpenoids against viral spike protein, >32% were flavonoids and their glycoside against Human transmembrane serine protease, >16% were flavonol glycosides and >16% were Anthocyanidine against viral main protease and >13% were flavonol glycoside against viral RNA dependet RNA polymerase. The primary concern about these PSM is their bioavailability. However, several PSM recorded higher bioavailability score and found fulfilling drug-likeness characters as per Lipinski's rule. Natural occurrence, biotransformation, bioavailability of selected PSM and their interaction with the target site of selected proteins were discussed in detail. Further, we hypothesized the use of selected PSM to cure the COVID-19 by inhibiting the process of viral host cell recognition and replication in host cell. However, these PSM needs thorough in vitro and in vivo evaluation before taking them to clinical trials.