Utility of monocyte distribution width for diagnosing colonic diverticulitis in the emergency department: a retrospective cohort study (preprint)

Background Colonic diverticulitis is a leading cause of abdominal pain. The monocyte distribution width (MDW) is a novel inflammatory biomarker with prognostic significance for coronavirus disease and pancreatitis; however, no study has assessed its correlation with the severity of colonic diverticulitis. Methods This single-center retrospective cohort study included patients older than 18 years who presented to the emergency department between November 1, 2020, and May 31, 2021, and received a diagnosis of acute colonic diverticulitis after abdominal computed tomography. The characteristics and laboratory parameters of patients with simple versus complicated diverticulitis were compared. The significance of categorical data was assessed using the chi-square or Fisher’s exact test. The Mann–Whitney U test was used for continuous variables. Multivariate regression analysis was performed to identify predictors of complicated colonic diverticulitis. Receiver operator characteristic (ROC) curves were used to test the efficacy of inflammatory biomarkers in distinguishing simple from complicated cases. Results Of the 160 patients enrolled, 21 (13.125%) had complicated diverticulitis. Although right-sided was more prevalent than left-sided colonic diverticulitis (70% versus 30%), complicated diverticulitis was more common in those with left-sided colonic diverticulitis (61.905%, p = 0.001). Age, white blood cell (WBC) count, neutrophil count, C-reactive protein (CRP) level, neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), and MDW were significantly higher in the complicated diverticulitis group (p < 0.05). Logistic regression analysis indicated that the left-sided location and the MDW were significant and independent predictors of complicated diverticulitis. The area under the ROC curve (AUC) was as follows: MDW, 0.870 (95% confidence interval [CI], 0.784–0.956); CRP, 0.800 (95% CI, 0.707–0.892); NLR, 0.724 (95% CI, 0.616–0.832); PLR, 0.662 (95% CI, 0.525–0.798); and WBC, 0.679 (95% CI, 0.563–0.795). The MDW had the largest AUC for diagnosing complicated diverticulitis; when the MDW cutoff was 20.38, the sensitivity and specificity were maximized to 90.5% and 80.6%, respectively. Conclusions Patients with complicated diverticulitis were significantly older and predominantly had left-sided colonic diverticulitis. A large MDW was a significant and independent predictor of complicated diverticulitis. The MDW may aid in planning antibiotic therapy for patients with colonic diverticulitis in the emergency department.

Impact of COVID-19 control measures on the spread of influenza

Objective: To analyze the effects of respiratory control measures before and after COVID-19 epidemic on influenza virus.

Identification of a conserved drug binding pocket in TMEM16 proteins (preprint)

The TMEM16 family of calcium-activated membrane proteins includes ten mammalian paralogs (TMEM16A-K) playing distinct physiological roles with some implicated in cancer and airway diseases. Their modulators with therapeutic potential include 1PBC, a potent inhibitor with anti-tumoral properties, and the FDA-approved drug niclosamide that targets TMEM16F to inhibit syncytia formation induced by SARS-CoV-2 infection. Here, we report cryo-EM structures of TMEM16F associated with 1PBC and niclosamide, revealing that both molecules bind the same drug binding pocket. We functionally and computationally validate this binding pocket in TMEM16A as well as TMEM16F, thereby showing that drug modulation also involves residues that are not conserved between TMEM16A and TMEM16F. This study establishes a much-needed structural framework for the development of more potent and more specific drug molecules targeting TMEM16 proteins.

Immune checkpoint blocking impact and nomogram prediction of COVID-19 inactivated vaccine seroconversion in patients with cancer: a propensity-score matched analysis.

BACKGROUND: Patients with cancer on active immune checkpoint inhibitors therapy were recommended to seek prophylaxis from COVID-19 by vaccination. There have been few reports to date to discuss the impact of progression cell death-1 blockers (PD-1B) on immune or vaccine-related outcomes, and what risk factors that contribute to the serological status remains to be elucidated. The study aims to find the impact of PD-1B on vaccination outcome and investigate other potential risk factors associated with the risk of seroconversion failure. METHODS: Patients with active cancer treatment were retrospectively enrolled to investigate the interaction effects between PD-1B and vaccination. Through propensity score matching of demographic and clinical features, the seroconversion rates and immune/vaccination-related adverse events (irAE and vrAE) were compared in a head-to-head manner. Then, a nomogram predicting the failure risk was developed with variables significant in multivariate regression analysis and validated in an independent cohort. RESULTS: Patients (n=454) receiving either PD-1B or COVID-19 vaccination, or both, were matched into three cohorts (vac+/PD-1B+, vac+/PD-1B-, and vac-/PD-1B+, respectively), with a non-concer control group of 206 participants. 68.1% (94/138), 71.3% (117/164), and 80.5% (166/206) were seropositive in vac+/PD-1B+cohort, vac+/PD-1B- cohort, and non-cancer control group, respectively. None of irAE or vrAE was observed to be escalated in PD-1B treatment except for low-grade rash.The vaccinated patients with cancer had a significantly lower rate of seroconversion rates than healthy control. A nomogram was thus built that encompassed age, pathology, and chemotherapy status to predict the seroconversion failure risk, which was validated in an independent cancer cohort of 196 patients. CONCLUSION: Although patients with cancer had a generally decreased rate of seroconversion as compared with the healthy population, the COVID-19 vaccine was generally well tolerated, and seroconversion was not affected in patients receiving PD-1B. A nomogram predicting failure risk was developed, including age, chemotherapy status, pathology types, and rheumatic comorbidity.

Diverse pathogens activate the host RIDD pathway to subvert BLOS1-directed immune defense (preprint)

The phagocytosis and destruction of pathogens in lysosomes constitute central elements of innate immune defense. Here, we show that Brucella , the causative agent of brucellosis, the most prevalent bacterial zoonosis globally, subverts this immune defense pathway by activating regulated IRE1α-dependent decay (RIDD) of mRNAs encoding BLOS1, a protein that promotes endosome-lysosome fusion. RIDD-deficient cells and mice harboring a RIDD-incompetent variant of IRE1α were resistant to infection. Non-functional Blos1 struggled to assemble the BLOC-1-related complex (BORC), resulting in differential recruitment of BORC-related lysosome trafficking components, perinuclear trafficking of Brucella -containing vacuoles (BCVs), and enhanced susceptibility to infection. The RIDD-resistant Blos1 variant maintains the integrity of BORC and a higher-level association of BORC-related components that promote centrifugal lysosome trafficking, resulting in enhanced BCV peripheral trafficking and lysosomal-destruction, and resistance to infection. These findings demonstrate that host RIDD activity on BLOS1 regulates Brucella intracellular parasitism by disrupting BORC-directed lysosomal trafficking. Notably, coronavirus MHV also subverted the RIDD-BLOS1 axis to promote intracellular replication. Our work therefore establishes BLOS1 as a novel immune defense factor whose activity is hijacked by diverse pathogens.

Targeting Stem-loop 1 of the SARS-CoV-2 5'UTR to suppress viral translation and Nsp1 evasion (preprint)

SARS-CoV-2 is a highly pathogenic virus that evades anti-viral immunity by interfering with host protein synthesis, mRNA stability, and protein trafficking. The SARS-CoV-2 nonstructural protein 1 (Nsp1) uses its C-terminal domain to block the mRNA entry channel of the 40S ribosome to inhibit host protein synthesis. However, how SARS-CoV-2 circumvents Nsp1-mediated suppression for viral protein synthesis and if the mechanism can be targeted therapeutically remain unclear. Here we show that N- and C-terminal domains of Nsp1 coordinate to drive a tuned ratio of viral to host translation, likely to maintain a certain level of host fitness while maximizing replication. We reveal that the SL1 region of the SARS-CoV-2 5 UTR is necessary and sufficient to evade Nsp1-mediated translational suppression. Targeting SL1 with locked nucleic acid antisense oligonucleotides (ASOs) inhibits viral translation and makes SARS-CoV-2 5 UTR vulnerable to Nsp1 suppression, hindering viral replication in vitro at a nanomolar concentration. Thus, SL1 allows Nsp1 to switch infected cells from host to SARS-CoV-2 translation, presenting a therapeutic target against COVID-19 that is conserved among immune-evasive variants. This unique strategy of unleashing a virus own virulence mechanism against itself could force a critical trade off between drug resistance and pathogenicity.

SARS-CoV-2 infects blood monocytes to activate NLRP3 and AIM2 inflammasomes, pyroptosis and cytokine release (preprint)

SARS-CoV-2 causes acute respiratory distress that can progress to multiorgan failure and death in a minority of patients. Although severe COVID-19 disease is linked to exuberant inflammation, how SARS-CoV-2 triggers inflammation is not understood. Monocytes and macrophages are sentinel immune cells in the blood and tissue, respectively, that sense invasive infection to form inflammasomes that activate caspase-1 and gasdermin D (GSDMD) pores, leading to inflammatory death (pyroptosis) and processing and release of IL-1 family cytokines, potent inflammatory mediators. Here we show that expression quantitative trait loci (eQTLs) linked to higher GSDMD expression increase the risk of severe COVID-19 disease (odds ratio, 1.3, p<0.005). We find that about 10% of blood monocytes in COVID-19 patients are infected with SARS-CoV-2. Monocyte infection depends on viral antibody opsonization and uptake of opsonized virus by the Fc receptor CD16. After uptake, SARS-CoV-2 begins to replicate in monocytes, as evidenced by detection of double-stranded RNA and subgenomic RNA and expression of a fluorescent reporter gene. However, infection is aborted, and infectious virus is not detected in infected monocyte supernatants or patient plasma. Instead, infected cells undergo inflammatory cell death (pyroptosis) mediated by activation of the NLRP3 and AIM2 inflammasomes, caspase-1 and GSDMD. Moreover, tissue-resident macrophages, but not infected epithelial cells, from COVID-19 lung autopsy specimens showed evidence of inflammasome activation. These findings taken together suggest that antibody-mediated SARS-CoV-2 infection of monocytes/macrophages triggers inflammatory cell death that aborts production of infectious virus but causes systemic inflammation that contributes to severe COVID-19 disease pathogenesis.

Disulfiram associated with lower risk of Covid-19: a retrospective cohort study (preprint)

In the global COVID-19 pandemic, there is a substantial need for effective, low-cost therapeutics. We investigated the potential effects of disulfiram on the incidence and outcomes of COVID-19 in an observational study in a large database of US Veterans Administration clinical records, the VA Corporate Data Warehouse (CDW). The study is motivated by the unique properties of disulfiram, which has been used as an anti-alcoholism drug since 1948, is non-toxic, easy to manufacture and inexpensive. Disulfiram reduces hyperinflammation in mammalian cells by inhibition of the gasdermin D pore. In a mouse model of sepsis, disulfiram reduced inflammatory cytokines and mortality. Disulfiram also is a low micromolar inhibitor of the Mpro and PLpro viral proteases of SARS-CoV-2. To investigate the potential effects of disulfiram on the incidence and severity of COVID-19, we carried out an epidemiological study in the CDW. The VA dataset used has 944,127 patients tested for SARS-Cov-2, 167,327 with a positive test, and 2,233 on disulfiram, of which 188 had a positive SARS-Cov-2 test. A multivariable Cox regression adjusted for age, gender, race/ethnicity, region, a diagnosis of alcohol use disorders, and Charlson comorbidity score revealed a reduced incidence of COVID-19 with disulfiram use with a hazard ratio of 0.66 and 95% confidence interval of 0.57 to 0.76 (P < 0.001). There were no deaths among the 188 SARS-Cov-2 positive patients treated with disulfiram. The expected number of deaths would have been 5-6 according to the 3% death rate among the untreated (P-value 0.03). Our finding of a lower hazard ratio and less severe outcomes for COVID-19 in patients treated with disulfiram compared to those not treated is a statistical association and does not prove any causative effect of disulfiram. However, the results of this study suggest that there is a pharmacological contribution to the reduced incidence and severity of COVID-19 with the use of disulfiram. Given the known anti-inflammatory and viral anti-protease effects of disulfiram, it is reasonable and urgent to initiate accelerated clinical trials to assess whether disulfiram reduces SARS-CoV-2 infection, disease severity and death.

SARS-CoV-2 infects blood monocytes to activate NLRP3 and AIM2 inflammasomes, pyroptosis and cytokine release (preprint)

SARS-CoV-2 causes acute respiratory distress that can progress to multiorgan failure and death in some patients. Although severe COVID-19 disease is linked to exuberant inflammation, how SARS-CoV-2 triggers inflammation is not understood. Monocytes are sentinel blood cells that sense invasive infection to form inflammasomes that activate caspase-1 and gasdermin D (GSDMD) pores, leading to inflammatory death (pyroptosis) and processing and release of IL-1 family cytokines, potent inflammatory mediators. Here we show that ~10% of blood monocytes in COVID-19 patients are dying and infected with SARS-CoV-2. Monocyte infection, which depends on antiviral antibodies, activates NLRP3 and AIM2 inflammasomes, caspase-1 and GSDMD cleavage and relocalization. Signs of pyroptosis (IL-1 family cytokines, LDH) in the plasma correlate with development of severe disease. Moreover, expression quantitative trait loci (eQTLs) linked to higher GSDMD expression increase the risk of severe COVID-19 disease (odds ratio, 1.3, p<0.005). These findings taken together suggest that antibody-mediated SARS-CoV-2 infection of monocytes triggers inflammation that contributes to severe COVID-19 disease pathogenesis.

Emotional exhaustion in front-line healthcare workers during the COVID-19 pandemic -- the effects of time pressure, social sharing and cognitive reappraisal: A cross-sectional study (preprint)

Background: With the increasing spread of COVID-19, healthcare workers, especially front-line medical staff, have become more vulnerable to emotional exhaustion.Objectives: This study aimed to determine the influence of time pressure on the emotional exhaustion of front-line healthcare workers, and explore the effects of social sharing and cognitive reappraisal on this.Methods: This cross-sectional study was conducted in March 2020. A total of 232 questionnaires were completed by front-line healthcare workers in Wuhan city, Hubei province, China. Hierarchical linear regression and conditional process analysis were performed to explore the relationships among time pressure, social sharing, cognitive reappraisal, and emotional exhaustion.Results: Time pressure was positively associated with social sharing and emotional exhaustion. Social sharing presented a dark side in terms of the impact on emotional exhaustion. Cognitive reappraisal negatively moderated the relationship between time pressure and social sharing, and it further indirectly influenced the relationship between time pressure and emotional exhaustion through social sharing.Conclusions: Our findings shed light on how time pressure influences the emotional exhaustion of healthcare workers during the COVID-19 period. Although social sharing is commonly regarded as a positive behavior, we identified a dark side in terms of its impact. We also identified that improving cognitive reappraisal may present a positive strategy toward alleviating emotional exhaustion.

Clinical Features and Death Risk Factors in COVID-19 Patients with Cancer: A Retrospective Study (preprint)

Purpose: Coronavirus disease 2019 (COVID-19) has spread around the world. This retrospective study aims to analyze the clinical features of COVID-19 patients with cancer and identify death outcome related risk factors.Methods: From February 10th to April 15th, 2020, 103 COVID-19 patients with cancer were enrolled. Difference analyses were performed between severe and non-severe patients. A propensity score matching analysis, including 103 COVID-19 patients with cancer and 206 matched non-cancer COVID-19 patients were performed. Next, we identified death related risk factors and developed a nomogram for predicting the probability.Results: In 103 COVID-19 patients with cancer, the main cancer categories were breast cancer, lung cancer and bladder cancer. Compared to non-severe patients, severe patients had a higher median age, and a higher proportion of smokers, diabetes, heart disease and dyspnea. In addition, most of the laboratory results between two groups were significant different. PSM analysis found that the proportion of dyspnea was much higher in COVID-19 patients with cancer. The severity incidence in two groups were similar, while a much higher mortality was found in COVID-19 patients with cancer compared to that in COVID-19 patients without cancer (11.7% vs. 4.4%, P = 0.028). Furthermore, we found that neutrophil-to-lymphocyte ratio (NLR) and C-reactive protein (CRP) were related to death outcome. And a nomogram based on the factors was developed.Conclusion: In COVID-19 patients with cancer, the clinical features and laboratory results between severe group and non-severe group were significant different. NLR and CRP were the risk factors that could predict death outcome.

The Spatiotemporal Interaction Effect of COVID-19 Transmission in the United States (preprint)

Background: Human mobility among geographic units is a possible cause of the widespread transmission of COVID-19 across regions. Due to the pressure of epidemic control and economic recovery, the states of the United States have adopted different policies for mobility limitations. Assessing the impact of these policies on the spatiotemporal interaction of COVID-19 transmission among counties in each state is critical to formulating the epidemic policies.Methods: The study utilized Moran’s I index and K-means clustering to investigate the time-varying spatial autocorrelation effect of 49 states (except the District of Colombia) with the daily new cases at the county level from Jan 22, 2020, to August 20, 2020. Based on the dynamic spatial lag model (SLM) and the SIR model with unreported infection rate (SIRu), the integrated SLM-SIRu model was constructed to estimate the inter-county spatiotemporal interaction coefficient of daily new cases in each state, which was further explored by Pearson correlation and stepwise OLS regression with socioeconomic factors.Results: The K-means clustering divided the time-varying spatial autocorrelation curves of 49 states into four types: continuous increasing, fluctuating increasing, weak positive, and weak negative. The Pearson correlation analysis showed that the spatiotemporal interaction coefficients in each state estimated by SLM-SIRu were significantly positively correlated with median age, population density, and the proportion of international immigrants and the highly educated population, but negatively correlated with the birth rate. The voting rate for Donald Trump in the 2016 U.S. presidential election showed a weak negative correlation. Further stepwise OLS regression retained only three positive correlated variables: poverty rate, population density, and the highly educated population proportion.Interpretation: This result suggests that various state policies in the U.S. have imposed different impacts on COVID-19 transmission among counties. All states should provide more protection and support for the low-income population, high-density populated states need to strengthen regional mobility restrictions, and the highly educated population should reduce unnecessary regional movement and strengthen self-protection.

Risk Factors Associated with Disease Severity and Clinical Outcomes for COVID-19 in Wuhan, China (preprint)

Background: A new type of pneumonia caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) appeared in Wuhan, China. However, the risk factors and characteristics related to the severity of the disease and its outcomes need to be further explored.Methods: In this retrospective study, we evaluated COVID-19 patients with severe disease and those who were critically ill, as diagnosed at Jinyintan Hospital (Wuhan, China). The demographic information, clinical characteristics, complications, and laboratory results for the patients were evaluated. Multivariate logistic regression methods were used to analyze risk factors related to hospital deaths.Results: The 235 COVID-19 patients included were divided into a severe group of 183 (78%) and a critical group of 52 (22%). Of these patients, 185 (79%) were discharged, and 50 (21%) died during hospitalization. In multivariate logistic analyses, age (OR=1.07, 95% CI 1.02-1.14, P=0.009), critical disease (OR=48.23, 95% CI 10.91-323.13, P<0.001), low lymphocyte counts (OR=15.48, 95% CI 1.98-176.49, P=0.015), elevated interleukin 6 (IL-6) (OR=9.11, 95% CI 1.69-67.75, P=0.017), and elevated aspartate aminotransferase (AST) (OR=8.46, 95% CI 2.16-42.60, P=0.004) were independent risk factors for adverse outcomes.Conclusions: The results show that advanced age (> 64 years), critical illness, low lymphocyte levels, and elevated IL-6 and AST were factors for the risk of death for COVID-19 patients who had severe disease and those who were critically ill.

The Spatiotemporal Interaction Effect of COVID-19 Transmission in the United States (preprint)

Background: Human mobility among geographic units is a possible cause of the widespread transmission of COVID-19 across regions. Due to the pressure of epidemic control and economic recovery, the states of the United States have adopted different policies for mobility limitations. Assessing the impact of these policies on the spatiotemporal interaction of COVID-19 transmission among counties in each state is critical to formulating the epidemic policies.Methods: The study utilized Moran’s I index and K-means clustering to investigate the time-varying spatial autocorrelation effect of 49 states (except the District of Colombia) with the daily new cases at the county level from Jan 22, 2020, to August 20, 2020. Based on the dynamic spatial lag model (SLM) and the SIR model with unreported infection rate (SIRu), the integrated SLM-SIRu model was constructed to estimate the inter-county spatiotemporal interaction coefficient of daily new cases in each state, which was further explored by Pearson correlation and stepwise OLS regression with socioeconomic factors.Results: The K-means clustering divided the time-varying spatial autocorrelation curves of 49 states into four types: continuous increasing, fluctuating increasing, weak positive, and weak negative. The Pearson correlation analysis showed that the spatiotemporal interaction coefficients in each state estimated by SLM-SIRu were significantly positively correlated with median age, population density, and the proportion of international immigrants and the highly educated population, but negatively correlated with the birth rate. The voting rate for Donald Trump in the 2016 U.S. presidential election showed a weak negative correlation. Further stepwise OLS regression retained only three positive correlated variables: poverty rate, population density, and the highly educated population proportion.Interpretation: This result suggests that various state policies in the U.S. have imposed different impacts on COVID-19 transmission among counties. All states should provide more protection and support for the low-income population, high-density populated states need to strengthen regional mobility restrictions, and the highly educated population should reduce unnecessary regional movement and strengthen self-protection. 

Prevalence and Influencing Factors of Anxiety and Depression Symptoms Amongst Surgical Nurses During COVID-19 Pandemic: A Large-Scale Cross-Sectional Study (preprint)

BackgroundThe outbreak of novel 2019 coronavirus disease (COVID-19) has imposed an enormous physical and psychological pressure on people across the world. This study focused on evaluating the prevalence and influencing factors of anxiety and depression symptoms in surgical nurses during the epidemic in China. MethodA cross-sectional, multicenter quantitative study was conducted in Anhui province (China) from March 3, 2020 to March 19, 2020, with a questionnaire package which consisted of general information questionnaire，Zung's self-rating anxiety scale (SAS), Zung's self-rating Depression Scale (SDS) and social support rating scale (SSRS). A total of 3600 surgical nurses participated in the survey by Wechat and QQ. Data were analysed using multiple linear regression models. ResultsA total of 3492 surgical nurses from 12tertiary hospitals and 12 secondary hospitals in one province of mainland China completed the survey. The prevalence rates of anxiety symptoms and depressive symptoms were 24.83% and 22.39%, respectively. The average level of anxiety and depression of surgical nurses were higher than that of the Chinese norm (P< 0.05).Levels of social support for surgical nurses were significantly negatively associated with the degree of anxiety (r = -0.630, P < 0.001) and depression (r = -0.578, P < 0.001). Fertility status (β = 1.469, P = 0.003), hospital (β = -0.611, P < 0.001), participation in care for COVID-19 patients (β = 2.229, P < 0.001), likelihood of being infected with COVID-19 (β = 1.146, P < 0.001), social support (β = -0.623, P < 0.001) were significantly influencing surgical nurses’ anxiety degree. Similarly, these characteristics were significantly associated with the odds of experiencing depression symptoms in surgical nurses. Divorce and widowed surgical nurses (β = -2.654, P < 0.001) were significantly more likely to experience depressive symptoms than single nurses. ConclusionIn this survey, we found that the surgical nurses had high anxiety and depression symptoms during the COVID-19 outbreak in China. The findings suggest that targeted psychological interventions to promote the mental health of surgical nurses with psychological problems need to be immediately implemented. 

Establishment of Routine Clinical Indicators-Based Nomograms for Predicting the Mortality in Patients with COVID-19 (preprint)

Background: The aim of the study was to establish and validate nomograms to predict the mortality risk of patients with COVID-19 using routine clinical indicators. Method: This retrospective study included a development cohort enrolled 2119 hospitalized COVID-19 patients and a validation cohort included 1504 COVID-19 patients. The demographics, clinical manifestations, vital signs and laboratory test results of the patients at admission and outcome of in-hospital death were recorded. The independent factors associated with death were identified by a forward stepwise multivariate logistic regression analysis and used to construct two prognostic nomograms. The models were then tested in an external dataset. Results: Nomogram 1 is a full model included nine factors identified in the multivariate logistic regression and nomogram 2 is built by selecting four factors from nine to perform as a reduced model. Nomogram 1 and nomogram 2 established showed better performance in discrimination and calibration than the MuLBSTA score in training. In validation, Nomogram 1 performed better than nomogram 2 for calibration. Conclusion: Nomograms we established performed better than the MuLBSTA score. We recommend the application of nomogram 1 in general hospital which provide robust prognostic performance but more cumbersome; nomogram 2 in mobile cabin hospitals which depend on less laboratory examinations and more convenient. Both nomograms can help clinicians in identifying patients at risk of death with routine clinical indicators at admission, which may reduce the overall mortality of COVID-19.

Whole Genome Sequencing for Revealing the Point Mutations of SARS-CoV-2 Genome in Bangladeshi Isolates and their Structural Effects on Viral Proteins (preprint)

Coronavirus disease-19 (COVID-19) is the recent global pandemic caused by the virus Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). The virus has already killed more than one million people worldwide and billions are at risk of getting infected. As of now, there is neither any drug nor any vaccine in sight with conclusive scientific evidence that it can cure or provide protection against the illness. Since novel coronavirus is a new virus, mining its genome sequence is of crucial importance for drug/vaccine(s) development. Whole genome sequencing is a helpful tool in identifying genetic changes that occur in a virus when it spreads through the population. In this study, we performed complete genome sequencing of SARS-CoV-2 to unveil the genomic variation and indel, if present. We discovered thirteen (13) mutations in Orf1ab, S and N gene where seven (7) of them turned out to be novel mutations from our sequenced isolate. Besides, we found one (1) insertion and seven (7) deletions from the indel analysis among the 323 Bangladeshi isolates. However, the indel did not show any effect on proteins. Our energy minimization analysis showed both stabilizing and destabilizing impact on viral proteins depending on the mutation. Interestingly, all the variants were located in the binding site of the proteins. Furthermore, drug binding analysis revealed marked difference in interacting residues in mutants when compared to the wild type. Our analysis also suggested that eleven (11) mutations could exert damaging effects on their corresponding protein structures. The analysis of SARS-CoV-2 genetic variation and their impacts presented in this study might be helpful in gaining a better understanding of the pathogenesis of this deadly virus.

Amilorides inhibit SARS-CoV-2 replication in vitro by targeting RNA structures (preprint)

The SARS-CoV-2 pandemic, and the likelihood of future coronavirus pandemics, has rendered our understanding of coronavirus biology more essential than ever. Small molecule chemical probes offer to both reveal novel aspects of virus replication and to serve as leads for antiviral therapeutic development. The RNA-biased amiloride scaffold was recently tuned to target a viral RNA structure critical for translation in enterovirus 71, ultimately uncovering a novel mechanism to modulate positive-sense RNA viral translation and replication. Analysis of CoV RNA genomes reveal many conserved RNA structures in the 5-UTR and proximal region critical for viral translation and replication, including several containing bulge-like secondary structures suitable for small molecule targeting. Following phylogenetic conservation analysis of this region, we screened an amiloride-based small molecule library against a less virulent human coronavirus, OC43, to identify lead ligands. Amilorides inhibited OC43 replication as seen in viral plaque assays. Select amilorides also potently inhibited replication competent SARS-CoV-2 as evident in the decreased levels of cell free virions in cell culture supernatants of treated cells. Reporter screens confirmed the importance of RNA structures in the 5-end of the viral genome for small molecule activity. Finally, NMR chemical shift perturbation studies of the first six stem loops of the 5-end revealed specific amiloride interactions with stem loops 4, 5a, and 6, all of which contain bulge like structures and were predicted to be strongly bound by the lead amilorides in retrospective docking studies. Taken together, the use of multiple orthogonal approaches allowed us to identify the first small molecules aimed at targeting RNA structures within the 5-UTR and proximal region of the CoV genome. These molecules will serve as chemical probes to further understand CoV RNA biology and can pave the way for the development of specific CoV RNA-targeted antivirals.