SARS-CoV-2 NSP12 utilizes various host splicing factors for replication and splicing regulation (preprint)

Background The RNA-dependent RNA polymerase (RdRp) is a crucial element in the replication and transcription of RNA viruses. Although the RdRps of lethal human coronaviruses SARS-CoV-2, SARS-CoV, and MARS-CoV have been extensively studied, the molecular mechanism of the catalytic subunit NSP12, which is involved in pathogenesis, remains unclear. Results In this study, the biochemical and cell biological results demonstrate the interactions between SARS-CoV-2 NSP12 and seven host proteins, including three splicing factors (SLU7, PPIL3, and AKAP8), suggesting that the polymerase activity and stability of SARS-CoV-2 RdRp were affected by them to varying degrees. Furthermore, the entry efficacy of SARS-CoV-2 pseudovirus considerably decreased when SLU7 or PPIL3 was knocked out, indicating that abnormal splicing of the host genome was responsible for this occurrence. In addition, NSP12 and its homologues from SARS-CoV and MARS-CoV suppressed thealternative splicing (AS) of cellular genes, which were influenced by the three splicing factors. Conclusions Overall, our research illustrates that SARS-CoV-2 NSP12 can engage with various splicing factors, thereby impacting virus entry, replication, and gene splicing. This not only improves our understanding of how viruses cause diseases but also lays the foundation for the development of antiviral therapies.

Guidelines for COVID-19 Laboratory Testing for Emergency Departments From the New Diagnostic Technology Team of the Taiwan Society of Emergency Medicine

COVID-19 tests have different turnaround times (TATs), accuracy levels, and limitations, which emergency physicians should be aware of. Nucleic acid amplification tests (NAATs) can be divided into standard high throughput tests and rapid molecular diagnostic tests at the point of care (POC). The standard NAAT has the advantages of high throughput and high accuracy with a TAT of 3–4 hours. The POC molecular test has the same advantages of high accuracy as standard high throughput PCR, but can be done in 13–45 minutes. Roche cobas Liat is the most commonly used machine in Taiwan, displaying 99%–100% sensitivity and 100% specificity, respectively. Abbott ID NOW is an isothermal PCR-based POC machine with a sensitivity of 79% and a specificity of 100%. A high rate of false positives and false negatives is associated with rapid antigen testing. Antibody testing is mostly used as part of public health surveys and for testing for immunity.

The Impact of Do-Not-Resuscitate Order in the Emergency Department on Respiratory Failure after ICU Admission.

(1) Background: It has been hypothesized that a discrepancy exists in the understanding of a do-not-resuscitate (DNR) order among physicians. We hypothesized that a DNR order signed in the emergency department (ED) could influence the patients' prognosis after intensive care unit (ICU) admission. (2) Methods: We included patients older than 17 years, who visited the emergency department for non-traumatic disease, who had respiratory failure, required ventilator support, and were admitted to the ICU between January 2010 and December 2016. The associations between DNR and mortality, hospital length of stay (LOS), and medical fees were analyzed. Prolonged hospital LOS was defined as hospital stay ≥75th percentile (≥26 days for the study). Patients were classified as those who did and did not sign a DNR order. A 1:4 propensity score matching was conducted for demographics, comorbidities, and etiology. (3) Results: The study enrolled a total of 1510 patients who signed a DNR and 6040 patients who did not sign a DNR. The 30-day mortality rates were 47.4% and 28.0% among patients who did and did not sign a DNR, respectively. A DNR order was associated with mortality after adjusting for confounding factors (hazard ratio, 1.9; confidence interval, 1.70-2.03). It was also a risk factor for prolonged hospital LOS in survivors (odds ratio, 1.2; confidence interval, 1.02-1.44). Survivors who signed a DNR order were charged higher medical fees than those who did not sign a DNR (217,159 vs. 245,795 New Taiwan Dollars, p < 0.001). (4) Conclusions: Signing a DNR order in the ED increased the ICU mortality rate among patients who had respiratory failure and needed ventilator support. It increased the risk of prolonged hospital LOS among survivors. Finally, signing a DNR order was associated with high medical fees among survivors.

[Variation Characteristics of Ambient Volatile Organic Compounds (VOCs) Volume Fraction During Hangzhou COVID-19 Period].

A continuous observation campaign was carried out with the Syntech Spectras GC955 volatile organics online monitoring system from December 1, 2019 to March 31, 2020 during the COVID-19 period in Hangzhou. Composition characteristics, diurnal variation, and atmospheric chemical reactivity of VOCs were analyzed. The results showed that φ(total VOCs) were the highest before the COVID-19 pandemic in different sites and the lowest during the first response period. The φ(total VOCs) at night was higher than that during the day. The daily variation in Wolongqiao φ(total VOCs) was less than that in Xiasha. The daily variation in φ(total VOCs) during the first level response period was less than that during the other three periods. The diurnal variation in the φ (total VOCs) in Xiasha showed a "V" shape, and that in Wolongqiao showed a typical bimodal structure. The OFP in Xiasha was higher than that in Wolongqiao. The OFP were the highest at the two sites before the COVID-19 pandemic. The OFP was the lowest during the first response period in Xiasha and the lowest during the second response period in Wolongqiao. The OFP of aromatics and olefins was higher, and the OFP of alkynes was the lowest in Xiasha. The OFP of olefin in Wolongqiao was much higher than that of the other three components, followed by alkane and alkyne.

Gut mycobiota alterations in patients with COVID-19 and H1N1 infections and their associations with clinical features.

The relationship between gut microbes and COVID-19 or H1N1 infections is not fully understood. Here, we compared the gut mycobiota of 67 COVID-19 patients, 35 H1N1-infected patients and 48 healthy controls (HCs) using internal transcribed spacer (ITS) 3-ITS4 sequencing and analysed their associations with clinical features and the bacterial microbiota. Compared to HCs, the fungal burden was higher. Fungal mycobiota dysbiosis in both COVID-19 and H1N1-infected patients was mainly characterized by the depletion of fungi such as Aspergillus and Penicillium, but several fungi, including Candida glabrata, were enriched in H1N1-infected patients. The gut mycobiota profiles in COVID-19 patients with mild and severe symptoms were similar. Hospitalization had no apparent additional effects. In COVID-19 patients, Mucoromycota was positively correlated with Fusicatenibacter, Aspergillus niger was positively correlated with diarrhoea, and Penicillium citrinum was negatively correlated with C-reactive protein (CRP). In H1N1-infected patients, Aspergillus penicilloides was positively correlated with Lachnospiraceae members, Aspergillus was positively correlated with CRP, and Mucoromycota was negatively correlated with procalcitonin. Therefore, gut mycobiota dysbiosis occurs in both COVID-19 patients and H1N1-infected patients and does not improve until the patients are discharged and no longer require medical attention.

The serum metabolome of COVID-19 patients is distinctive and predictive.

BACKGROUND: Metabolism is critical for sustaining life, immunity and infection, but its role in COVID-19 is not fully understood. METHODS: Seventy-nine COVID-19 patients, 78 healthy controls (HCs) and 30 COVID-19-like patients were recruited in a prospective cohort study. Samples were collected from COVID-19 patients with mild or severe symptoms on admission, patients who progressed from mild to severe symptoms, and patients who were followed from hospital admission to discharge. The metabolome was assayed using gas chromatography-mass spectrometry. RESULTS: Serum butyric acid, 2-hydroxybutyric acid, l-glutamic acid, l-phenylalanine, l-serine, l-lactic acid, and cholesterol were enriched in COVID-19 and COVID-19-like patients versus HCs. Notably, d-fructose and succinic acid were enriched, and citric acid and 2-palmitoyl-glycerol were depleted in COVID-19 patients compared to COVID-19-like patients and HCs, and these four metabolites were not differentially distributed in non-COVID-19 groups. COVID-19 patients had enriched 4-deoxythreonic acid and depleted 1,5-anhydroglucitol compared to HCs and enriched oxalic acid and depleted phosphoric acid compared to COVID-19-like patients. A combination of d-fructose, citric acid and 2-palmitoyl-glycerol distinguished COVID-19 patients from HCs and COVID-19-like patients, with an area under the curve (AUC) > 0.92 after validation. The combination of 2-hydroxy-3-methylbutyric acid, 3-hydroxybutyric acid, cholesterol, succinic acid, L-ornithine, oleic acid and palmitelaidic acid predicted patients who progressed from mild to severe COVID-19, with an AUC of 0.969. After discharge, nearly one-third of metabolites were recovered in COVID-19 patients. CONCLUSIONS: The serum metabolome of COVID-19 patients is distinctive and has important value in investigating pathogenesis, determining a diagnosis, predicting severe cases, and improving treatment.

Alterations of the Gut Microbiota in Patients With Coronavirus Disease 2019 or H1N1 Influenza.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is an emerging serious global health problem. Gastrointestinal symptoms are common in COVID-19 patients, and severe acute respiratory syndrome coronavirus 2 RNA has been detected in stool specimens. However, the relationship between the gut microbiome and disease remains to be established. METHODS: We conducted a cross-sectional study of 30 patients with COVID-19, 24 patients with influenza A(H1N1), and 30 matched healthy controls (HCs) to identify differences in the gut microbiota by 16S ribosomal RNA gene V3-V4 region sequencing. RESULTS: Compared with HCs, COVID-19 patients had significantly reduced bacterial diversity; a significantly higher relative abundance of opportunistic pathogens, such as Streptococcus, Rothia, Veillonella, and Actinomyces; and a lower relative abundance of beneficial symbionts. Five biomarkers showed high accuracy for distinguishing COVID-19 patients from HCs with an area under the curve (AUC) up to 0.89. Patients with H1N1 displayed lower diversity and different overall microbial composition compared with COVID-19 patients. Seven biomarkers were selected to distinguish the 2 cohorts (AUC = 0.94). CONCLUSIONS: The gut microbial signature of patients with COVID-19 was different from that of H1N1 patients and HCs. Our study suggests the potential value of the gut microbiota as a diagnostic biomarker and therapeutic target for COVID-19, but further validation is needed.

The faecal metabolome in COVID-19 patients is altered and associated with clinical features and gut microbes.

Although SARS-CoV-2 can invade the intestine, though its effect on digestion and absorption is not fully understood. In the present study, 56 COVID-19 patients and 47 age- and sex-matched healthy subjects were divided into a discovery cohort and a validation cohort. Blood, faeces and clinical information were collected from the patients in the hospital and at discharge. The faecal metabolome was analysed using gas chromatography-mass spectrometry, and Spearman's correlation analyses of clinical features, the serum metabolome, and the faecal micro- and mycobiota were conducted. The results showed that, the faeces of COVID-19 patients were enriched with important nutrients that should be metabolized or absorbed, such as sucrose and 2-palmitoyl-glycerol; diet-related components that cannot be synthesized by humans, such as 1,5-anhydroglucitol and D-pinitol; and harmful metabolites, such as oxalate, were also detected. In contrast, purine metabolites such as deoxyinosine and hypoxanthine, low-water-soluble long-chain fatty alcohols/acids such as behenic acid, compounds rarely occurring in nature such as D-allose and D-arabinose, and microbe-related compounds such as 2,4-di-tert-butylphenol were depleted in the faeces of COVID-19 patients. Moreover, these metabolites significantly correlated with altered serum metabolites such as oxalate and gut microbesincluding Ruminococcaceae, Actinomyces, Sphingomonas and Aspergillus. Although levels of several faecal metabolites, such as sucrose, 1,5-anhydroglucitol and D-pinitol, of discharged patients were not different from those of healthy controls (HCs), those of oxalate and 2-palmitoyl-glycerol did differ. Therefore, alterations in the faecal metabolome of COVID-19 patients may reflect malnutrition and intestinal inflammation and warrant greater attention. The results of present study provide new insights into the pathogenesis and treatment of COVID-19.

DABC-Net for robust pneumonia segmentation and prediction of COVID-19 progression on chest CT scans (preprint)

Currently, reliable, robust and ready-to-use CT-based tools for prediction of COVID-19 progression are still lacking. To address this problem, we present DABC-Net, a novel deep learning (DL) tool that combines a 2D U-net for intra-slice spatial information processing, and a recurrent LSTM network to leverage inter-slice context, for automatic volumetric segmentation of lung and pneumonia lesions. We evaluate DABC-Net on more than 10,000 radiologists-labeled CT slices from four different cohorts. Compared to state-of-the-art segmentation tools, DABC-Net is much faster, more robust, and able to estimate segmentation uncertainty. Based only on the first two CT scans within 3 days after admission from 656 longitudinal CT scans, the AUC of our DBAC-Net for disease progression prediction reaches 93%. We release our tool as a GUI for patient-specific prediction of pneumonia progression, to provide clinicians with additional assistance to triage patients at early days after the diagnosis and to optimize the assignment of limited medical resources, which is of particular importance in current critical COVID-19 pandemic.

Systematic profiling of SARS-CoV-2 specific IgG epitopes at single amino acid resolution (preprint)

SARS-CoV-2 specific IgG responses play critical roles for patients to recover from COVID-19, in-depth dissecting of the IgG responses on systems level is of great interest. Herein, we adopted a newly developed high-throughput epitope mapping technology (AbMap), analyzed 55 COVID-19 convalescent sera and 226 antibody samples enriched by specific proteins or peptides from these sera. We revealed three areas that are rich of IgG epitopes, two are on Spike protein but outside of RBD, and one is on Nucleocapsid protein. We identified 29 significant epitopes on Spike protein, from two of these significant epitopes, two critical epitope residues were found, i. e., D936 and P1263, which are highly related to the infectivity of SARS-CoV-2. In summary, we provided the first global map of IgG binding epitopes for SARS-CoV-2 at single amino acid resolution. This map will facilitate the precise development of therapeutic antibodies and vaccines.

Identification of Key Genes and Pathways in SARS-CoV-2 Infection using Bioinformatics Analysis (preprint)

Background: Coronavirus disease 2019 (COVID-19) is a disease that causes fatal disorders including severe pneumonia. Our study aimed to utilize bioinformatics method to analyze the expression profiling by high throughput sequencing in human bronchial organoids/primary human airway epithelial infected with SARS-CoV-2 to identify the potentially crucial genes and pathways associated with COVID-19.Methods: We analyzed microarray datasets GSE153970 and GSE150819 derived from the GEO database. Firstly, the Differentially expressed genes （DEGs） in human bronchial organoids/primary human airway epithelial infected with SARS-CoV-2. Next, the DEGs were used for GO and KEGG pathway enrichment analysis. Then, the PPI network was constructed and Cytoscape was used to find the key genes.Results: Gene expression profiles of GSE153970 and GSE150819, in all 12 samples were analyzed. A total of 145 DEGs and 5 hub genes were identified in SARS-CoV-2. Meanwhile, we found that the 145 genes are associated with immune responses and the top 5 hub genes including CXCL8, CXCL1, CXCL2, CCL20, and CSF2 were mainly related to leukocyte migration, endoplasmic reticulum lumen, receptor ligand activity. In addition, the results also showed that the hub genes were associated with Cytokine−cytokine receptor interaction, IL−17 signaling pathway, and Rheumatoid arthritis in SARS-CoV-2 infection.Conclusion: The five crucial genes consisting of CXCL8, CXCL1, CXCL2, CCL20, and CSF2 were considered as hub genes of SARS-CoV-2, which may be used as diagnostic biomarkers or molecular targets for the treatment of SARS-CoV-2. It is evidenced that bioinformatics analyses in SARS-CoV-2 can be useful for understanding the underlying molecular mechanism and exploring effective therapeutic targets.

The Trans-omics Landscape of COVID-19 (preprint)

The outbreak of coronavirus disease 2019 (COVID-19) has been causing a global health emergency. Although previous studies investigated COVID-19 at different omics levels, the molecular hallmarks of COVID-19, especially in those patients without comorbidities, have not been fully investigated. Here, we presented a trans-omics landscape for COVID-19 based on integrative analysis of genomic, transcriptomic, proteomic, metabolomic and lipidomic profiles from blood samples of 231 COVID-19 patients, ranging from asymptomatic to critically ill, importantly excluding those with any comorbidities. Notably, we found neutrophils heterogeneity existed between asymptomatic and critically ill patients. Expression discordance of inflammatory cytokines at mRNA and protein levels in asymptomatic patients could possibly be explained by post-transcriptional regulation by RNA binding proteins (RBPs) and microRNAs. Neutrophils over-activation, induced arginine depletion, and tryptophan metabolites accumulation contributed to T/NK cell dysfunction in critical patients. Anti-virus interferons were gradually suppressed along with disease severity. Overall, our study systematically revealed multi-omics characteristics of COVID-19, and the data we generated could hopefully help illuminate COVID-19 pathogenesis and provide valuable clues about potential therapeutic strategies for COVID-19.

Interferon-α2b spray inhalation did not shorten virus shedding time of SARS-CoV-2 in hospitalized patients: a preliminary matched case-control study.

BACKGROUND: Currently, there are no drugs that have been proven to be effective against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Because of its broad antiviral activity, interferon (IFN) should be evaluated as a potential therapeutic agent for treatment of coronavirus disease 2019 (COVID-19), especially while COVID-19-specific therapies are still under development. METHODS: Confirmed COVID-19 patients hospitalized in the First Affiliated Hospital, School of Medicine, Zhejiang University in Hangzhou, China, from January 19 to February 19, 2020 were enrolled in a retrospective study. The patients were separated into an IFN group and a control group according to whether they received initial IFN-α2b inhalation treatment after admission. Propensity-score matching was used to balance the confounding factors. RESULTS: A total of 104 confirmed COVID-19 patients, 68 in the IFN group and 36 in the control group, were enrolled. Less hypertension (27.9% vs. 55.6%, P=0.006), dyspnea (8.8% vs. 25.0%, P=0.025), or diarrhea (4.4% vs. 19.4%, P=0.030) was observed in the IFN group. Lower levels of albumin and C-reactive protein and higher level of sodium were observed in the IFN group. Glucocorticoid dosage was lower in the IFN group (median, 40 vs. 80 mg/d, P=0.025). Compared to the control group, fewer patients in the IFN group were ventilated (13.2% vs. 33.3%, P=0.015) and admitted to intensive care unit (ICU) (16.2% vs. 44.4%, P=0.002). There were also fewer critical patients in the IFN group (7.4% vs. 25.0%, P=0.017) upon admission. Although complications during admission process were comparable between groups, the discharge rate (85.3% vs. 66.7%, P=0.027) was higher and the hospitalization time (16 vs. 21 d, P=0.015) was shorter in the IFN group. When other confounding factors were not considered, virus shedding time (10 vs. 13 d, P=0.014) was also shorter in the IFN group. However, when the influence of other factors was eliminated using propensity score matching, virus shedding time was not significantly shorter than that of the control group (12 vs. 15 d, P=0.206). CONCLUSIONS: IFN-α2b spray inhalation did not shorten virus shedding time of SARS-CoV-2 in hospitalized patients.

Reducing the consumption of personal protective equipment by setting up a multifunctional sampling station in the emergency department to screen for COVID-19 infection in Taiwan.

In Taiwan, high-risk patients have been identified and tested for preventing community spread of COVID-19. Most sample collection was performed in emergency departments (EDs). Traditional sample collection requires substantial personal protective equipment (PPE), healthcare professionals, sanitation workers, and isolation space. To solve this problem, we established a multifunctional sample collection station (MSCS) for COVID-19 testing in front of our ED. The station is composed of a thick and clear acrylic board (2 cm), which completely separates the patient and medical personnel. Three pairs of gloves (length, 45 cm) are attached and fixed on the outside wall of the MSCS. The gloves are used to conduct sampling of throat/nasal swabs, sputum, and blood from patients. The gap between the board and the building is only 0.2 cm (sealed with silicone sealant). ED personnel communicate with patients using a small two-way broadcast system. Medical waste is put in specific trashcans installed in the table outside the MSCS. With full physical protection, the personnel conducting the sampling procedure need to wear only their N95 mask and gloves. After we activated the station, our PPE, sampling time, and sanitization resources were considerably conserved during the 4-week observation period. The MSCS obviously saved time and PPE. It elevated the efficiency and capacity of the ED for handling potential community infections of COVID-19.

The Trans-omics Landscape of COVID-19 (preprint)

System-wide molecular characteristics of COVID-19, especially in those patients without comorbidities, have not been fully investigated. We compared extensive molecular profiles of blood samples from 231 COVID-19 patients, ranging from asymptomatic to critically ill, importantly excluding those with any comorbidities. Amongst the major findings, asymptomatic patients were characterized by highly activated anti-virus interferon, T/natural killer (NK) cell activation, and transcriptional upregulation of inflammatory cytokine mRNAs. However, given very abundant RNA binding proteins (RBPs), these cytokine mRNAs could be effectively destabilized hence preserving normal cytokine levels. In contrast, in critically ill patients, cytokine storm due to RBPs inhibition and tryptophan metabolites accumulation contributed to T/NK cell dysfunction. A machine-learning model was constructed which accurately stratified the COVID-19 severities based on their multi-omics features. Overall, our analysis provides insights into COVID-19 pathogenesis and identifies targets for intervening in treatment.