A dual-labeled fluorescence quenching lateral flow assay based on one-pot enzyme-free isothermal cascade amplification for the rapid and sensitive detection of pathogens.

Rapid detection of nucleic acids is integral for clinical diagnostics, especially if a major public-health emergency occurs. However, such detection cannot be carried out efficiently in remote areas limited by medical resources. Herein, a dual-labeled fluorescence resonance energy transfer (FRET) lateral flow assay (LFA) based on one-pot enzyme-free cascade amplification was developed for rapid, convenient, and sensitive detection of open reading frame (ORF)1ab of severe acute respiratory syndrome-coronavirus-2. The catalyzed hairpin assembly (CHA) reaction of two well-designed hairpin probes was initiated by a target sequence and generated a hybridization chain reaction (HCR) initiator. Then, HCR probes modified with biotin were initiated to produce long DNA nanowires. After two-level amplification, the cascade-amplified product was detected by dual-labeled lateral flow strips. Gold nanoparticles (AuNPs)-streptavidin combined with the product and then ran along a nitrocellulose membrane under the action of capillary force. After binding with fluorescent microsphere-labeled-specific probes on the T line, a positive signal (red color) could be observed. Meanwhile, AuNPs could quench the fluorescence of the T line, and an inverse relationship between fluorescence intensity and the concentration of the CHA-HCR-amplified product was formed. The proposed strategy achieved a satisfactory limit of detection of 2.46 pM for colorimetric detection and 174 fM for fluorescent detection, respectively. Benefitting from the features of being one-pot, enzyme-free, low background, high sensitivity, and selectivity, this strategy shows great potential in bioanalysis and clinical diagnostics upon further development.

Analysis of research hotspots in COVID-19 genomics based on citespace software: Bibliometric analysis.

Introduction: To analyze the current state, hotspots, and cutting-edge trends of genomics research on the outbreak of Corona Virus Disease 2019 (COVID-19) from 2019 to the present (March 2022). Methods: Statistical and visual analysis of COVID-19 genomics results published in the 2019-2022 Web of Science Core Collection Database (WOSCC) was performed using CiteSpace software, including data on countries, institutions, authors, journals, co-citations, keywords, etc. Results: A total of 9133 English literature were included. The number of publications has significantly increased in 2021, and it is expected that this upward trend will last into the future. The research hotspots of COVID-19 revolve around quarantine, biological management, angiotensin-converting enzyme-2, RNA-dependent RNA polymerase, etc. Research frontiers and trends focus on molecular docking, messenger RNA, functional receptor, etc. Conclusion: The last two years have seen a significant increase in research interest in the field of novel coronavirus pneumonia genomics.

Sequential in vitro enzymatic N-glycoprotein modification reveals site-specific rates of glycoenzyme processing (preprint)

N-glycosylation is an essential eukaryotic post-translational modification that affects various glycoprotein properties, including folding, solubility, protein-protein interactions, and half-life. N-glycans are processed in the secretory pathway to form varied ensembles of structures, and diversity at a single site on a glycoprotein is termed microheterogeneity. To understand the factors that influence glycan microheterogeneity, we hypothesized that local steric and electrostatic factors surrounding each site influences glycan availability to enzymatic modification. We tested this hypothesis by expression of a panel of reporter N-linked glycoproteins in MGAT1-null HEK293 cells to produce immature Man5GlcNAc2 glycoforms (38 glycan sites total). These glycoproteins were then sequentially modified in vitro from high-mannose to hybrid and on to biantennary, core fucosylated, complex structures by a panel of N-glycosylation enzymes and each reaction time-course was quantified by LC-MS/MS. Substantial differences in rates of in vitro enzymatic modification were observed between glycan sites on the same protein and differences in modification rates varied depending on the glycoenzyme being evaluated. By comparison, proteolytic digestion of the reporters prior to N-glycan processing eliminated differences in in vitro enzymatic modification. Comparison of in vitro rates of enzymatic modification with the glycan structures found on the mature reporters expressed in wild type cells correlate well with the enzymatic bottlenecks found in vitro. These data suggest that higher-order local structures surrounding each glycosylation site contribute to the efficiency of modification both in vitro and in vivo to establish the spectrum of site-specific microheterogeneity found on N-linked glycoproteins.

A comparison of Remdesivir versus gold cluster in COVID-19 animal model: a better therapeutic outcome of gold cluster

While gold compound have been approved for Rheumatoid arthritis treatment as it well suppresses inflammatory cytokines of patients, no such treatment is currently available for COVID-19 treatment in vivo. We firstly disclose gold cluster yields better therapeutic outcome than Remdesivir in COVID-19 hamster treatments as it is armed with direct inhibition viral replication and intrinsic suppression inflammatory cytokines expression. Crystal data reveals that Au (I), released from gold cluster (GA), covalently binds thiolate of Cys145 of SARS-CoV-2 Mpro. GA directly decreases SARS-CoV-2 viral replication and intrinsically down-regulates NFκB pathway therefore significantly inhibiting expression of inflammatory cytokines in cells. The inflammatory cytokines in GA-treated COVID-19 transgenic mice are found to be significantly lower than that of control mice. When COVID-19 golden hamsters are treated by GA, the lung inflammatory cytokines levels are significantly lower than that of Remdesivir. The pathological results show that GA treatment significantly reduce lung inflammatory injuries when compared to that of Remdesivir-treated COVID-19 hamsters. Graphical

Virus-Receptor Interactions of Glycosylated SARS-CoV-2 Spike and Human Ace2 Receptor (preprint)

The current COVID-19 pandemic is caused by the SARS-CoV-2 betacoronavirus, which utilizes its highly glycosylated trimeric Spike protein to bind to the cell surface receptor ACE2 glycoprotein and facilitate host cell entry.  We utilized glycomics-informed glycoproteomics to characterize site-specific microheterogeneity of glycosylation for a recombinant trimer Spike mimetic immunogen and for a soluble version of human ACE2.  We combined this information with bioinformatic analyses of natural variants and with existing 3D-structures of both glycoproteins to generate molecular dynamics simulations of each glycoprotein alone and interacting with one another.  Our results highlight roles for glycans in sterically masking polypeptide epitopes and directly modulating Spike-ACE2 interactions.  Furthermore, our results illustrate the impact of viral evolution and divergence on Spike glycosylation, as well as the influence of natural variants on ACE2 receptor glycosylation that, taken together, can facilitate immunogen design to achieve antibody neutralization and inform therapeutic strategies to inhibit viral infection.

Virus-Receptor Interactions of Glycosylated SARS-CoV-2 Spike and Human ACE2 Receptor (preprint)

The current COVID-19 pandemic is caused by the SARS-CoV-2 betacoronavirus, which utilizes its highly glycosylated trimeric Spike protein to bind to the cell surface receptor ACE2 glycoprotein and facilitate host cell entry. We utilized glycomics-informed glycoproteomics to characterize site-specific microheterogeneity of glycosylation for a recombinant trimer Spike mimetic immunogen and for a soluble version of human ACE2. We combined this information with bioinformatic analyses of natural variants and with existing 3D-structures of both glycoproteins to generate molecular dynamics simulations of each glycoprotein alone and interacting with one another. Our results highlight roles for glycans in sterically masking polypeptide epitopes and directly modulating Spike-ACE2 interactions. Furthermore, our results illustrate the impact of viral evolution and divergence on Spike glycosylation, as well as the influence of natural variants on ACE2 receptor glycosylation that, taken together, can facilitate immunogen design to achieve antibody neutralization and inform therapeutic strategies to inhibit viral infection.

Distributions and risks of SARS-CoV-2 in hospital outdoor environment (preprint)

The outbreak of coronavirus infectious disease-2019 (COVID-19) pandemic has rapidly spread throughout over 200 countries, posing a global threat to human health. Till 15th May 2020, there are over 4.5 million confirmed cases, with roughly 300,000 death1. To date, most studies focus on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in indoor environment owing to its main transmission routes via human respiratory droplets and direct contact2,3. It remains unclear whether SARS-CoV-2 can spill over and impose transmission risks to outdoor environments despite potential threats to people and communities. Here, we investigated the presence of SARS-CoV-2 by measuring viral RNA in 73 samples from outdoor environment of three hospitals in Wuhan. We detected SARS-CoV-2 in soils (205-550 copies/g), aerosols (285-1,130 copies/m3) and wastewaters (255 to 18,744 copies/L) in locations close to hospital departments receiving COVID-19 patients or in wastewater treatment sectors. These findings reveal significant viral spillover in hospital outdoor environments that was possibly caused by respiratory droplets from patients or aerosolized particles from wastewater containing SARS-CoV-2. In contrast, SARS-CoV-2 was not detected in other areas or on surfaces with regular disinfection implemented. Soils may behave as viral warehouse through deposition and serve as a secondary source spreading SARS-CoV-2 for a prolonged time. For the first time, our findings demonstrate that there are high-risk areas in hospital outdoor environments to spread SARS-CoV-2, calling for sealing of wastewater treatment unit and complete sanitation to prevent COVID-19 transmission risks.

Outbreak analysis with a logistic growth model shows COVID-19 suppression dynamics in China (preprint)

China reported a major outbreak of a novel coronavirus, SARS-CoV2, from mid-January till mid-March 2020. The number of cases outside China is now growing fast, while in mainland China the virus outbreak is largely under control. We review the epidemic virus growth and decline curves in China using a phenomenological logistic growth model to summarize the outbreak dynamics using three parameters that characterize the epidemic's timing, rate and peak. During the initial phase, the number of virus cases doubled every 2.7 (range 2.2 - 4.4) days. The rate of increase in the number of reported cases peaked approximately 10 days after suppression measures were started on 23-25 January 2020. The peak in the number of reported sick cases occurred on average 18 days after the start of measures. From the time of starting measures till the peak, the number of cases increased by a factor 39 in the province Hubei, and by a factor 9.5 for all of China (range: 6.2-20.4 in the other provinces). Complete suppression took up to 2 months (range: 23-57d.), during which period severe restrictions, social distancing measures, testing and isolation of cases were in place. The suppression of the disease in China has been successful, demonstrating that suppression is a viable strategy to contain SARS-CoV2.

Epidemiological and clinical characteristics of 46 newly-admitted coronavirus disease 2019 cases in Beijing / 中华传染病杂志

Objective@#To investigate and analyze the epidemiological and clinical characteristics of some cases of coronavirus disease 2019 (COVID-19) in Beijing.@*Methods@#A retrospective study was conducted to analyze the data of 46 patients with COVID-19 in Beijing from 20th January 2020 to 8th February 2020 at the Fifth Medical Center of the PLA General Hospital. Features of clinical symptoms, laboratory inspections and imaging inspections were analyzed. Statistical analysis used Fisher exact test. If P<0.05, post-hoc test was used for pairwise comparison, and the statistics were corrected by Bonferroni test.@*Results@#Among the 46 patients included in this study, 27 were male and 19 were female. The age range was between 3 - 79 years old, and the mean age was (41.8 ± 16.3) years old. The average incubation period was (4.85 ± 3.00) days. A total of 26 cases (56.5%) were clustered patients, and 12 (26.1%), 23 (50.0%) and 11 patients (23.9%)were assigned to the mild group, common group, and sever group, respectively. Fever (39.8%), cough (27.6%), and fatigue (25.3%) was the main clinical symptom for these patients. The decrease in white blood cell counts occurred in 12 patients, four had the decrease in T lymphocyte counts, 17 had the decrease in CD4 + T lymphocyte counts, seven had the decrease in CD8 + T lymphocyte counts, 21 had the increase level of C-reactive protein (45.7%), and IL-6 level increased in 32 cases (69.6%), erythrocyte sedimentation rate increased in 20 cases(50.0%), serum ferritin level increased in 26 cases (56.5%), and blood lactate level increased in nine cases. There was a statistically significant difference in the number of cases in which the absolute value of T lymphocytes and of CD8 + T lymphocytes decreased among the mild, common and severe groups (all P<0.05). Comparing the number of cases in the three groups with elevated C-reactive protein, interleukin-6, erythrocyte sedimentation rate, serum ferritin and blood lactate levels, the differences were statistically significant (all P<0.05). The number of cases with elevated C-reactive protein levels was higher in severe group than that in mild and common group. The number of cases with elevated interleukin-6, erythrocyte sedimentation rate, and serum ferritin levels were higher in severe group than in mild group. The number of cases with elevated blood lactic acid levels was higher in severe group than in mild group. The differences between the above groups were statistically significant (both adjusted P<0.017). Analysis of chest X-rays showed that 34 patients (73.9%) had inflammation in the lungs.@*Conclusions@#The epidemiological characteristics of cases with COVID-19 in Beijing are mainly imported cases and clustered cases. The clinical manifestations are mainly fever , fatigue and cough. C-reactive protein, interleukin-6, red blood cell sedimentation rate, serum ferritin and blood lactate levels are higher in severe patients.

Epidemiological and clinical features of COVID-19 patients with and without pneumonia in Beijing, China (preprint)

Background:SARS-CoV-2-caused coronavirus disease (COVID-19) is posinga large casualty. The features of COVID-19patients withand without pneumonia,SARS-CoV-2 transmissibility in asymptomatic carriers, and factors predicting disease progression remain unknown. Methods: We collected information on clinical characteristics, exposure history, andlaboratory examinations of all laboratory-confirmed COVID-19 patients admitted to PLA General Hospital. Cox regression analysis was applied to identify prognostic factors. The last follow-up was February 18, 2020. Results:We characterized 55 consecutive COVID-19 patients. The mean incubation was 8.42(95% confidence interval [CI], 6.55-10.29) days. The mean SARS-CoV-2-positive duration from first positive test to clearance was 9.71(95%CI, 8.21-11.22) days. COVID-19 course was approximately 2 weeks. Asymptomatic carriers might transmit SARS-CoV-2. Compared with patients without pneumonia, those with pneumonia were 15 years older and had a higher rate of hypertension, higher frequencies of having a fever and cough, and higher levels of interleukin-6 (14.61 vs. 8.06pg/mL, P=0.040), B lymphocyte proportion (13.0% vs.10.0%, P=0.024), low account (<190/L) of CD8+ T cells (33.3% vs. 0, P=0.019). Multivariate Cox regression analysis indicated that circulating interleukin-6 andlactate independently predicted COVID-19 progression, with a hazard ratio (95%CI) of 1.052 (1.000-1.107) and 1.082 (1.013-1.155), respectively. During disease course,T lymphocytes were generally lower,neutrophils higher, in pneumonia patients than in pneumonia-free patients. CD8+ lymphocytes did not increase at the 20th days after illness onset. Conclusion: The epidemiological features areimportant for COVID-19 prophylaxis. Circulating interleukin-6 and lactateare independent prognostic factors. CD8+ T cell exhaustion might be critical in the development of COVID-19.