Remdesivir is an antiviral approved for COVID-19 treatment, but its wider use is limited by intravenous delivery. An orally bioavailable remdesivir analog may boost therapeutic benefit by ...facilitating early administration to non-hospitalized patients. This study characterizes the anti-SARS-CoV-2 efficacy of GS-621763, an oral prodrug of remdesivir parent nucleoside GS-441524. Both GS-621763 and GS-441524 inhibit SARS-CoV-2, including variants of concern (VOC) in cell culture and human airway epithelium organoids. Oral GS-621763 is efficiently converted to plasma metabolite GS-441524, and in lungs to the triphosphate metabolite identical to that generated by remdesivir, demonstrating a consistent mechanism of activity. Twice-daily oral administration of 10 mg/kg GS-621763 reduces SARS-CoV-2 burden to near-undetectable levels in ferrets. When dosed therapeutically against VOC P.1 gamma γ, oral GS-621763 blocks virus replication and prevents transmission to untreated contact animals. These results demonstrate therapeutic efficacy of a much-needed orally bioavailable analog of remdesivir in a relevant animal model of SARS-CoV-2 infection.
Reporter-expressing recombinant virus represents an excellent option and a powerful tool to investigate, among others, viral infection, pathogenicity, and transmission, as well as to identify ...therapeutic compounds that inhibit viral infection and prophylactic vaccines. To combat the ongoing coronavirus disease 2019 (COVID-19) pandemic, we have established a robust bacterial artificial chromosome (BAC)-based reverse genetics (RG) system to rapidly generate recombinant severe acute respiratory syndrome coronavirus 2 (rSARS-CoV-2) to study the contribution of viral proteins in viral pathogenesis. In addition, we have engineered reporter-expressing recombinant viruses in which we placed the reporter genes upstream of the viral nucleocapsid (N) gene to promote high levels of reporter gene expression, which facilitates the study of SARS-CoV-2
and
. To date, we have shared our BAC-based RG system with more than 100 laboratories around the world, which has helped to expedite investigations with SARS-CoV-2. However, genetic manipulation of the BAC containing the entire SARS-CoV-2 genome (~30,000 nt) is challenging. Herein, we provide the technical details to engineer rSARS-CoV-2 using the BAC-based RG approach. We describe (i) assembly of the full-length (FL) SARS-CoV-2 genome sequences into the empty pBeloBAC, (ii) verification of pBeloBAC-FL, (iii) cloning of a Venus reporter gene into pBeloBAC-FL, and (iv) recovery of the Venus-expressing rSARS-CoV-2. By following this protocol, researchers with knowledge of basic molecular biology and gene engineering techniques will be able to generate wild-type (WT) and reporter-expressing rSARS-CoV-2.
We have established a bacterial artificial chromosome (BAC)-based RG system to generate recombinant severe acute respiratory syndrome coronavirus 2 (rSARS-CoV-2) and to engineer reporter-expressing recombinant viruses to assess viral infection
and
. To date, we have shared our BAC-based RG system with more than 100 laboratories around the world, which has helped to expedite investigations with SARS-CoV-2. However, genetic manipulation of the BAC containing the full-length SARS-CoV-2 genome of ~30,000 nucleotides is challenging. Here, we provide all the detailed experimental steps required for the successful generation of wild-type (WT) recombinant SARS-CoV-2 (rSARS-CoV-2). Likewise, we provide a comprehensive protocol on how to generate and rescue rSARS-CoV-2 expressing high levels of a Venus fluorescent reporter gene from the locus of the viral nucleocapsid (N) protein. By following these protocols, researchers with basic knowledge in molecular biology will be able to generate WT and Venus-expressing rSARS-CoV-2 within 40 days.
Infectious coronavirus (CoV) disease 2019 (COVID-19) emerged in the city of Wuhan (China) in December 2019, causing a pandemic that has dramatically impacted public health and socioeconomic ...activities worldwide. A previously unknown coronavirus, severe acute respiratory syndrome CoV-2 (SARS-CoV-2), has been identified as the causative agent of COVID-19. To date, there are no U.S. Food and Drug Administration (FDA)-approved vaccines or therapeutics available for the prevention or treatment of SARS-CoV-2 infection and/or associated COVID-19 disease, which has triggered a large influx of scientific efforts to develop countermeasures to control SARS-CoV-2 spread. To contribute to these efforts, we have developed an infectious cDNA clone of the SARS-CoV-2 USA-WA1/2020 strain based on the use of a bacterial artificial chromosome (BAC). Recombinant SARS-CoV-2 (rSARS-CoV-2) was readily rescued by transfection of the BAC into Vero E6 cells. Importantly, BAC-derived rSARS-CoV-2 exhibited growth properties and plaque sizes in cultured cells comparable to those of the natural SARS-CoV-2 isolate. Likewise, rSARS-CoV-2 showed levels of replication similar to those of the natural isolate in nasal turbinates and lungs of infected golden Syrian hamsters. This is, to our knowledge, the first BAC-based reverse genetics system for the generation of infectious rSARS-CoV-2 that displays features
similar to those of a natural viral isolate. This SARS-CoV-2 BAC-based reverse genetics will facilitate studies addressing several important questions in the biology of SARS-CoV-2, as well as the identification of antivirals and development of vaccines for the treatment of SARS-CoV-2 infection and associated COVID-19 disease.
The pandemic coronavirus (CoV) disease 2019 (COVID-19) caused by severe acute respiratory syndrome CoV-2 (SARS-CoV-2) is a major threat to global human health. To date, there are no approved prophylactics or therapeutics available for COVID-19. Reverse genetics is a powerful approach to understand factors involved in viral pathogenesis, antiviral screening, and vaccine development. In this study, we describe the feasibility of generating recombinant SARS-CoV-2 (rSARS-CoV-2) by transfection of a single bacterial artificial chromosome (BAC). Importantly, rSARS-CoV-2 possesses the same phenotype as the natural isolate
and
This is the first description of a BAC-based reverse genetics system for SARS-CoV-2 and the first time that an rSARS-CoV-2 isolate has been shown to be phenotypically identical to a natural isolate in a validated animal model of SARS-CoV-2 infection. The BAC-based reverse genetics approach will facilitate the study of SARS-CoV-2 and the development of prophylactics and therapeutics for the treatment of COVID-19.
Autophagy is an essential component of host innate and adaptive immunity. Viruses have developed diverse strategies for evading or utilizing autophagy for survival. The response of the autophagy ...pathways to virus invasion is poorly documented. Here, we report on the induction of autophagy initiated by the pathogen receptor HSP90AA1 (heat shock protein 90 kDa α cytosolic, class A member 1) via the AKT-MTOR (mechanistic target of rapamycin)-dependent pathway. Transmission electron microscopy and confocal microscopy revealed that intracellular autolysosomes packaged avibirnavirus particles. Autophagy detection showed that early avibirnavirus infection not only increased the amount of light chain 3 (LC3)-II, but also upregulated AKT-MTOR dephosphorylation. HSP90AA1-AKT-MTOR knockdown by RNA interference resulted in inhibition of autophagy during avibirnavirus infection. Virus titer assays further verified that autophagy inhibition, but not induction, enhanced avibirnavirus replication. Subsequently, we found that HSP90AA1 binding to the viral protein VP2 resulted in induction of autophagy and AKT-MTOR pathway inactivation. Collectively, our findings suggest that the cell surface protein HSP90AA1, an avibirnavirus-binding receptor, induces autophagy through the HSP90AA1-AKT-MTOR pathway in early infection. We reveal that upon viral recognition, a direct connection between HSP90AA1 and the AKT-MTOR pathway trigger autophagy, a critical step for controlling infection.
Full text
Available for:
BFBNIB, GIS, IJS, KISLJ, NUK, PNG, UL, UM, UPUK
More demand-side flexible resources (DFRs) are participating in the frequency regulation of renewable power systems, whose heterogeneous characteristics have a significant impact on the system ...frequency response. Consequently, selecting suitable DFRs poses a formidable challenge for independent system operators (ISO). In this paper, a reserve allocation methodology for heterogeneous DFRs is proposed to manage the risk of power system frequency. Firstly, a performance curve is developed to describe the cost, capacity, and response speed of DFRs. Moreover, a clustering method for multiple distributed DFRs is conducted to calculate the aggregated performance curves and uncertainty coefficients. Then, the frequency security criterion considering DFRs’ performance is constructed, whose linearity makes it can be easily coupled into the system scheduling model and solved. Furthermore, a risk management model for DFRs considering frequency-chance-constraint is proposed to make a trade-off between cost and frequency security. Finally, the model is transformed into mixed integer second-order cone programming (MISOCP) and solved by the commercial solver. The proposed model is validated by the IEEE 30 and IEEE 118 bus systems.
Full text
Available for:
EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
•We have developed a rapid, accurate, and highly reproducible plaque reduction microneutralization (PRMNT) assay for SARS-CoV-2.•Our PRMNT assay allows to identify and characterize antibodies with ...neutralizing activity against SARS-CoV-2.•Likewise, our PRMNT assay can be used to find compounds with antiviral activity against SARS-CoV-2.•The PRMNT assay can be developed using peroxidase or infrared staining readouts.•Our PRMNT assay can be adapted to HTS settings to interrogate complex library of SARS-CoV-2 inhibitors.
Towards the end of 2019, a novel coronavirus (CoV) named severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), genetically similar to severe acute respiratory syndrome coronavirus (SARS-CoV), emerged in Wuhan, Hubei province of China, and has been responsible for coronavirus disease 2019 (COVID-19) in humans. Since its first report, SARS-CoV-2 has resulted in a global pandemic, with over 10 million human infections and over 560,000 deaths reported worldwide at the end of June 2020. Currently, there are no United States (US) Food and Drug Administration (FDA)-approved vaccines and/or antivirals licensed against SARS-CoV-2. The high economical and health impacts of SARS-CoV-2 has placed global pressure on the scientific community to identify effective prophylactic and therapeutic treatments for SARS-CoV-2 infection and associated COVID-19 disease. While some compounds have been already reported to reduce SARS-CoV-2 infection and a handful of monoclonal antibodies (mAbs) have been described that neutralize SARS-CoV-2, there is an urgent need for the development and standardization of assays which can be used in high through-put screening (HTS) settings to identify new antivirals and/or neutralizing mAbs against SARS-CoV-2. Here, we described a rapid, accurate, and highly reproducible plaque reduction microneutralization (PRMNT) assay that can be quickly adapted for the identification and characterization of both neutralizing mAbs and antivirals against SARS-CoV-2. Importantly, our MNA is compatible with HTS settings to interrogate large and/or complex libraries of mAbs and/or antivirals to identify those with neutralizing and/or antiviral activity, respectively, against SARS-CoV-2.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Although the immune adaptor adhesion and degranulation-promoting adaptor protein (ADAP) acts as a key mediator of integrin inside-out signaling leading to T cell adhesion, the regulation of this ...adaptor during integrin activation and clustering remains unclear. We now identify Ubc9, the sole small ubiquitin-related modifier E2 conjugase, as an essential regulator of ADAP where it is required for TCR-induced membrane recruitment of the small GTPase Rap1 and its effector protein RapL and for activation of the small GTPase Rac1 in T cell adhesion. We show that Ubc9 interacted directly with ADAP in vitro and in vivo, and the association was increased in response to anti-CD3 stimulation. The Ubc9-binding domain on ADAP was mapped to a nuclear localization sequence (aa 674-700) within ADAP. Knockdown of Ubc9 by short hairpin RNA or expression of the Ubc9-binding-deficient ADAP mutant significantly decreased TCR-induced integrin adhesion to ICAM-1 and fibronectin, as well as LFA-1 clustering, although it had little effect on the TCR proximal signaling responses and TCR-induced IL-2 transcription. Furthermore, downregulation of Ubc9 impaired TCR-mediated Rac1 activation and attenuated the membrane targeting of Rap1 and RapL, but not Rap1-interacting adaptor molecule. Taken together, our data demonstrate for the first time, to our knowledge, that Ubc9 acts as a functional binding partner of ADAP and plays a selective role in integrin-mediated T cell adhesion via modulation of Rap1-RapL membrane recruitment and Rac1 activation.
With the development of advanced metering infrastructure (AMI), electrical data are collected frequently by smart meters. Consequently, the load data volume and length increase dramatically, which ...aggravates the data storage and transmission burdens in smart grids. On the other hand, for event detection or market-based demand response applications, load service entities (LSEs) want smart meter readings to be classified in specific and meaningful types. Considering these challenges, a stacked auto-encoder (SAE)-based load data mining approach is proposed. First, an innovative framework for smart meter data flow is established. On the user side, the SAEs are utilized to compress load data in a distributed way. Then, centralized classification is adopted at remote data center by softmax classifier. Through the layer-wise feature extracting of SAE, the sparse and lengthy raw data are expressed in compact forms and then classified based on features. A global fine-tuning strategy based on a well-defined labeled subset is embedded to improve the extracted features and the classification accuracy. Case studies in China and Ireland demonstrate that the proposed method is more capable to achieve the minimum of error and satisfactory compression ratios (CR) than benchmark compressors. It also significantly improves the classification accuracy on both appliance and house level datasets.
Full text
Available for:
IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
With the booming of electric vehicles (EVs) across the world, their increasing charging demands pose challenges to urban distribution networks. Particularly, due to the further implementation of ...time-of-use prices, the charging behaviors of household EVs are concentrated on low-cost periods, thus generating new load peaks and affecting the secure operation of the medium- and low-voltage grids. This problem is particularly acute in many old communities with relatively poor electricity infrastructure. In this paper, a novel two-stage charging scheduling scheme based on deep reinforcement learning is proposed to improve the power quality and achieve optimal charging scheduling of household EVs simultaneously in active distribution network (ADN) during valley period. In the first stage, the optimal charging profiles of charging stations are determined by solving the optimal power flow with the objective of eliminating peak-valley load differences. In the second stage, an intelligent agent based on proximal policy optimization algorithm is developed to dispatch the household EVs sequentially within the low-cost period considering their discrete nature of arrival. Through powerful approximation of neural network, the challenge of imperfect knowledge is tackled effectively during the charging scheduling process. Finally, numerical results demonstrate that the proposed scheme exhibits great improvement in relieving peak-valley differences as well as improving voltage quality in the ADN.
PDF
