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    Development and validation of cost-effective one-step multiplex RT-PCR assay for detecting the SARS-CoV-2 infection MESHD using SYBR Green melting curve analysis

    Authors: Shovon Lal Sarkar; A. S. M. Rubayet Ul Alam; Prosanto Kumar Das; Md. Hasan Ali Pramanik; Hassan M. Al-Emran; Iqbal Kabir Jahid; Md. Anwar Hossain

    doi:10.1101/2021.05.06.21256629 Date: 2021-05-08 Source: medRxiv

    TaqMan probe-based expensive commercial real-time (RT) PCR kits are being used in COVID-19 MESHD diagnosis. The unprecedented scale of SARS-CoV-2 infections MESHD has urgently needed to meet the challenge of testing more persons at a reasonable cost. This study developed a rapid, simple, and cost-effective alternative diagnostic method based on melting curve analysis of SYBR green multiplex assay with a host-specific internal control. A total of 90 randomly selected samples were used for comparing the assay with an available commercial kit to analyse the variation and validity of this in-house developed method. Our customized designed primers specifically detected the virus as similar to commercial kit manufactured by Sansure Biotech Inc. We optimized separately the N, E, S, and RdRp PROTEIN genes by SYBR Green RT-PCR method based on melting curve analysis. Afterwards, a multiplex COVID-19 MESHD diagnosis method targeting N and E genes PROTEIN of the virus along with the { beta}-actin HGNC gene of the host as an internal control has been established. The total run-time of our proposed method was less than 90 minutes. The cost of each sample processing was less than $2. Overall, this one-step and one-tube method can revolutionize the COVID-19 MESHD diagnosis in developing countries.

    Screening of HLA-A HGNC restricted T cell epitopes of SARS-CoV-2 and induction of CD8+ T cell responses in HLA-A HGNC transgenic mice

    Authors: Xiaoxiao Jin; Yan Ding; Shihui Sun; Xinyi Wang; Zining Zhou; Xiaotao Liu; Miaomiao Li; Xian Chen; Anran Shen; Yandan Wu; Bicheng Liu; Jianqiong Zhang; Jian Li; Yi Yang; Haibo Qiu; Chuanlai Shen; Yuxian He; Guangyu Zhao

    doi:10.1101/2021.04.01.438020 Date: 2021-04-01 Source: bioRxiv

    While SARS-CoV-2-specific T cells have been characterized to play essential roles in host immune protection in COVID-19 MESHD patients, few researches focus on the functional validation of T cell epitopes and development of vaccines inducing specific T cell responses. In this study, 120 CD8 T cell epitopes from E, M, N, S and RdRp PROTEIN proteins of SARS-CoV-2 were validated by on-silicon prediction, DC-peptide-PBL costimulation with PBMCs of healthy donors and HLA-A HGNC molecule competitive binding experiments. Among them, 110, 15, 6, 14 and 12 epitopes were highly homologous with SARS-CoV MESHD, OC43, NL63, HKU1, and 229E, respectively. Thirty-one epitopes restricted by HLA-A2 molecule were used to generate peptide cocktail vaccines in combination with Poly(I:C), R848 or polylactic-co-glycolic acid nanoparticles, which elicited robust specific CD8 T cell responses in wild-type and HLA-A2/DR1 transgenic mice. Seven of the 31 epitopes were found to be cross-presented by HLA-A2 and H-2K/Db molecules. These data have provided a library of SARS-CoV-2 CD8 T cell epitopes which restricted by a series of high-frequency HLA-A HGNC allotypes and covered broad population in Asia, and initially confirmed the feasibility of human MHC class I molecule-restricted SARS-CoV2 epitope peptide cocktail vaccines, thus will facilitate the development of T cell epitope vaccines and specific cellular function detection kits.

    Protein-primed RNA synthesis in SARS-CoVs MESHD and structural basis for inhibition by AT-527

    Authors: Ashleigh Shannon; Veronique Fattorini; Bhawna Sama; Barbara Selisko; Mikael Feracci; Camille Falcou; Pierre Gauffre; Priscila El Kazzi; Etienne Decroly; Nadia Rabah; Karine Toulon; Cecilia Eydoux; Jean-Claude Guillemot; Mathieu Noel; Francoise Debart; Jean-Jacques Vasseur; Adel Moussa; Steven Good; Kai Lin; Jean-Pierre Sommadossi; Yingxiao Zhu; Xiaodong Yan; Hui Shi; Francois Ferron; Bruno Canard

    doi:10.1101/2021.03.23.436564 Date: 2021-03-23 Source: bioRxiv

    How viruses from the Coronaviridae family initiate viral RNA synthesis is unknown. Here we show that the SARS-CoV-1 and -2 Nidovirus RdRp PROTEIN-Associated Nucleotidyltransferase (NiRAN) domain on nsp12 uridylates the viral cofactor nsp8, forming a UMP- Nsp HGNC8 covalent intermediate that subsequently primes RNA synthesis from a poly(A) template; a protein-priming mechanism reminiscent of Picornaviridae enzymes. In parallel, the RdRp PROTEIN active site of nsp12 synthesizes a pppGpU primer, which primes (-)ssRNA synthesis at the precise genome-poly(A) junction. The guanosine analogue 5'-triphosphate AT-9010 (prodrug: AT-527) tightly binds to the NiRAN and inhibits both nsp8-labeling and the initiation of RNA synthesis. A 2.98 A resolution Cryo-EM structure of the SARS-CoV-2 nsp12-nsp7-(nsp8)2 /RNA/NTP quaternary complex shows AT-9010 simultaneously binds to both NiRAN and RdRp PROTEIN active site of nsp12, blocking their respective activities. AT-527 is currently in phase II clinical trials, and is a potent inhibitor of SARS-CoV-1 and -2, representing a promising drug for COVID-19 MESHD treatment.

    Dual targeting of cytokine storm and viral replication in COVID-19 MESHD by plant-derived steroidal pregnanes in silico

    Authors: Gideon A. Gyebi; Oludare M. Ogunyemi; Ibrahim M. Ibrahim; Saheed O. Afolabi; Joseph O. Adebayo

    doi:10.21203/rs.3.rs-329239/v1 Date: 2021-03-14 Source: ResearchSquare

    The high morbidity and mortality rate of Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV-2) infection MESHD arises majorly from the Acute Respiratory Distress Syndrome MESHD and “cytokine storm” syndrome, which is sustained by an aberrant systemic inflammatory response and elevated pro-inflammatory cytokines. Thus, phytocompounds with broad-spectrum anti-inflammatory activity that target multiple SARS-CoV-2 proteins will enhance the development of effective drugs against the disease. In this study, an in-house library of 106 steriodal plant-derived pregnanes (PDPs) was docked in the active regions of human glucocorticoid receptors (hGRs) in a comparative molecular docking analysis. Based on the minimal binding energy and a comparative dexamethason binding mode analysis, a list of top twenty ranked PDPs docked in the agonist conformation of hGR, with binding energies ranging between -9.8 and -11.2 Kcal/mol, was obtained and analyzed for interactions with the human Janus kinases 1 and Interleukins-6 and SARS-CoV-2 3-chymotrypsin- like protease, Papain PROTEIN-like protease and RNA-dependent RNA polymerase PROTEIN. For each target protein, the top three ranked PDPs were selected. Eight PDPs (bregenin, hirundigenin, anhydroholantogenin, atratogenin A, atratogenin B, glaucogenin A, glaucogenin C and glaucogenin MESHD D) with high binding tendencies to the catalytic residues of multiple targets were identified. A high degree of structural stability was observed from the 100 ns molecular dynamics simulation analyses of glaucogenin C MESHD and hirundigenin complexes of hGR. The selected top-eight ranked PDPs demonstrated favourable druggable and in silico ADMET properties. Thus, the therapeutic potentials of glaucogenin C MESHD and hirundigenin can be explored for further in vitro and in vivo studies.

    Atorvastatin effectively inhibits late replicative cycle steps of SARS-CoV-2 in vitro

    Authors: Maria Isabel Zapata-Cardona; Lizdany Flórez-Álvarez; Wildeman Zapata-Builes; Ariadna Guerra-Sandoval; Carlos Guerra-Almonacid; Jaime Hincapié-García; Maria Teresa Rugeles; Juan Carlos Hernández

    doi:10.1101/2021.03.01.433498 Date: 2021-03-03 Source: bioRxiv

    Introduction: SARS-CoV-2 has caused a pandemic of historic proportions and continues to spread worldwide. Currently, there is no effective therapy against this virus. This article evaluated the in vitro antiviral effect of Atorvastatin against SARS-CoV-2 and also identified the interaction affinity between Atorvastatin and three SARS-CoV-2 proteins MESHD, using in silico structure-based molecular docking approach. Materials and methods: The antiviral activity of Atorvastatin against SARS-CoV-2 was evaluated by three different treatment strategies using a clinical isolate of SARS-CoV-2. The interaction of Atorvastatin with Spike, RNA-dependent RNA polymerase PROTEIN ( RdRp PROTEIN) and 3C-like protease ( 3CLpro PROTEIN) was evaluated by molecular docking. Results: Atorvastatin showed anti-SARS-CoV-2 activity of 79%, 54.8%, 22.6% and 25% at 31.2, 15.6, 7.9, and 3.9 {micro}M, respectively, by pre-post-treatment strategy. In addition, atorvastatin demonstrated an antiviral effect of 26.9% at 31.2 {micro}M by pre-infection treatment. This compound also inhibited SARS-CoV-2 in 66.9%, 75%, 27.9% and 29.2% at concentrations of 31.2, 15.6, 7.9, and 3.9 {micro}M, respectively, by post-infection treatment. The interaction of atorvastatin with SARS-CoV-2 Spike PROTEIN SARS-CoV-2 Spike MESHD, RdRp PROTEIN and 3CL protease PROTEIN yielded a binding affinity of -8.5 Kcal/mol, -6.2 Kcal/mol, and -7.5 Kcal/mol, respectively. Conclusion: Our study demonstrated the in vitro anti-SARS-CoV-2 activity of Atorvastatin, mainly against the late steps of the viral replicative cycle. A favorable binding affinity with viral proteins by bioinformatics methods was also shown. Due to its low cost, availability, well-established safety and tolerability, and the extensive clinical experience of atorvastatin, it could prove valuable in reducing morbidity and mortality from COVID-19 MESHD.

    Harnessing recombinase polymerase amplification for rapid detection of SARS-CoV-2 in resource-limited settings

    Authors: Dounia Cherkaoui; Da Huang; Benjamin Miller; Rachel A McKendry

    doi:10.1101/2021.02.17.21251732 Date: 2021-02-19 Source: medRxiv

    The COVID-19 pandemic MESHD has challenged testing capacity worldwide. The mass testing needed to stop the spread of the virus requires new molecular diagnostic tests that are faster and with reduced equipment requirement, but as sensitive as the current gold standard protocols based on polymerase chain reaction. We developed a fast (25-35 minutes) molecular test using reverse transcription recombinase polymerase amplification for simultaneous detection of two conserved regions of the virus, targeting the E and RdRP PROTEIN genes. The diagnostic platform offers two complementary detection methods: real-time fluorescence or visual dipstick. The analytical sensitivity of the test by real-time fluorescence was 9.5 (95% CI: 7.0-18) RNA copies per reaction for the E gene PROTEIN and 17 (95% CI: 11-93) RNA copies per reaction for the RdRP PROTEIN gene. The analytical sensitivity for the dipstick readout was 130 (95% CI: 82-500) RNA copies per reaction. The assay showed high specificity with both detection methods when tested against common seasonal coronaviruses, SARS-CoV and MERS-CoV MESHD model samples. The dipstick readout demonstrated potential for point-of-care testing, with simple or equipment-free incubation methods and a user-friendly prototype smartphone application was proposed with data capture and connectivity. This ultrasensitive molecular test offers valuable advantages with a swift time-to-result and it requires minimal laboratory equipment compared to current gold standard assays. These features render this diagnostic platform more suitable for decentralised molecular testing.

    In silico exploration of phytoconstituents from Phyllanthus emblica and Aegle marmelos as potential therapeutics against SARS-CoV-2 RdRp MESHD RdRp PROTEIN

    Authors: Khushboo Pandey; Kiran Bharat Lokhande; K. venkateswara Swamy; Shuchi Nagar; Manjusha Dake

    doi:10.21203/rs.3.rs-225174/v1 Date: 2021-02-09 Source: ResearchSquare

    Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) worldwide has increased the importance of computational tools to design a drug or vaccine in reduced time with minimum risk. Earlier studies have emphasized the important role of RNA-dependent RNA polymerase PROTEIN ( RdRp PROTEIN) in SARS-CoV-2 replication as a potential drug target. In our study, comprehensive computational approaches were applied to identify potential compounds targeting RdRp PROTEIN of SARS-CoV-2. To study the binding affinity and stability of the phytocompounds from Phyllanthus emblica and Aegel marmelos within the defined binding site of SARS-CoV-2 RdRp MESHD RdRp PROTEIN, they were subjected to molecular docking, 100ns molecular dynamics ( MD MESHD) simulation followed by post-simulation analysis. Further, to assess the importance of features involved in the strong binding affinity, molecular field-based similarity analysis was performed. Based on comparative molecular docking and simulation studies of the selected phytocompounds with SARS-CoV-2 RdRp PROTEIN revealed that, EBDGp possess stronger binding affinity (-23.32 kcal/mol) and stability than other phytocompounds and reference compound, Remdesivir (-19.36 kcal/mol). Molecular field-based similarity profiling has supported our study in the validation of the importance of the presence of hydroxyl groups in EBDGp, involved in increasing its binding affinity towards SARS-CoV-2 RdRp MESHD RdRp PROTEIN. Molecular docking and dynamic simulation results confirmed that EBDGp has better inhibitory potential than Remdesivir and can be an effective novel drug for SARS-CoV-2 RdRp MESHD RdRp PROTEIN. Furthermore, binding free energy calculations confirmed the higher stability of the SARS-CoV-2 RdRp PROTEIN-EBDGp complex. These results suggest that the EBDGp compound may emerge as a promising drug against SARS-CoV-2 and hence requires further experimental validation.

    Existence of SARS-CoV-2 RNA on ambient particulate matter samples: A nationwide study in Turkey

    Authors: Ozgecan Kayalar; Akif Ari; Gizem Babuccu; Nur Konyalilar; Ozlem Dogan; Fusun Can; Ulku Alver Sahin; Eftade Gaga; Levent Kuzu; Pelin Ari; Mustafa Odabasi; Yucel Tasdemir; Siddik Cindoruk; Fatma Esen; Egemen Sakin; Burak Caliskan; Lokman Tecer; Merve Ficici; Ahmet Altin; Burcu Onat; Coskun Ayvaz; Burcu Uzun; Arslan Saral; Tuncay Dogeroglu; Semra Malkoc; Ozlem Uzmez; Fatma Kunt; Senar Aydin; Melik Kara; Baris Yaman; Guray Dogan; Bihter Olgun; Ebru Dokumaci; Gulen Gullu; Elif Uzunpinar; Hasan Bayram

    doi:10.1101/2021.01.24.21250391 Date: 2021-01-25 Source: medRxiv

    Coronavirus disease 2019 MESHD ( COVID-19 MESHD) is caused by the SARS-CoV-2 virus MESHD and has been affecting the world since the end of 2019. Turkey is severely affected with the first case being reported on March 11th 2020. Ambient particulate matter (PM) samples in various size ranges were collected from 13 sites including urban and urban background locations and hospital gardens in 10 cities across Turkey between the 13th of May and the 14th of June, 2020 to investigate a possible presence of SARS-CoV-2 RNA on ambient PM. A total of 155 daily samples (TSP, n=80; PM2.5, n=33; PM2.5-10, n=23; PM10, n=19; and 6 size segregated, n=48) were collected using various samplers in each city. The N1 gene and RdRP PROTEIN gene expressions were analyzed for the presence of SARS-CoV-2 as suggested by the Centers for Disease Control and Prevention (CDC). According to RT-PCR and 3D-RT-PCR analysis, dual RdRP PROTEIN and N1 gene positivity were detected in 20 (9.8 %) of the samples. The highest percentage of virus detection on PM samples was from hospital gardens in Tekirda[g], Zonguldak, and [I]stanbul--especially in PM2.5 mode. Samples collected from two urban sites were also positive. Findings of this study have suggested that SARS-CoV-2 may be transported by ambient particles especially at sites close to the infection hot-spots. However, whether this has an impact on the spread of the virus infection MESHD remains to be determined. Significance StatementAlthough there are several studies reporting the existence of SARS-CoV-2 in indoor aerosols is established, it remains unclear whether the virus is transported by ambient atmospheric particles. The presence of the SARS-CoV-2 RNA in ambient particles collected from characteristic sites within various size ranges was investigated, and positive results were found in urban sites especially around Turkish hospitals. In this context, this study offers a new discussion on the transmission of the virus via ambient particles.

    DINC-COVID: A webserver for ensemble docking with flexible SARS-CoV-2 proteins MESHD

    Authors: Sarah Hall-Swan; Dinler A Antunes; Didier Devaurs; Mauricio M Rigo; Lydia E Kavraki; Geancarlo Zanatta; Mohit Kumar Divakar; Panyam Suresh; Disha Sharma; Nambi Rajesh; Rahul C Bhoyar; Dasari Ankaiah; Sanaga Shanthi Kumari; Gyan Ranjan; Valluri Anitha Lavanya; Mercy Rophina; S. Umadevi; Paras Sehgal; Avula Renuka Devi; A. Surekha; Pulala Chandra; Rajamadugu Hymavathy; P R Vanaja; Vinod Scaria; Sridhar Sivasubbu; Chloe Simela; Veronica French; Rachel Harris; Sharon A.M. Stevelink; Simon Wessely

    doi:10.1101/2021.01.21.427315 Date: 2021-01-22 Source: bioRxiv

    Motivation: Recent efforts to computationally identify inhibitors for SARS-CoV-2 proteins have largely ignored the issue of receptor flexibility. We have implemented a computational tool for ensemble docking with the SARS-CoV-2 proteins, including the main protease PROTEIN ( Mpro PROTEIN), papain-like protease PROTEIN ( PLpro PROTEIN) and RNA-dependent RNA polymerase PROTEIN ( RdRp PROTEIN). Results: Ensembles of other SARS-CoV-2 proteins are being prepared and made available through a user-friendly docking interface. Plausible binding modes between conformations of a selected ensemble and an uploaded ligand are generated by DINC, our parallelized meta-docking tool. Binding modes are scored with three scoring functions, and account for the flexibility of both the ligand and receptor. Additional details on our methods are provided in the supplementary material. Availability: dinc-covid.kavrakilab.org . Supplementary information: Details on methods for ensemble generation and docking are provided as supplementary data online.

    In vitro screening of anti-viral and virucidal effects against SARS-CoV-2 by Hypericum perforatum and Echinacea.

    Authors: Leena Hussein Bajrai; Sherif Ali El-kafrawy; Rabie Saleh Alnahas; Esam Ibraheem Azhar; Ayush Jain; Roman Sarkar; Abhishek Dubey; Syed Azeez Tehseen; Sharvan Sehrawat; Florian Douam; Nicholas Crossland; Madison M Hebert; Scott W Benzinger; Koushik T Sinha; Keith T Gagnon; Rafael Rezende; Eduardo Cilli; Guilherme Malafaia; Nicholas Thomson; Caroline Buckee; Firdausi Qadri; Tahmina Shirin

    doi:10.1101/2021.01.11.426295 Date: 2021-01-13 Source: bioRxiv

    Special Infectious Agent Unit in King Fahd Medical Research Center at King Abdulaziz University, Jeddah, Saudi Arabia, has pursed the anti-viral project field to optimize the group of medicinal plants for human-infectious diseases. We have begun virtually in this field since COVID-19 pandemic MESHD, besides our divergence in the infectious agents. In this study and based on the previous review, Hypericum perforatum (St. Johns Wort) and Echinacea (gaia HERBS(R)) were tested in vitro using Vero E6 cells for their anti-viral effects against the newly identified Severe Acute Respiratory Syndrome Coronavirus-2 MESHD (SARS-CoV-2) through its infectious cycle from 0 to 48 hours post infection. The hypericin (0.9 mg) of H. perforatum and the different parts (roots, seeds, aerial) of two types of Echinacea species (Echinacea purpurea and Echinacea angustifolia) were examined their efficacy in certain concentration and under light-dependent anti-viral activities to measure the inhibition of the SARS-CoV-2 mRNA expression of RNA-dependent RNA polymerase PROTEIN ( RdRP PROTEIN) gene and the viral load with quantitative real-time polymerase chain reaction (qRT-PCR), and to assess the neutralization of the SARS-CoV-2 spike PROTEIN receptor binding on cell culture assay. Interestingly, the mixture (H.E.) of 100 mg/mL of H. perforatum and Echinacea was tested too on SARS-CoV-2 and showed crucial anti-viral activity competing H. perforatum then Echinacea effects as anti-viral treatment. Therefore, the results of gaia HERBS(R) products, H. perforatum and Echinacea species, applied in this study showed significant anti-viral and virucidal effects in the following order of potency: H. perforatum, H.E., and Echinacea on SARS-CoV-2 infectious cycle; and will definitely required a set up of clinical trial with specific therapeutic protocol based on the outcome of this study. Author SummaryAfter an outbreak of Rift Valley Fever MESHD in the Southern region of Saudi Arabia, particularly in May 2003, Special Infectious Agents Unit (SIAU) was established and founded by Prof. Esam Ibraheem Azhar. This unit contains a full range of facilities including Biosafety Level 3, allows him and his research groups to ambulate and culture risk group 3 viruses in Saudi Arabia & Gulf States for the first time. Since that time, SIAU MESHD and our international collaboration have been extended to implement a standard protocols in the infectious agents diagnostics procedure through different mode of collaboration including exchange of expertise, joint research program and more recently a technology transfer agreements with number of international institute sharing same interests. Furthermore, we have been engaged in number of researches related to Hajj & Umrah plus number of national services with the Ministry of Health (MOH) through which, we utilize our Mobile biosafety level 3 Lab to enhance the diagnostics of MERS CoV in the Holly sites during Hajj since 2014. In our SIAU and with a powerful team, we have excellent researches made valuable contributions through in vivo and in vitro animal and human studies, and several human viral pathogens which are a threat to global health security due to millions of pilgrims visiting Saudi Arabia every year from 182 countries: with particular areas of interests in: Alkhurma Viral Hemorrhagic Fever MESHD, Dengue Hemorrhagic Fever Viruses, Rift Valley Fever Virus, MERS-CoV MESHD and more recently the new global infectious diseases threat, Sever Acute Respiratory Syndrome Coronavirus-2 MESHD (SARS-CoV-2).

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MeSH Disease
HGNC Genes
SARS-CoV-2 Proteins

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