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    Different selection dynamics of S and RdRp PROTEIN between SARS-CoV-2 genomes with and without the dominant mutations

    Authors: Necla Koçhan; Doğa Eskier; Asli Suner; Gökhan Karakülah; Yavuz Oktay

    doi:10.1101/2021.01.03.20237602 Date: 2021-01-05 Source: medRxiv

    SARS-CoV-2 is a betacoronavirus responsible for the COVID-19 pandemic MESHD that has affected millions of people worldwide, with no dedicated treatment or vaccine currently available. As pharmaceutical research against and the most frequently used tests for SARS-CoV-2 infection MESHD both depend on the genomic and peptide sequences of the virus for their efficacy, understanding the mutation rates and content of the virus is critical. Two key proteins for SARS-CoV-2 infection MESHD and replication are the S protein PROTEIN, responsible for viral entry into the cells, and RdRp PROTEIN, the RNA polymerase responsible for replicating the viral genome. Due to their roles in the viral cycle, these proteins are crucial for the fitness MESHD and infectiousness of the virus. Our previous findings had shown that the two most frequently observed mutations in the SARS-CoV-2 genome, 14408C>T in the RdRp PROTEIN coding region, and 23403A>G in the S gene, are correlated with higher mutation density over time. In this study, we further detail the selection dynamics and the mutation rates of SARS-CoV-2 genes, comparing them between isolates carrying both mutations, and isolates carrying neither. We find that the S gene and the RdRp PROTEIN coding region show the highest variance between the genotypes, and their selection dynamics contrast each other over time. The S gene displays higher positive selection in mutant isolates early on, and undergoes increasing negative selection over time, whereas the RdRp PROTEIN region in the mutant isolates shows strong negative selection throughout the pandemic.

    Global surveillance of potential antiviral drug resistance in SARS-CoV-2: proof of concept focussing on the RNA-dependent RNA polymerase PROTEIN

    Authors: Alfredo Mari; Tim-Christoph Roloff; Madlen Stange; Kirstine Kobberoee Soegaard; Erblin Asllanaj; Gerardo Tauriello; Leila Tamara Alexander; Michael Schweitzer; Karoline Leuzinger; Alexander Gensch; Aurelien Martinez; Julia Bielicki; Hans Pargger; Martin Siegemund; Christian Nickel; Roland Bingisser; Michael Osthoff; Stefano Bassetti; Parham Sendi; Manuel Battegay; Catia Marzolini; Helena Seth-Smith; Torsten Schwede; Hans H. Hirsch; Adrian Egli

    doi:10.1101/2020.12.28.20248663 Date: 2021-01-04 Source: medRxiv

    Antiviral treatments for COVID-19 MESHD have involved many repurposed drugs. Currently, SARS-CoV-2 RNA-dependent RNA polymerase PROTEIN ( RdRp PROTEIN, encoded by nsp12-nsp7-nsp8) has been targeted by numerous inhibitors with debated clinical impact. Among these, remdesivir has been conditionally approved for the treatment of COVID-19 MESHD patients. Although the emergence of antiviral resistance, an indirect proxy for antiviral efficacy, poses a considerable healthcare threat, an evolutionary perspective on emerging resistant mutants is still lacking. Here we show that SARS-CoV-2 RdRp PROTEIN is under purifying selection, that potential escape mutations are rare, and unlikely to lead to viral fitness loss MESHD. In more than 56,000 viral genomes from 105 countries dating from December 2019 to July 2020 we found negative selective pressure affecting nsp12 (Tajimas D = -2.62), with potential antiviral escape mutations in only 0.3% of sequenced genomes. Those affected known key residues, such as Nsp12:Val473 and Nsp12:Arg555. Of the potential escape mutations found globally, in silico structural models show that this rarely implies loss of stability in RdRp PROTEIN. No potential escape mutation were found in our local cohort of remdesivir treated patients from the first wave (n=8). Our results indicate that RdRp PROTEIN is a suitable drug target, and that remdesivir does not seem to exert high selective pressure. Our study could be the starting point of a larger monitoring effort of drug resistance throughout the COVID-19 pandemic MESHD. We recommend the application of repetitive genome sequencing of SARS-CoV-2 from patients treated with antivirals to provide early insights into the evolution or antiviral resistance.

    Comparative analysis of loop-mediated isothermal amplification (LAMP)-based assays for rapid detection of SARS-CoV-2 genes

    Authors: Daniel Urrutia-Cabrera; Roxanne Hsiang-Chi Liou; Jianxiong Chan; Sandy Shen-Chi Hung; Alex W Hewitt; Keith Martin; Patrick Kwan; Raymond Ching-Bong Wong

    doi:10.1101/2020.12.21.20248288 Date: 2020-12-22 Source: medRxiv

    The COVID-19 pandemic MESHD caused by SARS-CoV-2 has infected millions worldwide and there is an urgent need to increase our diagnostic capacity to identify infected cases. Although RT-qPCR remains the gold standard for SARS-CoV-2 detection, this method requires specialised equipment in a diagnostic laboratory and has a long turn-around time to process the samples. To address this, several groups have recently reported development of loop-mediated isothermal amplification (LAMP) as a simple, low cost and rapid method for SARS-CoV-2 detection. Herein we present a comparative analysis of three LAMP-based assays that target different regions of the SARS-CoV-2: ORF1ab PROTEIN RdRP PROTEIN, ORF1ab PROTEIN nsp3 HGNC and Gene N PROTEIN. We perform a detailed assessment of their sensitivity, kinetics and false positive rates for SARS-CoV-2 diagnostics in LAMP or RT-LAMP reactions, using colorimetric or fluorescent detection. Our results independently validate that all three assays can detect SARS-CoV-2 in 30 minutes, with robust accuracy at detecting as little as 1000 RNA copies and the results can be visualised simply by color changes. We also note the shortcomings of these LAMP-based assays, including variable results with shorter reaction time or lower load of SARS-CoV-2, and false positive results in some experimental conditions. Overall for RT-LAMP detection, the ORF1ab PROTEIN RdRP PROTEIN and ORF1ab PROTEIN nsp3 HGNC assays have higher sensitivity and faster kinetics for detection, whereas the Gene N PROTEIN assay exhibits no false positives in 30 minutes reaction time. This study provides validation of the performance of LAMP-based assays for SARS-CoV-2 detection, which have important implications in development of point-of-care diagnostic for SARS-CoV-2.

    Routine SARS-CoV-2 wastewater surveillance results in Turkey to follow Covid-19 MESHD outbreak

    Authors: Bilge Alpaslan Kocamemi; Halil Kurt; Ahmet Sait; Hamza Kadi; Fahriye Sarac; Ismail Aydin; Ahmet Mete Saatci; Bekir Pakdemirli

    doi:10.1101/2020.12.21.20248586 Date: 2020-12-22 Source: medRxiv

    A global pandemic of Coronavirus Disease 2019 MESHD ( Covid-19 MESHD) caused by severe acute respiratory syndrome coronavirus 2 MESHD (SAR-CoV-2) declared by WHO in March 2019 is still ongoing. As of 13th of December 2020, 70 million people were infected by SARS-CoV-2 and 1.5 million people lost their lives globally (WHO, 2020). Since March 2019, diagnosis of Covid-19 MESHD cases has been done through PCR test of samples from nasopharyngeal and throat swabs. However, in March 2019, it was reported that the faeces [1] and urine [2] of all infected people contain SARS-CoV-2 MESHD. Later, numerous researchers [3-7] detected SARS-CoV-2 in faeces of both symptomatic and asymptomatic patients. Moreover, some studies [1,4,8-12] suggested the possibility of extended duration of viral shedding in faeces after the patients respiratory samples tested negative. In this respect, SARS-CoV-2 wastewater-based epidemiology (WBE), i.e., wastewater surveillance, aiming to estimate the distribution of asymptomatic and symptomatic individuals in a specific region has received worldwide attention. Various research groups worldwide [1, 13-54] have started SARS-CoV-2 detection in wastewater since WBE provides tracking whole population by testing a small number of wastewater samples in a specific region and can predict SARS-CoV-2 RNA in human faeces a few days to a week before onset of symptoms. This makes WBE quite economic tool for continual tracking of decreasing or increasing trend of the Covid-19 MESHD in a particular region. However, up to date, almost all of the WBE studies have been performed with samples from a few treatment plants. There was no reported nationwide wastewater surveillance study that has been integrated into a national Covid-19 MESHD management strategy by decision makers. Nationwide, SARS-CoV-2 surveillance studies have great potential to reflect the actual distribution of Covid-19 MESHD cases in a community by accounting not only symptomatic patients tested but also asymptomatic patients having no or mild symptoms and not been tested. As opposed to clinical surveillance studies, wastewater-based surveillance studies will reflect the number of cases in a community by testing one sample from a treatment plant serving this community instead of performing individual swab tests. Turkey, which is among the few countries that started wastewater based surveillance studies at the early stages of pandemic is a leading country, performing a nationwide surveillance study. The distribution of Covid-19 MESHD cases throughout the country via SARS-CoV-2 measurements in influent, effluent and sludge samples of wastewater treatment plants (WWTPs) located in 81 cities through May 2020- June 2020 was conducted [36, 51, 52]. In June 2020, nationwide routine sampling through 22 regional identified cities has been started. However, from June to September 2020 all samples were detected negative due to problems with RT-pCR primer targeting RdRp PROTEIN gene of SARS-CoV-2 genome. Since September 2020, routine sampling from 22 cities of Turkey with 2 weeks sampling period (weekly for mega city Istanbul) has been continued and regional Covid-19 MESHD distributions have been reported as viral loads on color-scale maps. To the best our knowledge, this is the first routine nationwide surveillance study indicating Covid-19 MESHD distribution regularly using color-scale presentation on a map.

    Mutation Landscape of SARS COV2 in Africa

    Authors: Angus A Nassir; Clarisse Musanabaganwa; Ivan Mwikarago; Vanessa Blanc; Joan Gispert; Bonventura Clotet; Nuria Izquierdo-Useros; Darryl Hill; Paolo Madeddu; Shawn A Abbasi; Whitney Pickens; Katia George; Daniel R Boutz; Dalton M Towers; Jonathan R McDaniel; Daniel Billick; Jule Goike; Lori Rowe; Dhwani Batra; Jan Pohl; Justin Lee; Shivaprakash Gangappa; Suryaprakash Sambhara; Michelle Gadush; Nianshuang Wang; Maria D Person; Brent L Iverson; Jimmy D Gollihar; John Dye; Andrew Herbert; Ralph S Baric; Jason S McLellan; George Georgiou; Jason J Lavinder; Gregory C Ippolito; Fergus Gleeson; Yper Hall; Simon G. P. Funnell; Sally Sharpe; Francisco Javier Salguero; Andrew R Gorringe; Miles Carroll

    doi:10.1101/2020.12.20.423630 Date: 2020-12-21 Source: bioRxiv

    COVID-19 MESHD disease has had a relatively less severe impact in Africa. To understand the role of SARS CoV2 mutations on COVID-19 MESHD disease in Africa, we analysed 282 complete nucleotide sequences from African isolates deposited in the NCBI Virus Database. Sequences were aligned against the prototype Wuhan sequence (GenBank accession: NC_045512.2) in BWA v. 0.7.17. SAM and BAM files were created, sorted and indexed in SAMtools v. 1.10 and marked for duplicates using Picard v. 2.23.4. Variants were called with mpileup in BCFtools v. 1.11. Phylograms were created using Mr. Bayes v 3.2.6. A total of 2,349 single nucleotide polymorphism (SNP) profiles across 294 sites were identified. Clades associated with severe disease in the United States, France, Italy, and Brazil had low frequencies in Africa (L84S=2.5%, L3606F=1.4%, L3606F/V378I/=0.35, G251V=2%). Sub Saharan Africa (SSA) accounted for only 3% of P323L and 4% of Q57H mutations in Africa. Comparatively low infections MESHD in SSA were attributed to the low frequency of the D614G clade in earlier samples (25% vs 67% global). Higher disease burden occurred in countries with higher D614G frequencies (Egypt=98%, Morocco=90%, Tunisia=52%, South Africa) with D614G as the first confirmed case. V367F, D364Y, V483A and G476S mutations associated with efficient ACE2 HGNC receptor binding and severe disease were not observed in Africa. 95% of all RdRp PROTEIN mutations were deaminations leading to CpG depletion and possible attenuation of virulence. More genomic and experimental studies are needed to increase our understanding of the temporal evolution of the virus in Africa, clarify our findings, and reveal hot spots that may undermine successful therapeutic and vaccine interventions.

    Genetic Conservation of SARS-CoV-2 RNA Replication Complex in Globally Circulating Isolates from Humans and Minks Predicts Minimal Pre-Existing Resistance to Remdesivir

    Authors: Ross Martin; Jason Perry; Tomas Cihlar; Hongmei Mo; Danielle P Porter; Evguenia S Svarovskaia; Véronique Albanèse; Kristina Dietert; Michael Mülleder; Vadim Farztdinov; Benedikt Obermayer; Sandra-Maria Wienhold; Sandro Andreotti; Thomas Höfler; Birgit Sawitzki; Christian Drosten; Leif Erik Sander; Norbert Suttorp; Markus Ralser; Dieter Beule; Achim Dieter Gruber; Christine Goffinet; Markus Landthaler; Jakob Trimpert; Martin Witzenrath

    doi:10.1101/2020.12.19.423600 Date: 2020-12-19 Source: bioRxiv

    Remdesivir (RDV) exhibits potent antiviral activity against SARS-CoV-2 and is currently the only drug approved for the treatment of COVID-19 MESHD. However, little is currently known about the potential for pre-existing resistance to RDV and the possibility of SARS-CoV-2 genetic diversification that might impact RDV efficacy as the virus continue to spread globally. In this study, > 90,000 SARS-CoV-2 sequences from globally circulating clinical isolates and >300 from mink isolates collected through early September 2020 were analyzed for genetic diversity in the RNA replication complex (nsp7, nsp8, nsp10, nsp12, nsp13, and nsp14) with a focus on the RNA-dependent RNA polymerase PROTEIN (nsp12), the molecular target of RDV. Overall, low genetic variation was observed with only 12 amino acid substitutions present in the entire RNA replication complex in [≥]0.5% of analyzed sequences with the highest overall frequency (82.2%) observed for nsp12 P323L that consistently increased over time. Low sequence variation in the RNA replication complex was also observed among the mink isolates. Importantly, the coronavirus Nsp12 mutations previously selected in vitro in the presence of RDV were identified in only 2 isolates (0.002%) within all the analyzed sequences. In addition, among the sequence variants observed in [≥]0.5% clinical isolates, including P323L, none were located near the established polymerase active site or sites critical for the RDV mechanism of inhibition. In summary, the low diversity and high genetic stability of the RNA replication complex observed over time predicts a minimal global risk of pre-existing SARS-CoV-2 resistance to RDV.

    A recombinant fragment of Human surfactant protein D HGNC binds Spike protein PROTEIN and inhibits infectivity and replication of SARS-CoV-2 in clinical samples

    Authors: Taruna Madan; Barnali Biswas; Praveen Varghese; Rambhadur Subedi; Hrishikesh Pandit; Susan Idicula-Thomas; Indra Kundu; Sheetalnath Babasaheb Rooge; Reshu Aggarwal; Dinesh Tripathi; Savneet Kaur; Ekta Gupta; Sanjeev Gupta

    doi:10.1101/2020.12.18.423415 Date: 2020-12-18 Source: bioRxiv

    Rationale COVID-19 MESHD is an acute infectious disease MESHD caused by the Severe Acute Respiratory Syndrome Coronavirus 2 MESHD (SARS-CoV-2). Human surfactant protein D HGNC ( SP-D HGNC) is known to interact with spike protein PROTEIN of SARS-CoV, but its immune-surveillance against SARS-CoV-2 is not known. ObjectiveThis study aimed to examine the potential of a recombinant fragment of human SP-D HGNC (rfhSP-D) as an inhibitor of replication and infection of SARS-CoV-2 MESHD. MethodsrfhSP-D interaction with spike protein PROTEIN of SARS-CoV-2 and hACE-2 receptor was predicted via docking analysis. The inhibition of interaction between spike protein PROTEIN and ACE-2 HGNC by rfhSP-D was confirmed using direct and indirect ELISA. The effect of rfhSP-D on replication and infectivity of SARS-CoV-2 from clinical samples was studied by measuring the expression of RdRp PROTEIN gene of the virus using qPCR. Measurements and Main ResultsIn-silico interaction studies indicated that three amino acid residues in the RBD of spike of SARS-CoV-2 PROTEIN were commonly involved in interacting with rfhSP-D and ACE-2 HGNC. Studies using clinical samples of SARS-CoV-2 positive cases (asymptomatic, n=7 and symptomatic, n=8 and negative controls n=15) demonstrated that treatment with 5M rfhSP-D inhibited viral replication by ~5.5 fold and was more efficient than Remdesivir (100 M). Approximately, a 2-fold reduction in viral infectivity was also observed after treatment with 5M rfhSP-D. ConclusionsThese results conclusively demonstrate that the calcium independent rfhSP-D mediated inhibition of binding between the receptor binding domain of the S1 subunit of the SARS-CoV-2 spike PROTEIN protein and human ACE-2 HGNC, its host cell receptor, and a significant reduction in SARS-CoV-2 infection MESHD and replication in-vitro.

    Remdesivir is a delayed translocation inhibitor of SARS CoV-2 replication in vitro

    Authors: Jack PK Bravo; Tyler L Dangerfield; David W Taylor; Kenneth A Johnson

    doi:10.1101/2020.12.14.422718 Date: 2020-12-16 Source: bioRxiv

    Remdesivir is a nucleoside analog approved by the FDA for treatment of COVID-19 MESHD. Here, we present a 3.9-[A]-resolution cryoEM reconstruction of a remdesivir-stalled RNA-dependent RNA polymerase PROTEIN complex, revealing full incorporation of three copies of remdesivir monophosphate (RMP) and a partially incorporated fourth RMP in the active site. The structure reveals that RMP blocks RNA translocation after incorporation of three bases following RMP, resulting in delayed chain termination, which can guide the rational design of improved antiviral drugs.

    Computational Analysis of Dynamic Allostery and Control in the three SARS-CoV- 2 non-structural proteins PROTEIN

    Authors: Igors Dubanevics; Charles Heaton; Carlos Riechmann; Tom C B McLeish; Theresa A Ramelot; Thomas B. Acton; Elena Moreno; Thomas Kehrer; Catherine A. Royer; Adolfo Garcia-Sastre; Robert M Krug; Gaetano T. Montelione

    doi:10.1101/2020.12.12.422477 Date: 2020-12-14 Source: bioRxiv

    Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which caused the COVID-19 pandemic MESHD, has no vaccine or antiviral drugs available to the public, at the time of writing. The virus non-structural proteins are promising drug targets because of their vital role in the viral cycle. A significant body of work has been focused on finding inhibitors which covalently and competitively bind the active site of the non-structural proteins, but little has been done to address regions other than the active site, i.e. for non-competitive inhibition. Here we extend previous work on the SARS-CoV-2 Mpro PROTEIN ( nsp5 HGNC) to three other SARS-CoV-2 proteins: host shutoff factor PROTEIN ( nsp1 HGNC), papain-like protease PROTEIN ( nsp3 HGNC, also known as PLpro PROTEIN) and RNA-dependent RNA-polymerase PROTEIN (nsp12, also known as RdRp) in complex PROTEIN with nsp7 and nsp8 cofactors. Using open-source software (DDPT) to construct Elastic Network Models (ENM) of the chosen proteins we analyse their fluctuation dynamics and thermodynamics, as well as using this protein family to study convergence and robustness of the ENM. Exhaustive 2-point mutational scans of the ENM and their effect on fluctuation free energies suggest several new candidate regions, distant from the active site, for control of the proteins function, which may assist the drug development based on the current small molecule binding screens. The results also provide new insights, including non-additive effects of double-mutation or inhibition, into the active biophysical research field of protein fluctuation allostery and its underpinning dynamical structure.

    Generation of a SARS-CoV-2 Replicon as a Model System to Dissect Virus Replication and Antiviral Inhibition

    Authors: Xi He; Shuo Quan; Min Xu; Silveria Rodriguez; Shih Lin Goh; Jiajie Wei; Arthur Fridman; Kenneth A Koeplinger; Steve S Carroll; Jay A Grobler; Amy S Espeseth; David B Olsen; Daria J Hazuda; Dai Wang; Ilya Levental; Florian Douam; Robert F. Padera; Bruce D. Levy; Clifford P. Brangwynne

    doi:10.1101/2020.12.12.422532 Date: 2020-12-13 Source: bioRxiv

    SARS-CoV-2 research and antiviral discovery are hampered by the lack of a cell-based virus replication system that can be readily adopted without biosafety level 3 (BSL-3) restrictions. Here, the construction of a non-infectious SARS-CoV-2 reporter replicon and its application in deciphering viral replication mechanisms and evaluating SARS-CoV-2 inhibitors are presented. The replicon genome is replication competent but does not produce progeny virions. Its replication can be inhibited by RdRp PROTEIN mutations or by known SARS-CoV-2 antiviral compounds. Using this system, a high-throughput antiviral assay has also been developed. Significant differences in potencies of several SARS-CoV-2 inhibitors in different cell lines were observed, which highlights the challenges of discovering antivirals capable of inhibiting viral replication in vivo and the importance of testing compounds in multiple cell culture models. The generation of a SARS-CoV-2 replicon provides a powerful platform to expand the global research effort to combat COVID-19 MESHD.

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MeSH Disease
HGNC Genes
SARS-CoV-2 Proteins

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