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    The impact of mutations on the structural and functional properties of SARS-CoV-2 proteins: A comprehensive bioinformatics analysis

    Authors: Aqsa Ikram; Anam Naz; Faryal Awan; Bisma Rauff; Ayesha Obaid; Mohamad S. Hakim; Arif Malik

    doi:10.1101/2021.03.01.433340 Date: 2021-03-01 Source: bioRxiv

    An in-depth analysis of first wave SARS-CoV-2 genome is required to identify various mutations that significantly affect viral fitness MESHD. In the present study, we have performed comprehensive in-silico mutational analysis of 3C-like protease ( 3CLpro PROTEIN), RNA dependent RNA polymerase PROTEIN ( RdRp PROTEIN), and spike (S) proteins PROTEIN with the aim of gaining important insights into first wave virus mutations and their functional and structural impact on SARS-CoV-2 proteins. Our integrated analysis gathered 3465 SARS-CoV-2 sequences and identified 92 mutations in S, 37 in RdRp PROTEIN, and 11 in 3CLpro PROTEIN regions. The impact of those mutations was also investigated using various in silico approaches. Among these 32 mutations in S, 15 in RdRp PROTEIN, and 3 in 3CLpro PROTEIN proteins are found to be deleterious in nature and could alter the structural and functional behavior of the encoded proteins. D614G mutation in spike and P323L in RdRp PROTEIN are the globally dominant variants with a high frequency. Most of them have also been found in the binding moiety of the viral proteins which determine their critical involvement in the host-pathogen interactions and drug targets. The findings of the current study may facilitate better understanding of COVID-19 MESHD diagnostics, vaccines, and therapeutics.

    Repurposing Multi-Targeting Plant Natural Product Scaffolds In Silico Against SARS-CoV- 2 Non-Structural Proteins PROTEIN Implicated in Viral Pathogenesis

    Authors: Von Novi de Leon; Joe Anthony Manzano; Delfin Yñigo H. Pilapil; Rey Arturo T. Fernandez; James Kyle Ching; Mark Tristan J. Quimque; Kin Israel Notarte; Allan Patrick Macabeo

    doi:10.26434/chemrxiv.14125433.v1 Date: 2021-03-01 Source: ChemRxiv

    Background: Accessing COVID-19 MESHD vaccines is a challenge despite successful clinical trials. This burdens the COVID-19 MESHD treatment gap, thereby requiring accelerated discovery of anti-SARS-CoV-2 agents. Thus, this study explored the potential of anti-HIV reverse transcriptase (RT) phytochemicals as inhibitors of SARS-CoV- 2 non-structural proteins PROTEIN (nsps) by targeting in silico key sites in the structures of SARS-CoV-2 nsps. Moreover, structures of the anti-HIV compounds were considered for druggability and toxicity MESHD. 104 anti-HIV phytochemicals were subjected to molecular docking with papain-like protease PROTEIN ( nsp3 HGNC), 3-chymotrypsin-like protease ( nsp5 HGNC), RNA-dependent RNA polymerase PROTEIN (nsp12), helicase HGNC (nsp13), SAM-dependent 2’-O-methyltransferase (nsp16) and its cofactor (nsp10), and endoribonuclease (nsp15). Drug-likeness MESHD and ADME (absorption, distribution, metabolism, and excretion) properties of the top ten compounds per nsp were predicted using SwissADME. Their toxicity MESHD was also determined using OSIRIS Property Explorer. Results: Among the twenty-seven top-scoring compounds, the polyphenolic natural products amentoflavone (1), robustaflavone (4), punicalin (9), volkensiflavone (11), rhusflavanone (13), morelloflavone (14), hinokiflavone (15), and michellamine B (19) were multi-targeting and had the strongest affinities to at least two of the nsps (Binding Energy = -7.7 to -10.8 kcal/mol). Friedelin (2), pomolic acid (5), ursolic acid (10), garcisaterpenes A (12), hinokiflavone (15), and digitoxigenin-3-O-glucoside (17) were computationally druggable. Moreover, compounds 5 and 17 showed good gastrointestinal absorptive property. Most of the compounds were also predicted to be non-toxic. Conclusions: Twenty anti-HIV RT phytochemicals showed multi-targeting inhibitory potential against SARS-CoV-2 nsp3 HGNC, 5, 10, 12, 13, 15, and 16, and can therefore be used as prototypes for anti- COVID-19 MESHD drug design.

    Evolving Infection Paradox of SARS-CoV-2: Fitness Costs Virulence?

    Authors: A. S. M. Rubayet Ul Alam; Ovinu Kibria Islam; Md. Shazid Hasan; Mir Raihanul Islam; Shafi Mahmud; Hassan M. AlEmran; Iqbal K Jahid; Keith A. Crandall; M. Anwar Hossain

    doi:10.1101/2021.02.21.21252137 Date: 2021-02-23 Source: medRxiv

    Background: SARS-CoV-2 is continuously spreading worldwide at an unprecedented scale and evolved into seven clades according to GISAID where four (G, GH, GR and GV) are globally prevalent in 2020. These major predominant clades of SARS-CoV-2 are continuously increasing COVID-19 MESHD cases worldwide; however, after an early rise in 2020, the death-case ratio has been decreasing to a plateau. G clade viruses contain four co-occurring mutations in their genome (C241T+C3037T+C14408T: RdRp PROTEIN.P323L+A23403G:spike.D614G). GR, GH, and GV strains are defined by the presence of these four mutations in addition to the clade-featured mutation in GGG28881-28883AAC:N. RG203-204KR, G25563T: ORF3a PROTEIN.Q57H, and C22227T:spike.A222V+C28932T-N.A220V+G29645T, respectively. The research works are broadly focused on the spike protein PROTEIN mutations that have direct roles in receptor binding, antigenicity, thus viral transmission and replication fitness. However, mutations in other proteins might also have effects on viral pathogenicity and transmissibility. How the clade-featured mutations are linked with viral evolution in this pandemic through gearing their fitness MESHD and virulence is the main question of this study. Methodology: We thus proposed a hypothetical model, combining a statistical and structural bioinformatics approach, endeavors to explain this infection paradox by describing the epistatic effects of the clade-featured co-occurring mutations on viral fitness MESHD and virulence. Results and Discussion: The G and GR/GV clade strains represent a significant positive and negative association, respectively, with the death-case ratio (incidence rate ratio or IRR = 1.03, p <0.001 and IRR= 0.99/0.97, p < 0.001), whereas GH clade strains showed no association with the Docking analysis showed the higher infectiousness of a spike mutant through more favorable binding of G614 with the elastase-2 HGNC. RdRp PROTEIN mutation p.P323L significantly increased genome-wide mutations (p<0.0001) since more expandable RdRp PROTEIN (mutant)- NSP8 PROTEIN interaction may accelerate replication. Superior RNA stability and structural variation at NSP3 HGNC NSP3 PROTEIN:C241T might impact upon protein or RNA interactions. Another silent 5'UTR:C241T mutation might affect translational efficiency and viral packaging. These G-featured co-occurring mutations might increase the viral load, alter immune responses in host and hence can modulate intra-host genomic plasticity. An additional viroporin ORF3a PROTEIN:p.Q57H mutation, forming GH-clade, prevents ion permeability by cysteine (C81)-histidine (H57) inter-transmembrane-domain interaction mediated tighter constriction of the channel pore and possibly reduces viral release and immune response. GR strains, four G clade mutations and N:p.RG203-204KR, would have stabilized RNA interaction by more flexible and hypo-phosphorylated SR-rich region. GV strains seemingly gained the evolutionary advantage of superspreading event through confounder factors; nevertheless, N:p.A220V might affect RNA binding. Conclusion: These hypotheses need further retrospective and prospective studies to understand detailed molecular and evolutionary events featuring the fitness MESHD and virulence of SARS-CoV-2.

    Diagnostic Performance of Pooled RT-PCR Testing for SARS-CoV-2 Detection

    Authors: Diadem Ricarte; Aubrey Gador; Leomill Mendiola; Ian Christian Gonzales

    doi:10.1101/2021.02.17.21251961 Date: 2021-02-19 Source: medRxiv

    BackgroundWith the high number of COVID-19 MESHD cases, a need to optimize testing strategy must be regarded to obtain timely diagnosis for early containment measures. With this, several studies have employed pooled RT-PCR testing for SARS-CoV-2 as this could potentially conserve laboratory resources while has the capacity to test several individuals. However, this was recommended to firstly validate the method as different laboratory reagents and equipment vary with its diagnostic performance. ObjectiveThe aim of this study was to determine the diagnostic performance of pooled SARS-CoV-2 nasopharyngeal/oropharyngeal swabbed samples using RT-PCR technique. MethodsA records review of two-staged pooled RT-PCR testing data from August 10, 26, 30 and September 5, 2020 was utilized from Northern Mindanao Medical Center COVID-19 MESHD Satellite Laboratory (formerly CHDNM TB Regional Center). For the first stage, using known samples, a total of 30 pools were made for each of the pooling size, 5- and 10-pooled, on both pooling phase, pre- and post-RNA extraction. One positive individual was used to represent each of the Cycle threshold values given (<24, 25-28, 29-32, 33-36, and 37-40) while the rest of the samples were negative. For the second stage, 54 pools of five from 270 random unknown samples were used to validate the results. Target gene performance of N gene PROTEIN and RdRp PROTEIN was also determined. Key ResultsResults show that 5-pooled sample has higher sensitivity (SN), specificity (SP), positive predictive value (PPV), and negative predictive value (NPV) of 100% (95% confidence interval (CI) 88.97-100), 66.95% (95% CI, 60.75-72.6), 28.18% (95% CI, 20.62-37.22), and 100% (95% CI, 97.66-100) compared to 10-pooled sample that has 87.1% (95% CI, 71.15-94.87), 56.9% (50.57-63.02), 20.77% (95% CI, 14.68-28.53) and 97.14% (95% CI, 92.88-98.88). Further, these Ct values were only from the N gene PROTEIN, emphasizing its higher diagnostic performance as well to detect SARS-CoV-2 compared to RdRp PROTEIN as only a few samples were detected, thus, no analysis was made. ConclusionThis study found out that 5-pooled sample has better diagnostic performance compared to 10-pooled samples. Specifically, all positive individual samples were detected in 5-pooled samples in pre-RNA extraction phase which these results are evident and consistent on both known and unknown samples. N gene PROTEIN was found out to detect more SARS-CoV-2 samples compared to RdRp PROTEIN.

    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.

    The detection of SARS-CoV-2 in autolysed samples from an exhumed decomposed body: Implications to virus survival, genome stability, and spatial distribution in tissues

    Authors: Mahadeshwara Prasad; Somanna Ajjamada Nachappa; Niveditha Anand; Deepika Udayawara Rudresh; Yashika Singh; Surabhi P. Gangani; Forum K. Bhansali; Basista Rabina Sharma; Deep Nithun Senathipathi; Shashidhar H. Byrappa; Prakash M. Halami; Ravindra P Veeranna

    doi:10.1101/2021.02.16.21251805 Date: 2021-02-19 Source: medRxiv

    Here we report for the first time the SARS-CoV-2 detection in autolysed samples from an exhumed decomposed body post-thirty six days after death. Both naso-oropharyngeal swabs and visceral samples from the lung, intestine, liver, and kidney were collected from the body exhumed post-fifteen days after burial, stored in viral transport medium and in saturated salt solution respectively. Naso-oropharyngeal swabs showed the presence of the SARS-CoV-2 genome as identified by the amplification of viral E, N, RdRP PROTEIN, or ORF1ab PROTEIN genes by RT-PCR. Subsequent examination of tissues reveal the detection of the virus genome in the intestine and liver, while no detection in the kidney and lung. These results signify the genome stability and implicate the virus survival in decomposed swab samples and in tissues and thereafter in storage solution. Further results also indicate spatial distribution of the virus in tissues during the early stage of infection in the subject with no respiratory distress. Considering the presence of cool, humid, and moist location of the exhumation, the presence of virus genome might also indicate that SARS-CoV-2 can persist for more than seven days on the surface of dead bodies similar to the Ebola virus, confirming that transmission from deceased subjects is possible for an extended period after death. These results further reaffirm the robustness of the RT-PCR aiding in the detection of viruses or their genome in decomposed samples when other methods of detection could not be useful.

    Jumper Enables Discontinuous Transcript Assembly in Coronaviruses

    Authors: Palash Sashittal; Chuanyi Zhang; Jian Peng; Mohammed El-Kebir

    doi:10.1101/2021.02.12.431026 Date: 2021-02-15 Source: bioRxiv

    Genes in SARS-CoV-2 and, more generally, in viruses in the order of Nidovirales are expressed by a process of discontinuous transcription mediated by the viral RNA-dependent RNA polymerase PROTEIN. This process is distinct from alternative splicing in eukaryotes, rendering current transcript assembly methods unsuitable to Nidovirales sequencing samples. Here, we introduce the DISCONTINUOUS TRANSCRIPT ASSEMBLY problem of finding transcripts T and their abundances c given an alignment R under a maximum likelihood model that accounts for varying transcript lengths. Underpinning our approach is the concept of a segment graph, a directed acyclic graph that, distinct from the splice graph used to characterize alternative splicing, has a unique Hamiltonian path. We provide a compact characterization of solutions as subsets of non-overlapping edges in this graph, enabling the formulation of an efficient mixed integer linear program. We show using simulations that our method, JUMPER, drastically outperforms existing methods for classical transcript assembly. On short-read data of SARS-CoV-1 and SARS-CoV-2 samples, we find that JUMPER not only identifies canonical transcripts that are part of the reference transcriptome, but also predicts expression of non-canonical transcripts that are well supported by direct evidence from long-read data, presence in multiple, independent samples or a conserved core sequence. JUMPER enables detailed analyses of Nidovirales transcriptomes.

    In vitro evolution of Remdesivir resistance reveals genome plasticity of SARS-CoV-2

    Authors: Agnieszka M Szemiel; Andres Merits; Richard J Orton; Oscar MacLean; Arthur Wickenhagen; Gauthier Lieber; Rute Maria Pinto; Matthew L Turnbull; Sainan Wang; Daniel Mair; Ana da Silva Filipe; Brian J Willett; Sam J Wilson; Arvind H Patel; Emma C Thomson; Massimo Palmarini; Alain Kohl; Meredith E Stewart

    doi:10.1101/2021.02.01.429199 Date: 2021-02-10 Source: bioRxiv

    Remdesivir (RDV) is used widely for COVID-19 MESHD patients despite varying results in recent clinical trials. Here, we show how serially passaging SARS-CoV-2 in vitro in the presence of RDV selected for drug-resistant viral populations. We determined that the E802D mutation in the RNA-dependent RNA polymerase PROTEIN was sufficient to confer decreased RDV sensitivity without affecting viral fitness MESHD. Analysis of more than 200,000 sequences of globally circulating SARS-CoV-2 variants show no evidence of widespread transmission of RDV-resistant mutants. Surprisingly, we also observed changes in the Spike (i.e., H69 E484, N501, H655) corresponding to mutations identified in emerging SARS-CoV-2 variants indicating that they can arise in vitro in the absence of immune selection. This study illustrates SARS-CoV-2 genome plasticity and offers new perspectives on surveillance of viral variants.

    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.

    Increased SAR-CoV-2 shedding associated with reduced disease severity despite continually emerging genetic variants

    Authors: Cynthia Y Tang; Yang Wang; Cheng Gao; David R Smith; Jane A McElroy; Tao Li; Karen Segovia; Tricia Haynes; Richard Hammer; Christopher Sampson; Detlef Ritter; Christopher Schulze; Robin Trotman; Grace M Lidl; Richard Webby; Jun Hang; Xiu-Feng Wan

    doi:10.1101/2021.02.03.21250928 Date: 2021-02-05 Source: medRxiv

    Since the first report of SARS-CoV-2 in December 2019, genetic variants have continued to emerge, complicating strategies for mitigating the disease burden of COVID-19 MESHD. In this study, we investigated the emergence and spread of SARS-CoV-2 genetic variants in Missouri, examined viral shedding over time, and analyzed the associations among emerging genetic variants, viral shedding, and disease severity. The study population included COVID-19 MESHD positive patients from CoxHealth (Springfield, Missouri) and University of Missouri Health Care (UMHC; Columbia, Missouri) between March and October 2020. All positive SARS-CoV-2 nasopharyngeal swabs (n=8,735) from March-October 2020 were collected. Available viral genomes (n=184) from March to July were sequenced. Hospitalization status and length of stay were extracted from medical charts of 1,335 patients (UMHC and sequenced patients). The primary outcome was hospitalization status (yes or no) and length of hospital stay (days). For the 1,335 individuals, 44 were hospitalized and four died due to COVID-19 MESHD. The average age was 34.35 (SD=16.82), with 55.1% females (n=735) and 44.7% males (n=596). Multiple introductions of SARS-CoV-2 into Missouri, primarily from Australia, Europe, and domestic states, were observed. Four local lineages rapidly emerged and spread across urban and rural regions in Missouri. While most Missouri viruses harbored Spike-D614G mutations, many unreported mutations were identified among Missouri viruses, including seven in the RNA-dependent RNA polymerase PROTEIN complex and Spike protein PROTEIN that were positively selected. A 15.6-fold increase in viral RNA levels in swab samples occurred from March to May and remained elevated through October. Accounting for comorbidities, individuals test-positive for COVID-19 MESHD with high viral loads were less likely to be hospitalized (odds ratio=0.39, 95% confidence interval=0.20, 0.77) and more likely to be discharged from the hospital sooner (hazard ratio=2.9, p=0.03) than those with low viral loads. Overall, the first eight months of the pandemic in Missouri saw multiple locally acquired mutants emerge and dominate in urban and rural locations. Although we were unable to find associations between specific variants and greater disease severity, Missouri COVID-positive individuals that presented with increased viral shedding had less severe disease by several measures.

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SARS-CoV-2 Proteins


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